Abstract Holzapfel, Genevieve

ABSTRACT

HOLZAPFEL, GENEVIEVE ANNE. Structural Analysis of Active Site Conformations in Ras and Rap1 GTPases. (Under the direction of Carla Mattos).

Ras and Rap1 are monomeric GTPases in the Ras subfamily of the Ras superfamily that despite high sequence homology, have distinct diverse functions in the cell including involvement in cell proliferation and cell adhesion. GTPases undergo conformational changes associated with the transition from GTP to GDP predominately in the switch I and switch II regions. Switch I and switch II are dynamic regions that can take many different conformations. Here we examine the effect that small molecules can have on the equilibrium between different switch II conformations. We show that binding of calcium acetate in Ras to an allosteric site results in a catalytically active conformation that we call “allosteric on”. We also show that binding of DTT or DTE in the interlobal region result in a different Ras catalytically inactive conformation we call “allosteric off”. This conformation was also seen in oncogenic Ras mutants.

The intrinsic hydrolysis mechanism of Ras is currently debated. Based on the allosteric on structure we propose a two water model. Our proposed mechanism can be supported by examining the protonation state of the active site residues. We have pursued neutron crystallography as a means to visualize hydrogen positions in Ras to support our hypothesis of intrinsic hydrolysis. Deuteration and perdeuteration are utilized in neutron crystallography to improve the signal to noise ratio in the neutron data collected. Resulting crystals were grown to a large volume and when mounted on a neutron beam diffraction spots were detected. This proves feasibility of collecting neutron diffraction data on Ras.

Rap1, as the closest homologue to Ras in the Ras superfamily, provides a comparison protein for specificity determinants in Ras. The structure of Rap1 alone has not been solved. Additionally, specific analysis of Rap1 small molecule binding has also not been shown. The structure of Rap1 bound to GTP and GppNHp are also presented. These results show novel active site conformations in Rap1. The resulting active site conformations provide comparison to Ras with both differences and similarities. Furthermore, data are presented that examine small molecule binding to Rap1 using the Multiple Solvent Crystal Structures (MSCS) technique. Combined with the MSCS data of Ras, these data can disambiguate the subtle differences between Ras and Rap1.

by Genevieve Anne Holzapfel

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Biochemistry

Raleigh, North Carolina

2012

APPROVED BY:

______Carla Mattos Robert Rose Committee Chair

______Paul Wollenzien Jason Haugh ii

DEDICATION This thesis is dedicated to my husband Joe who is always willing to lend support if I need it and Ariana whose smile makes it all worth it.

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BIOGRAPHY Genevieve was born and spent her early childhood in the suburbs outside of Chicago. Her parents were divorced very early in her life. Her father worked various jobs in the financial market. Her mother had various careers including respiratory therapist and social worker. The financial situation was always tight; however, Genevieve’s grandparents were generous and provided a house for her to live in throughout her childhood. While in the Chicago area, Genevieve attended a wonderful elementary school that exposed her to many things including Argonne National Lab and Fermi Lab. After elementary school Genevieve moved to Colorado, a big change from the metro area. Colorado was a great place to spend the remainder of her youth. While in high school Genevieve was able to secure a student position with the United States Department of Agriculture as a biological lab aide. This provided a glimpse into what working in a laboratory environment is like and also provided an alternative to working as a hostess. Attending high school near Colorado State University provided an opportunity to enroll in college classes while still in high school. Genevieve attended CSU and graduated in 2003 with a degree in biochemistry and a minor in chemistry. Genevieve received the Peg Baker memorial scholarship provided initially by Spear, Kellog and Logan which was later Goldman Sachs. While in school, Genevieve had the opportunity to study abroad to the University of Glasgow in Scotland. This opportunity provided a new perspective. After graduating college Genevieve moved to North Carolina to attend NCSU and study with Carla Mattos. She was awarded an NIH biotechnology training fellowship for her second and third years in graduate school. Here she met her husband, Joe, and had her daughter, Ariana.

ACKNOWLEDGMENTS I would like to acknowledge my advisor Carla Mattos for her mentoring and support during even the worst of times. I would also like to acknowledge my committee for the assistance and advice. Special thanks for Greg Buhrman and Paul Swartz for teaching me the basics of crystallography and for many helpful discussions. I would also like to acknowledge past and present students that have helped with parts of the work presented included Kelly Daughtry, Chris Miller, Glenna Wink, J. Alex Fetzer, Chelsea Woodhall, and Phoebe Cruz.

TABLE OF CONTENTS LIST OF TABLES ...... vii LIST OF FIGURES ...... viii CHAPTER 1...... 1 Introduction ...... 1 Ras Effectors ...... 3 Rap Effectors ...... 5 Structural Regions of Ras GTPases ...... 6 Ras Hydrolysis ...... 9 Rap Hydrolysis ...... 10 Posttranslational Modification and Cellular Localization ...... 13 Ras Oncogenesis ...... 14 MSCS/FT-Map Analysis of Ras...... 16 CHAPTER 2...... 17 Allosteric Modulation of Ras Positions Q61 for a Direct Role in Catalysis ...... 17 Introduction ...... 18 Materials and Methods ...... 20 Results ...... 20 Discussions ...... 28 Contribution ...... 33 CHAPTER 3...... 34 Small Molecules Selectively Shift the Equilibrium Between On and Off Allosteric States in Ras-GppNHp Crystals ...... 34 Introduction ...... 34 Results ...... 38 Conclusions ...... 55 Materials and Methods ...... 57 CHAPTER 4 Neutron Diffraction of Ras ...... 59

Introduction ...... 59 Methods ...... 72 Results ...... 77 Discussion...... 93 CHAPTER 5 Structural Analysis of Uncomplexed Rap1A ...... 99 Introduction ...... 99 Experimental Procedures ...... 104 Results ...... 107 Discussion...... 122 CHAPTER 6 The Multiple Solvent Crystal Structures Method Performed on Rap1a ...... 128 Introduction ...... 128 Methods ...... 134 Results and Discussion ...... 136 REFERENCES ...... 145

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LIST OF TABLES CHAPTER 2 Table 2.1: Data collection and refinement statistics are presented for the four structures discussed in this article ...... 21 CHAPTER 3 Table 3.1: Data refinement and collection statistics for Chapter 3 ...... 40 CHAPTER 4

Table 4.1: Data collection and refinement statistics for perdeuterated Ras crystals

grown in D2O conditions in the P3221 space group ...... 81 Table 4.2: Data collection and refinement statistics are presented for several structures of perdeuterated Ras in the R32 space group...... 87 CHAPTER 5 Table 5.1: Data collection and refinement statistics for Rap1A bound to GTP or GppNHp ...... 108 CHAPTER 6 Table 6.1: Data collection and refinement statistics are presented for the Rap1A MSCS collected ...... 137

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LIST OF FIGURES CHAPTER 1 Figure 1.1: Phylogenic tree of the Ras superfamily ...... 2 Figure 1.2: Sequence alignment of the Ras subfamily members H-Ras, N-Ras, K-Ras, Rap1a, Rap1b and Rap2 from homo sapien ...... 3 Figure 1.3: Ribbon diagram of the structural regions in Ras ...... 7 Figure 1.4: Active site comparison of Ras-RasGAP ...... 11 CHAPTER 2 Figure 2.1: The allosteric switch in Ras ...... 23 Figure 2.2: H-bonding networks 1 and 2 linking the allosteric site to Q61 ...... 25 Figure 2.3: Switch I and switch II in the well-ordered active site in Ras-GppNHp 26 Figure 2.4: Allosteric site in structures resulting from soaked crystals ...... 27 Figure 2.5: Proposed mechanism of intrinsic hydrolysis in Ras ...... 29 CHAPTER 3 Figure 3.1: Difference density for bound DTE and DTT in the switch II/ helix 3 pocket ...... 43 Figure 3.2: Allostreric off active site comparison ...... 44 Figure 3.3: View of the allosteric site in the Ras High PEG CaAcetate soaked structure (885) ...... 45 Figure 3.4: Soak Flow Chart ...... 47 Figure 3.5: Mixed conformation electron density ...... 48 Figure 3.6: Zinc cyclen soaked switch II/helix 3 pocket ...... 52 Figure 3.7: Allosteric “on” active site comparison ...... 55 CHAPTER 4 Figure 4.1: Cartoon of Ras state 1 and state 2 ...... 61 Figure 4.2: Density map difference between deuteration levels ...... 70 Figure 4.3: Optical photographs of Ras crystals ...... 78

Figure 4.4: Neutron Laue diffraction patterns for Ras at different exposure lengths ...... 69 Figure 4.5: SDS-PAGE separation of perdeuterated Ras...... 80

Figure 4.6: Room temperature data set of Ras in P3221 crystal form ...... 82

Figure 4.7: The active site of the perdeuterated Ras grown in the P3221 crystal form collected at room temperature ...... 83 Figure 4.8: Pre-switch I variation ...... 84 Figure 4.9: Pre-switch I hydrogen bonding networks ...... 85 Figure 4.10: Room temperature comparison of Ras in the R32 crystal form ...... 91 Figure 4.11: Room temperature electron density ...... 92 Figure 4.12: Perdeuterated active and allosteric site conformations ...... 94 Figure 4.13: Room temperature perdeuterated conformations ...... 95 CHAPTER 5 Figure 5.1: The active site of Raps in complex with Raf ...... 103 Figure 5.2: Chromatogram of Rap1 run on a QHP column ...... 105 Figure 5.3: Global comparison of Rap1a ...... 110 Figure 5.4: T61 interacts with the catalytic water molecule ...... 111 Figure 5.5: Pre-bridging and bridging water molecule conformations ...... 114 Figure 5.6: T61 replaces the bridging water molecule ...... 115 Figure 5.7: A comparison of the conformations adopted by switch II in Rap1 .. 117 Figure 5.8: Comparison of Rap1 helix 3 and helix 4 residues with Ras in the allosteric on state ...... 118 Figure 5.9: Two different conformations of loop 7 are observed in the presented Rap1 structures ...... 119 Figure 5.10: Calcium Acetate binding site ...... 122 CHAPTER 6 Figure 6.1: MSCS results for H-Ras-GppNHp in the “off” state of the allosteric switch ...... 131

Figure 6.2: Electron density for GOL molecules placed in the Rap1 soaked structure ...... 143 Figure 6.3: The binding sites seen in the Rap1-GOL structure are compared to the data obtained in the Ras MSCS set ...... 143 Figure 6.4: GOL-I from Rap1 GOL soaked structure ...... 144

CHAPTER ONE

Introduction:

The Ras superfamily is a family of GTPases which act as molecular switches in a wide variety of signal transduction pathways. This superfamily is comprised of the Ras, Rho, Rab, Arf, and GNA subfamilies (Figure .1) [2]. Members of the Ras superfamily undergo conformational changes upon transition from the GTP-bound active form to GDP-bound inactive form [11]. These changes are greatest at the switch I (residues 30-40) and switch II (residues 60-73) regions near the active site and allow for protein binding partners to distinguish between GTP and GDP bound protein. In the GTP bound form a conformational change occurs which activates the GTPase to interact with downstream effector molecules. The transition from GDP to GTP is facilitated by Guanine Nucleotide Exchange Factors (GEFs) which bind to the GDP-bound form. This interaction loosens the active site and allows for the release of GDP. Once GDP is released, the GTPase binds GTP, as it is encountered at higher concentration in the cell [12]. To turn the signal off, GTP is hydrolyzed to GDP. GTPases possess an intrinsic GTP hydrolysis rate that is typically slow. The binding of GTPase Activating Proteins (GAPs) speeds up the intrinsic hydrolysis rate. Maintaining the proper balance between an active or inactive GTPase is essential in the cell because the balance is used to filter, amplify or time upstream signals [13].

The Ras subfamily is comprised of several members including Ras, Rap, Ral, R-Ras, and Rheb [2] (Figure .1). Ras has served as a model protein to compare proteins in both the Ras sub and superfamily owing to it being characterized early and to its prominent role in about 30% of human cancers. Ras has three isoforms, H-Ras, K-Ras, and N-Ras which are sequentially similar (>95%) with much of the variation occurring at the C-terminus. Rap1 is the closest homolog to Ras having a >50% sequence identity. Rap consists of Rap1a, Rap1b, Rap2a, Rap2b and Rap2c. Rap1 and Rap2 share >60% sequence identity. The sequence identity for

Rap1a and Rap1b is also high at >90% and like the isoforms of Ras most of the variation occurs in the C-terminal hyper variable region (HVR) (Figure 1.2). Rap1 and Ras have effector binding regions that have a high sequence similarity (switch I). The similarity between Ras and Rap1 makes Rap1 a good model for determining Ras specificity.

Figure 1.1: Phylogenic tree of the Ras superfamily. The Ras superfamily consists of five subfamilies, Rho (green) GNA (brown), ARF (yellow), Rab (cyan), and Ras (magenta), shown as an unrooted phylogenetic tree ( From Colicelli 2004) .[2]

Figure 1.2: Sequence alignment of the Ras subfamily members H-Ras, N-Ras, K-Ras, Rap1a, Rap1b and Rap2 from homo sapien. The important structural regions the P-loop (green), switch I (magenta), switch II (cyan), the guanine nucleotide binding pocket (yellow) and the HVR (grey) are highlighted. An important residue difference in Ras and Rap is position 61 which is highlighted in red and is part of switch II. Sequence alignment was performed via ClustalW2.

Ras Effectors:

Ras has many known effectors with which it interacts in a GTP dependent manner including Raf, PI3K, RalGDS, RASSF (NORE1), Rin1, Tiam, Af6, PLCε, PKCζ and functionally related isoforms. Fortunately, there is structural information available for Ras effector complexes with the complexes of Ras(G12V)-PI3K [14], Ras(E31K)-RalGDS [15], Ras(D30E,E31K)-

NORE1A-RBD (K302D) [16], and Raps-RafRBD [17] having been solved by X-ray crystallography. The Ras binding domain (RBD) is a putative binding region for Ras on the effector protein. There are three classifications of RBD: Raf and Tiam1; PI3K; and the Ras association (RA) domain in RalGDS and AF6. While switch I is implicated in the binding of all known Ras effectors, secondary binding sites are possible as revealed by mutagenesis, NMR, and X-ray crystallography studies. For example, RafRBD binds switch I, but the Ras cystine rich domain (CRD) also interacts with Ras at a region composed of residues immediately preceding and following switch I [18]. PI3K, RalGDS and NORE1A all form interactions with switch II as well as switch I.

Much focus has been placed on the role of Ras in Raf activation as part of the Mitogen- Activated Protein Kinase (MAPK) signal cascade. This pathway exists in all eukaryotes and leads to cell differentiation, proliferation, survival, apoptosis and thus is implicated in oncogenesis [19]. Ras is activated in this pathway where it binds and activates Raf, a MAPK Kinase Kinase (MAPKKK). Raf in turn phosphorylates and activates MEK (MAPKK). The signal is then propagated through MEK phosphorylation and activation of ERK (MAPK) which once activated can interact with transcription factors. The Ras-Raf interaction has a high affinity of 3.5nM [20]. This is compared with the weaker affinity of PI3K and RalGDS for Ras, which is in the µM range [14, 21]. Another Ras effector with a high affinity is NORE1A, which functions as a tumor suppressor. Though NORE1A has a low Kon similar to weak Ras -1 protein binders, it has a slow Koff of 0.1s which gives it a similar affinity for Ras as that of Raf [16]. RasGAP (p120) has an affinity for Ras in the µM range similar to RalGDS and PI3K. Both Raf and NORE1A have a much higher affinity for Ras than does RasGAP. This casts doubt as to whether GAP could compete for Ras binding with high affinity proteins such as Raf, especially given the concentrations for these proteins in the cell [22].

Rap1 Effectors:

Rap1 has similar effector binding regions to Ras and can interact with some of the same effector proteins including Raf, RASSF, PI3K and RalGDS [23]. Rap1a, like Ras, gained notoriety for its ability to bind Raf. It was shown that Rap1 (originally named Krev-1) was able to revert K-Ras transformed NIH3T3 cells [24]. It was proposed that Rap1 acted as an antagonist to Ras by binding Raf and sequestering away. However, more recent studies with either full-length transfected or endogenous Rap1 do not show that Rap1 is able to affect the functioning of Raf in vivo [25]. Given that Ras and Rap1 are localized to different areas of the cell it is possible that though both are capable of binding the same proteins those interactions are regulated by factors associated with spatial localization.

Much study of Rap1 now focuses on its role in cell-cell adhesion and cell-cell junction formation involving interactions with effector proteins such as AF-6, Krit1, RAPL (a RASSF splice form), Riam, RacGEFs (Tiam1 and Vav2) and RhoGAPs (RA-RhoGAP and Arap3) [26]. Through these interactions, Rap1 plays an important role in the specialized cells of the cardiovasculature reviewed in Jeyaraj et al [27]. Using knockout mice, animal models and experiments in cells it has been shown that Rap1 has a significant role in blood vessel formation and permeability, platelet aggregation, and cardiac myocyte growth and survival [28-31]. It was also shown that Rap1 is involved in migration, adhesion, or development of hematopoietic cells [27, 32, 33]. Information on the involvement of Rap1 in disease progression in the cardiovasculature is currently being investigated [27]. While much structural information is available for Ras in complex with effectors, there is little information available for Rap1 or Rap2. The Rap1-Raf structure is the only effector complex solved to date.

Structural Regions of Ras GTPases:

Ras GTPases consist of a catalytic core (1-166) and a HVR (167-189) that is post translationally modified. The catalytic core consists of six β strands, five α helices and 10 loop regions [34]. Ras can be split into two global regions; the N-terminal lobe 1 (residues 1-86) and the C-terminal lobe 2 (87-171) (Figure 1.) [35]. Lobe 1 consists of the active site components including switch I, switch II, the P-loop and most of the nucleotide binding pocket. This lobe can be termed the effector lobe as it contains the sites of protein-protein interaction. Lobe 2 contains the membrane-interacting portion of the protein and can be termed the allosteric lobe [5]. Amongst Ras isoforms the effector lobe is 100% conserved and the allosteric lobe has 95% sequence identity. There is also higher sequence identity in effector lobe compared with the allosteric lobe between Ras and Rap1. The important structural regions in Ras, discussed below, are highlighted in Figure 1..

The Ras family contains two highly dynamic regions, switch I and switch II which undergo conformational changes upon GDP/GTP binding [34]. Switch I, comprised of residues (30- 40), is the primary effector binding region and is involved in downstream effector binding as well as interaction with GAPs. This region is highly similar in Ras and Rap1 with the only variation seen at residues 30 (Ras has a Asp and Rap has a Glu) and 31 (Ras has a Glu and Rap has a Lys). The similarity in the primary effector binding region makes it unsurprising that Ras and Rap1 are able to interact with many of the same downstream effectors. It is believed that the charge reversal between Ras and Rap1 relates to effector specificity [36].

Figure 1.3: Ribbon diagram of the structural regions in Ras. Structure of H-Ras bound to the nonhydrolyzable GTP analog, GppNHp (grey). The effector lobe is shown in blue with switch I, switch II and the P-loop highlighted in cyan. The allosteric lobe is shown in green with the guanine nucleotide binding pocket shown in yellow.

Switch I functions not only in facilitating interactions with downstream effectors, but it also comprises part of active site with the P-loop and switch II. Switch I has been shown through NMR to adopt two states, state 1 and state 2, where state 1 corresponds to conformations where Y32 is away from the nucleotide in what we call an “open conformation” and state 2 is where Y32 is found in near the nucleotide in a “closed conformation” [37, 38]. It has

been shown that the ratio between state 1 and state 2 varies in the Ras family. The state 1 population is 36±2% in H-Ras, 93±2% in M-Ras and 5±1% in Rap1 [39]. In both Rap1 and Ras state 2 is the state that interacts with downstream effectors [17]. State 1 is the Ras switch I conformation adopted in the Ras-RasGAP complex [40]. Conversely, in the Rap1-RapGAP complex switch I is in the state 2 conformation [41]. It was proposed that Y32 is a nucleotide sensing residue and that it helps stabilize switch I in the state 2 conformation when bound to GTP [13]. Another switch I residue, T35, is a conserved residue that makes direct interactions with both the ɣ-phosphate and the active site Mg2+ and is necessary for stabilization of switch I in the GTP-bound form. Mutation of T35 to Ser or Ala results in a disordered switch I as observed in NMR experiments [37].

Switch II is comprised of residues 60-73 which NMR shows adopts multiple conformations [42]. This is reflected in Ras crystals structures, where switch II tends to either be stabilized by crystal contacts or disordered. While switch I is the primary effector binding region, switch II can also interact with effectors such as NORE1A, PI3K and RalGDS. Switch II also forms interactions with GAP and with GEF proteins. Residue 64 (Ras Y64, Rap1 F64) is important in binding to downstream effectors for both Ras and Rap1. In Ras and Rap1 mutation of these residues affects binding to NORE1A [16] and to their respective GAPs [41, 43]. It is also proposed that residue 64 is a specificity determinant for GAP binding Ras or Rap1. Like switch I, switch II undergoes conformational changes upon GTP binding. G60 is a nucleotide sensing residue with its backbone amide forming a hydrogen bond to the ɣ- phosphate. Another important switch II residue is residue 61 which is crucial for hydrolysis in Ras [44]. This residue varies in Ras and Rap1 where in Ras residue 61 is a Gln and in Rap1 residue 61 is a Thr. The importance of this residue is detailed in the Ras Hydrolysis and Rap Hydrolysis sections below.

The P-loop (10-17) and nucleotide binding pocket complete the nucleotide binding regions. The P-loop helps stabilize the negative charge of the phosphates comprising the bound

nucleotide through interactions with the backbone amides of residues 14-17. The negative charge is also partially stabilized by a positively charged magnesium ion. The coordination of this conserved magnesium ion involves residues S17 and T35, the β and ɣ-phosphates, and two water molecules stabilized by D33, T58, and D57. Magnesium is required for hydrolysis and can affect the affinity for GTP or GDP. Mutation of P-loop residues G12 and G13 are commonly seen in Ras oncogenesis and result in disruption of the active site. The guanine nucleotide binding pocket is comprised of residues 116-119 (NKXD) and 145-147 (SAK). The three nucleotide binding regions are connected through hydrogen bonding involving N116 [45].

Ras Hydrolysis:

Hydrolysis in Ras proceeds through two mechanisms: intrinsic and GAP-stimulated. The intrinsic mechanism is slow and is dependent on Q61, where mutation of Q61 reduces the intrinsic rate of hydrolysis by ten-fold [44]. Residue Q61 is also important in GAP stimulated hydrolysis. Both the mechanism of intrinsic and GAP stimulated hydrolysis has been debated for years. Evidence now supports a loose, dissociative-like transition state for both intrinsic and GAP stimulated hydrolysis [46]. It was observed in the canonical P3221 crystal form and in the Ras-RasGAP structure that Q61 interacts with the catalytic water molecule

(Figure 1.4). The positioning of Q61 in the P3221 space group lead to the proposal that Q61 activates a catalytic water molecule for nucleophilic attack on the ɣ-phosphate of GTP [47]. However, Gln is a weak base for such a function. Given that Q61 is the only base in the vicinity of the catalytic water molecule, it was proposed that GTP can act as a general base and abstract a proton from the catalytic water molecule [48, 49].

Our lab solved Ras in the R32 space group where switch I is in a state 2 conformation and developed an alternative mechanism of intrinsic hydrolysis [50]. In this mechanism the O1G of the ɣ-phosphate acts as a general base and abstracts a proton from the catalytic water

molecule. This proton is stabilized by a bridging water molecule located between Y32 and the ɣ-phosphate. This mechanism was based on a closed Y32 conformation seen in effector complexes. Y32 would serve to help position the bridging water molecule and it would accept a H-bond from the bridging water molecule making this water molecule more prone to accept a H from the ɣ-phosphate.

In Rho, Y32 has been shown to be important in GAP stimulated hydrolysis where mutation to Ala, Phe, Ser, Glu, or Lys reduces the rate of hydrolysis [51]. In both Rho and Ras Y32 (Y34 in Rho) is away from the active site in the GAP complex allowing for insertion of the Arginine (Arg) finger (R789) [43]. Y32 participates in hydrophobic interactions with Ras residues P34, I36 and Y64 with RasGAP residues L902 and L910 [52]. In the P3221 crystal form Y32 is similarly away from the active site in a state 1 conformation and interacts with a symmetry related nucleotide. The insertion of a symmetry related Y32 into the active site overlays with the position of the Arg finger in the Ras-RasGAP complex (Figure 1.4). In GAP stimulated hydrolysis the Arg finger helps stabilize the negative charge that develops between the β and ɣ-phosphate as the transition state is formed [46]. Kinetic isotope experiments suggest a change in electrostatic potential of the leaving group oxygen atoms when Q61 is mutated to His [46]. This suggested Q61 has a role as an active site stabilizer. The Ras-GAP complex structure shows Q61 interacting with both the catalytic water and the backbone of the Arg finger. This lead to the postulation that Q61 may help orient both the catalytic water and the Arg finger for hydrolysis [46].

Rap Hydrolysis:

Rap has a Q61T substitution when compared to Ras which gives Rap a slow rate of intrinsic hydrolysis similar to the rate observed in the Ras Q61T mutation [53]. While overall little is known about the mechanism of intrinsic hydrolysis in Rap1, mutational analysis provides clues. This slow rate of hydrolysis in Rap can be rescued by the T61Q mutation which has a

Figure 1.4: Active site comparison of Ras-RasGAP. Ras bound to GppNHp solved in the P3221 (PDB code: 1ctq warm pink) space group compared to the conformation in the Ras-RasGAP complex with GDP and AlF3 (PDB code: 1wq1 teal). Y32 is swung away from the active site (Y32-A) allowing for insertion of the Arg finger, R789, or Y32 from a symmetry related molecule, Y32-B (black). Superposition was based on the bound nucleotides which are shown in grey. Q61 is near the catalytic water in both the P3221 and GAP structure. Water is red in the P3221 structure and orange in the GAP structure. hydrolysis rate similar for that observed in Ras [53]. Hydrolysis experiments suggest that T61 is not involved in the intrinsic hydrolysis reaction. Mutation to either T61L or T61A does not significantly alter the intrinsic hydrolysis rate [53]. Unlike in the case of Ras, mutation of residue 61 in Rap1 does not change the rate of GAP stimulated hydrolysis, though mutation of residue 61 was shown to affect the affinity of the Rap1-Rap1GAP complex [53]. GAP stimulated hydrolysis also proceeds differently in Rap1 than in Ras.

Rap1 specific GAPs contain several Arg residues, however, mutation of these residues affects binding between Rap1 and Rap1GAP and not the hydrolysis rate. Instead RapGAPs

employ an Asn (D290) which was termed the Asn thumb, keeping with the Ras terminology. This Asn thumb in Rap is inserted in a different orientation from the Arg finger in Ras and overlays the position of Q61 in Ras in the Ras-RasGAP complex. It is postulated that the Asn thumb in Rap replaces Q61 in Ras and performs a similar function, in orienting the catalytic water molecule in the active site [54]. The structure of Rap1 in complex with GAP also demonstrates one other major structural difference when compared to Ras. In Rap1 in complex with GAP, Y32 is in direct interaction with the ɣ-phosphate (state 2). This is contrary to Ras where Y32 was swung away from the active site to allow insertion of the Arg finger. The position of Y32 in Rap1 would sterically clash with an inserted Arg finger. Mutation of Y32 also affects Rap1 GAP stimulated hydrolysis [41]. The Y32F mutant shows a two-fold reduction in hydrolysis rate. The Y32A mutant has a significantly impaired hydrolysis rate. It was postulated that Y32 helps stabilize the state 2 conformation of switch I in Rap1 necessary for GAP binding and that the hydroxyl group of Y32 is essential for this function [41]. In early structures of Rap2 an active site where switch I was stabilized in the state 2 conformation was revealed and it was proposed that Y32 was also a nucleotide sensitive residue that helps stabilize switch I in Rap [13].

Interestingly, certain Ras GAPs can also stimulate Rap1 GTP hydrolysis [55]. A series of mutagenesis experiments sought to examine how Ras GAP stimulated Rap1 GTP hydrolysis in the absence of Q61 [56]. It was shown that Arg finger from GAP1IP4BP (a Ras GAP) was not essential for complex formation with Rap1 but did significantly affect the rate of GTP hydrolysis. This indicates that in Rap1 the Arg finger from GAP may be playing a similar role as it does in Ras hydrolysis and that Rap1 would subsequently be in the state 1 conformation to allow for insertion of the Arg finger. Mutagenesis experiments also examined the impact of Asn and Gln residues from GAP in the active site region. These mutations did not affect the hydrolysis rate, indicating that Ras GAP stimulation of Rap1 does not utilize an Asn thumb as is in the case of Rap specific GAPs [56]. It is theorized that

the binding of RasGAP may stabilize Rap1 in a conformation that promotes hydrolysis, although the specific details of that conformation is currently elusive.

Post Translational Modification and Cellular Localization:

Amongst both Ras and Rap isoforms much of the sequence diversity occurs at the C- terminal HVR that is post translationally modified and is thought to be related to the proteins cellular localization [57]. Ras was originally observed at the plasma membrane [58], although more recent work has established its presence in some internal cellular membrane systems [59]. Rap1 is predominantly localized perinuclearly in the Golgi apparatus and late endosomes [60, 61]. Rap1 can also be located at small levels at the plasma membrane [62]. Proper targeting of Ras to the plasma membrane is important for proper cellular function including plasma membrane recruitment and activation of both Raf and RalGDS [63-65]. It was originally proposed that Rap1 could compete with Ras for these proteins. While experimental evidence clearly shows in vitro that Rap1 shares effector proteins with Ras (RalGDS, Raf for example) there is less evidence to support these interactions in vivo. This is thought to be a result of the different cellular localization of Rap1 and Raf [26].

The differences in Rap1 and Ras cellular localization is at least in part due to differences in post-translational modification. In the process of post-translational modification in Ras a farnesyl group and in Rap1 a geranylgeranyl group is added to a Cys on the C-terminus. The addition of the different moieties in K-Ras and in Rap1 is sufficient in conjunction with the poly-basic region at the C-terminus HVR in both proteins for proper membrane targeting. In H-Ras an additional palmityl group is required for proper plasma membrane targeting as H-Ras lacks a C-terminal poly-basic region. The process of Rap1 targeting is less understood than Ras targeting. It was discovered that residues 85-89 (found in a loop between β sheet 4 and helix 3) played a role in targeting Rap1a to the perinuclear region [61]. Substitution of

Rap1 residues 85-89 to mimic Ras resulted in a ubiquitously expressed protein. The reverse substitution where H-Ras residues 85-89 were substituted for Rap1 resulted in a chimera protein that was perinuclearly expressed and not located at the plasma membrane despite proper post translational modification. How this sequence functions in maintaining the perinuclear distribution in Rap1 despite the post translational modification and poly basic region is not understood, but it may involve protein-protein interactions [61].

Ras Oncogenesis:

Ras is mutated in around 30% of human cancers including mutations in three of the most lethal cancers, (pancreatic (90%), lung (30%) and colon (50%)) [66]. The high incidence of Ras mutants in cancer makes Ras a prime target for drug targeting. This therapy, however, remains elusive. There are three mutations in Ras that account for almost 97-99% of Ras mutations in cancer, G12V, G13V, and Q61L [67]. These mutations affect the hydrolysis of GTP. The G12V and Q61L mutations are GAP insensitive (where the rate of GTP hydrolysis is unaffected by the addition of GAP). In the G12V mutant, GAP insensitivity likely results from steric hindrance that prevents proper the proper active site conformation for catalysis, including placement of the Arg finger [43, 68]. To better understand mutation of position 61 seventeen Q61 mutants were created. All mutation had a similar 10-fold reduction in intrinsic hydrolysis rate, however, there was large variation in transformation efficiencies (1000X) [44]. The results showed that Q61E, Q61P and Q61G were poorly transforming. The moderately transforming mutants were Q61T, Q61W, and Q61N. Strongly transforming mutations included Q61L, Q61R, Q61K, Q61A, Q61C [44].

Work from our lab examined the different structural properties of Ras Q61 mutants with varying transformation efficiencies. The structures of Q61L, Q61V, Q61K (strongly transforming), Q61I (moderately transforming) and Q61G (weakly transforming; which was previously solved by Ford et. al [69]) were compared. Crystals were grown in CaCl2 and had

symmetry of the R32 space group. The Ras conformation seen in this space group is similar to the structure seen in the Raps-Raf complex and a Ca2+ ion is positioned to take the place of K84 in Raf. Crystal contacts stabilized switch I in the closed conformation (state 2). Switch II, on the other hand, was free of crystal contacts and was disordered in the wild type structure. In the wild type structure a water molecule (wat 189) between Y32 and the γ-phosphate was observed and was termed the bridging water molecule. This water molecule was seen in the Raps-Raf complex but not in the Rap-Raf complex. The weakly transforming Ras mutant, Q61G, revealed an active site that was similar to wild type. Switch II was also similarly disordered [50].

The moderately and strongly transforming mutants adopted a different conformation where switch II was ordered outside of crystal contacts. There was no bridging water molecule present and Y32 moved making a direct interaction with the nucleotide. This conformation was stabilized by hydrophobic interactions between Y32, P35, I36, L/V/K/I61 and Y64 and overlayed with the conformation seen in Ran-Importin-β. The Ran-Importin-β complex has been shown to impair hydrolysis of GTP and thus the conformation of the highly and moderately transforming mutants was designated to be noncatalytic [70]. Here a correlation between the stabilization of a noncatalytic conformation and transformation efficiencies was observed. Since it is thought that mutation of Q61 in Ras works through the Raf pathway the effect of Raf was examined. It was shown that both wild type and Q61L in the absence of Raf hydrolyzed GTP as shown by low levels of Ras-GTP at the end of the experimental time frame, assayed by the amount of complex with Raf as visualized by gel filtration. In wild type Ras addition of Raf before or after incubation with Ras-GTP resulted in similar results. In Q61L, however, addition of Raf during hydrolysis incubation period nearly abolished GTP hydrolysis and a higher level of complex was observed [50].

MSCS/FT-Map Analysis of Ras:

The Multiple Solvents Crystal Soaks (MSCS) is a technique where different crystals of a protein are soaked in various organic solvents. Typically, organic solvents are quite damaging and, therefore, the crystals are crosslinked before transfer into organic solvents. This technique provides insight into protein binding sites, plasticity, and hydration. In model systems such as elastase and RNAse A organic solvent positions overlay with known “hot spots” or protein interaction sites [71, 72]. In RNAse A hot spots in the active site overlayed with the binding site for known inhibitors. The ability of MSCS to pick out binding site hot spots makes this technique valuable in gathering information that could be used in drug design. FT-Map is a computational version of MSCS where molecular probes are clustered at druggable sites based on their free energy of interaction with the protein [73]. Like MSCS this technique can also garner information on sites of protein-protein interaction. Both MSCS and FT-Map were performed on Ras and gave complimentary results [5]. In the MSCS set, those structures solved in the presence of trifluoroethanol, hexane, cyclopentanol, and glycerol show an ordered of switch II [5]. The ordering of switch II overlayed well with the noncatalytic conformation seen in the highly and moderately transforming mutants as well as the Ran-Importin-β complex. We are interested in advancing the MSCS technique to determine if differences in organic solvent binding can be observed between two highly similar proteins. Rap1 in comparison to Ras would provide such a comparison given the high homology between the two proteins, similarity in binding regions and the propensity to be able to interact with the same proteins.

CHAPTER TWO

Allosteric Modulation of Ras Positions Q61 for a Direct Role in Catalysis Greg Buhrman, Genevieve Holzapfel, Susan Fetics, and Carla Mattos PNAS. 2010 March 16; 107(11): 4931–4936.

Summary:

Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the γ-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild- type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the γ-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca2+ and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.

Introduction:

The Ras/Raf/MEK/ERK signaling pathway is the most well studied of five known mitogen activated protein kinase (MAPK) cascades involved in the mediation and timing of signaling events in the cell [74]. This pathway is activated by Ras GTPase in response to extracellular signals and is involved in the control of cell proliferation, differentiation, and survival [75]. In its resting state Ras is bound to GDP and is in a conformation in which it does not interact with Raf or other effector proteins [76]. Guanine nucleotide exchange factors facilitate the release of GDP [77]. Once the more abundant GTP binds, the Ras switch I (residues 30–40) and switch II (residues 60–76) regions become poised for interaction with effector proteins, leading to the propagation of signal transduction cascades. Ras has a low intrinsic rate of GTPase activity that is enhanced by 3–5 orders of magnitude in the presence of GTPase activating proteins (GAPs), resulting in depletion of Ras-GTP as the switch is turned off [43]. The biochemical properties of Ras and its oncogenic mutants have been well characterized in the absence of Raf or other factors [78, 79], and numerous structures of wild-type and oncogenic Ras mutants have been used to study the possible mechanisms through which Ras becomes defective in its ability to hydrolyze GTP [80-84]. The switch regions in these structures, solved from the crystal form with symmetry of space group P3221, are modulated by crystal contacts to resemble the switch I and switch II conformations found in the Ras/RasGAP complex [43, 85]. Since the structure of this complex has elucidated the mechanism through which GAP enhances the GTPase activity of Ras [43], it seems reasonable, given its similarity to the canonical structure of the uncomplexed form, that it could also serve as a framework for the mechanism of intrinsic hydrolysis in Ras [47, 86]. Based on this assumption, a mechanism for intrinsic hydrolysis has been proposed with a two-water model: The γ-phosphate of GTP abstracts a proton from W189, which in turn activates the catalytic W175 for nucleophilic attack on the γ-phosphate during the hydrolysis reaction [4].

More recently, we proposed an alternative mechanism based on the structure of Ras from crystals with symmetry of space group R32 [50], where switch I and water molecules in the active site are as observed in the Raps*/Raf complex [36] and switch II is unhindered by crystal contacts. It is clear that the R32 crystal form is an excellent mimic for the Ras active site in the complex with Raf [50] and could model events that may occur in this complex in the absence of GAPs. Raf interacts with Ras-GTP through two domains: the Ras-binding domain (RBD) and the cystein-rich domain (CRD) [87]. The crystal structure of Raf-RBD in complex with Raps-GppNHp shows the conserved switch I residue Y32 with its hydroxyl group interacting with a γ-phosphate oxygen atom through a bridging water molecule [88], exactly as we see in our structure [50]. In our proposed mechanism a proton from the catalytic water molecule is shuttled via the γ-phosphate to the water molecule bridging the γ-phosphate to Y32 (which we have also named W189 due to its proximity to the γ- phosphate, although it does not overlap with the position of W189 in the P3221 crystal form) and is eventually delivered to the GDP leaving group. The fact that switch II is disordered with no electron density for Q61 means that a key catalytic residue was left out of this mechanism. In the present paper, we resolve this issue with analysis of a crystal structure of wild-type Ras in which switch II is ordered through an allosteric switch. We show that the remote allosteric site binds Ca2+ but not Mg2+ in the crystals. The result is a more complete picture of our proposed mechanism of intrinsic hydrolysis in Ras, and a paradigm shift for future studies of regulation in the Ras/Raf//MEK/ERK pathway.

*Raps refers to the Ras homologue Rap with the switch I mutations E30D,K31E that results in a switch I region identical in sequence to that of Ras.

Materials and Methods:

Ras-GppNHp Crystallization

The Ras-GppNHp protein solution consisted of 20 mg/mL protein in a stabilization buffer

(20mM Hepes pH 7.5, 50mM NaCl, 20mM MgCl2, and 10mM dithioerythritol (DTE). Crystals were grown at 18°C for 1 week using sitting drop crystallization trays containing 5μL protein solution and 5μL reservoir solution. The reservoir solution was composed of 500μL of 200mM calcium acetate hydrate, 20%w/v PEG 3350, and 0.05% n-Octyl-β-D- glucopyranoside (NOG) diluted with 50μL of stabilization buffer. Synchrotron data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamline at the Advanced Photon Source (APS).

Soaks in Calcium Acetate and Magnesium Acetate Solutions

For the calcium acetate and magnesium acetate soaks, Ras-GppNHp crystals grown as above were transferred to a solution containing 10mM Hepes pH 7.5, 30%w/v PEG 3350,

10mM MgCl2, 25mM NaCl, and either 200mM Ca(OAc)2 or 200mM Mg(OAc)2 and flash frozen after 1 or 2h for data collection. After soaking in Mg(OAc)2 for 3 days, a crystal was then transferred back to the solution containing 200mM Ca(OAc)2 and soaked for 24h before data collection. Datasets for the soaked crystals were collected on our home MAR345 area detector mounted on a Rigaku RuH3R rotating anode generator.

Results:

H-Ras with 23 residues truncated at the C-terminus is used for the structural studies and includes the entire catalytic domain, residues 1–166 (referred to as Ras throughout the paper). The structure of Ras in which switch II is disordered was obtained from crystals

grown in 200mM calcium chloride, with symmetry of the space group R32 [50]. More recently we found that the crystals grew larger if calcium acetate was used instead of calcium chloride, and the resulting model revealed structural features that are explored here. Wild-type Ras-GppNHp was solved in the presence of calcium acetate to 1.3Å resolution from a crystal with R32 symmetry. Crystals removed from the mother liquor and soaked either in 200mM calcium acetate or 200mM magnesium acetate yielded structures solved to 1.85 and 1.86Å resolution, respectively, and crystals transferred to calcium acetate after a magnesium acetate soak resulted in a 2.1Å resolution structure. Data collection and refinement statistics for the four structures are presented in Table 2.1.

Table 2.1: Data collection and refinement statistics are presented for the four structures discussed in this article. Synchrotron data were collected at 100 K for wild-type Ras-GppNHp crystals grown in the presence of calcium acetate taken directly from the mother liquor. The three datasets from crystals soaked in Ca(OAc)2, Mg(OAc)2, or backsoaked from Mg(OAc)2 to Ca(OAc)2 were collected at our home institution at 120 K on a MAR345 area detector mounted on a Rigaku RuH3R generator operating at 50 kV and 100 mA. Data were processed using Denzo HKL or HKL2000 [3] and refined with PHENIX [8] and Coot [10].

Structure of Wild-Type Ras in the Presence of Calcium Acetate Reveals Allosteric Modulation of Switch II

The overall structure of wild-type Ras in the presence of 200mM calcium acetate is similar to that in the presence of 200mM calcium chloride (PBD ID code 2RGE), with one striking exception: There is a shift of loop7, helix 3, and the C-terminal end of switch II, which culminates in the ordering of the N-terminal portion of switch II and placement of Q61 in the active site near the bridging water molecule that interacts with Y32 and the O1G atom of GppNHp (Figure 2.1). This shift is modulated by the binding of calcium acetate at a site remote from the catalytic center. The structural elements involved in the shift include the C- terminal end of helix 3 (residues 98–103) and the sequentially adjacent loop 7 (residues 104–108). Anchoring residues on either end of this shifted region interact directly with the bound acetate molecule: The side chain of R97 interacts with one of the acetate oxygen atoms and the main chain amide of V109 donates an H-bond to the other (Figure 2.1). Accompanying the shift in residues 98–108 is also a shift in residues 69–75 comprising the C-terminal portion of switch II, with R68 nestled between helix 3 and residues 60–67 at the N-terminal portion of the switch.

The Ca2+ ion binds between helix 4 and loop 7. It is hexacoordinated, interacting closely with one of the acetate oxygen atoms, three water molecules, W82, W392, W366, and the main chain carbonyl oxygen atoms of D107 and Y137, which are opposite each other in the coordination sphere (Figure 2.1). The observed shift appears to be a result of a decrease in distance between these two residues due to their involvement in coordinating the Ca2+ ion. While the carbonyl of Y137 on helix 4 changes positions only slightly, D107 shifts significantly toward Y137, bringing with it loop 7 and the C-terminal portion of helix 3. The shift creates a set of conditions leading to formation of two connected H-bonding networks, network 1 and network 2 described below, that have the effect of ordering switch II and placing Q61 in what we propose is its precatalytic conformation at the beginning of the

Figure 2.1 The allosteric switch in Ras. Ribbon diagram of Ras-GppNHp showing the shift in helix 3 and loop 7 due to the binding of calcium acetate in the allosteric site. Ras bound to calcium acetate is shown in green with a semitransparent surface. Note the presence of switch II in the model. Ras in the absence of calcium acetate is in yellow (PBD ID code 2RGE). The GppNHp and acetate (with van der Waals dot surface) are shown in stick. (Upper Left) Details of the calcium acetate binding site with a 2Fo-Fc electron density map contoured at the 1σ level. Dashed lines indicate H bonds and calcium ion coordination. All figures were generated in PyMol. hydrolysis reaction. These H-bonding networks comprise the elements of an allosteric switch, which in the present structure is in the “on” conformation, and which is “off” in our previously published structures of wild-type Ras and of the RasQ61L oncogenic mutant [50]. Network 1 includes the calcium acetate with R97 at the center. It involves H94, R97, E98, and K101 (on the side of helix 3 facing away from switch II), the side chains of D107 (loop 7), and Y137 (helix 4) (Figure 2.2A). This network, consisting entirely of protein atoms and the calcium acetate, clusters in a space that is largely occupied by crystallographic water molecules in the structure of Ras in the presence of calcium chloride. With the helix 3 residues that face helix 4 placed within network 1, the helix 3 residues Y96, Q99, and R102

facing switch II are in position to contribute, along with switch II residues M72, Y71, D69, R68, S65, E62, and several water molecules, to network 2 centered around R68 (Figure 2.2B). This leads to an extension of the α-helix found in the C-terminal half of switch II. The extended switch II helical structure places key elements important in ordering the N- terminal half of the switch, from which the side chain of catalytic residue Q61 extends. For example, S65 in the ordered switch II conformation makes a good H bond with the main chain carbonyl group of E62, while its carbonyl group connects to Q99 on helix 3 through water molecule W391 (Figure 2.2B). Q99 in turn links to R68 through W384. Surprisingly, the side chain of E62, which is disordered in all other available structures of Ras, is very well ordered and interacts through water molecule W176 with the side chain oxygen atom of Q61. In addition to interacting with the side chain of E62 through W176, the side chain of Q61 H-bonds to the active site water molecule, W189, through its other lone pair electrons. Water molecule W176 also links Q61 to the side chain of R68 as well as to helix 3 residues D92 and Y96 through an H-bonding network involving W9, W28, and W367 (Figure 2.3). While the side chain of Q61 is involved in this network, its carbonyl group is bridged to the side chain of R68 through water molecule W372. Due to resonance stabilization involving the aromatic ring, the Y32 hydroxyl oxygen atom has its two lone pairs confined to the plane of the ring. Thus, in addition to its interaction with the active site bridging water molecule W189, it is involved in a Hydrogen bonding network through which it is linked to the side chain of N86 at the beginning of helix 3. The C-terminal end of helix 3 is at the center of the allosteric switch. This network is shown in detail in Figure 2.3. While the side chain of Q61 is involved in this network, its carbonyl group is bridged to the side chain of R68 through water molecule W372. The net effect of all these interactions is that Q61 is positioned through both backbone and side chain H bonds that place it in the active site and that connect it through networks 1 and 2 to the remote allosteric site, occupied in our structure by the calcium acetate as a mimic to a yet undiscovered natural modulator.

Figure 2.2: H-bonding networks 1 and 2 linking the allosteric site to Q61. The structures in the presence and absence of calcium acetate are shown in green and yellow, respectively. Dashed lines indicate H- bonding interactions involved in the network. (A) Network 1 centered on R97. Shift in helix 3 not visible from this orientation. (B) Network 2 centered on R68, showing the interactions that stabilize switch II. In this orientation the shift in helix 3 is clearly visible. Note the steric overlap between the ordered switch II in the calcium acetate bound structure (green) with helix 3 residues in the structure with an empty allosteric site (yellow). R68 is connected through single water molecules to Y96 (W367) and Q99 (W384) on helix 3, and the carbonyl group of Gly60 (W373) and Q61 (W372) on switch II. All four water molecules are unique to the calcium acetate-bound structure.

The active site features reported for the wild-type Ras structure solved in the presence of calcium chloride [50] are also observed in the calcium acetate bound structure, with the added feature of the ordered switch II with placement of the Q61 near the bridging water molecule W189. Thus, in both structures, all of switch I is in the same conformation as found in the Raps/Raf-RBD complex with Y32 closed over the nucleotide and interacting with it through the bridging water molecule. Two water molecules interact closely with the Y32 side chain oxygen atom: On one side is W396, which links to N86 at the N-terminal end of helix 3 through an H-bonding network involving W162 and W101, and on the other is the bridging water molecule, W189, which in turn H-bonds to the O1G atom of the γ-phosphate of GppNHp (Figure 2.3). The T35 residue maintains its well-known interactions within the active site: Its side-chain hydroxyl group coordinates the Mg2+ ion and its main-chain carbonyl oxygen atom makes a good H bond to the catalytic water molecule (W175). In

Figure 2.3 Switch I and switch II in the well-ordered active site in Ras-GppNHp. The 2Fo-Fc electron density map drawn around protein atoms and the nucleotide is contoured at the 1σ level and depicted in gray. The map showing the network of water molecules connecting switch II and the active site with helix 3 is contoured at the 1.3σ level and depicted in blue for clarity. The residue numbers are given in the corresponding color scheme. addition to donating an H bond to the carbonyl oxygen atom of T35 and to the O1G atom of the nucleotide analogue as observed in the calcium chloride condition, in the presence of calcium acetate the catalytic water molecule also accepts an H bond from the backbone N atom of the Q61 residue, which in our previously published structure (PBD ID code 2RGE) is disordered.

Ras Binds Ca2+ but not Mg2+ at the Allosteric Site

As an initial test of the specificity of the allosteric site for Ca2+, crystals grown in the presence of calcium acetate were removed from the mother liquor and soaked in stabilization solutions containing either 200mM calcium acetate or 200mM magnesium acetate. The soak in 200mM calcium acetate is a control to make sure that a transfer to stabilization solution in itself does not remove the Ca2+ ion and that comparisons are made

between calcium acetate and magnesium acetate solutions that are otherwise identical. Figure 2.4A and B shows the allosteric binding site in the two resulting structures. The structure soaked in calcium acetate is the same as that described above from crystals taken directly from the mother liquor, with clear electron density for the calcium acetate in the allosteric site. The soak in magnesium acetate, however, resulted in a structure with an “empty” allosteric site and with an alternate conformation for R97 typical of the off state of the allosteric switch, overlapping with the acetate binding site as seen in crystals grown in calcium chloride. Although there are other features of the off state, such as an alternate position of Y96 relative to R68 and a small shift of loop 7 toward the off state, the full shift is not made, probably due to constraints of the crystal packing. Crystals soaked in magnesium acetate and then transferred back to calcium acetate show a bound Ca2+ ion and acetate molecule as seen in Figure 2.4C, regaining all of the features of the on state of the allosteric switch.

Figure 2.4: Allosteric site in structures resulting from soaked crystals. (A) Soak in calcium acetate. (B) Soak in magnesium acetate. (C) Backsoak from magnesium acetate to calcium acetate. The three structures are superimposed on the Ras structure from crystals taken directly from the mother liquor containing calcium acetate, depicted in gray. The 2Fo-Fc electron density maps were contoured at the 1σ level.

Discussion:

Proposed Mechanism of Intrinsic Hydrolysis

It is now established that intrinsic hydrolysis in Ras occurs through a loose, dissociative-like transition state (TS) with significant concerted character, as does the GAP-catalyzed reaction [89]. This does not rule out a mechanism in which the GTP itself receives a proton from the catalytic water molecule [90]. Within these parameters, we propose a general outline for catalysis deduced from our structure of the ground state of Ras with an activated allosteric switch, augmenting the mechanism we previously published [50]. As the reaction proceeds, a proton is shuttled from the catalytic water molecule via the O1G atom of the γ- phosphate of GTP to the bridging water molecule, which is particularly prone to accept an H bond, as it can donate H bonds to both Y32 and Q61 (Figure 2.5). This would promote development of a partial positive charge in the active site near the β-γ bridging oxygen atom of GTP to stabilize a dissociative-like TS creating a counterpart, albeit weaker, to the arginine finger provided by GAPs. This is the key element lacking in our previously published mechanism. It is consistent with kinetic isotope effect experiments in which it was concluded that Q61 must mediate stabilization of negative charges on the leaving group oxygen during the reaction [89]. We envision that as the reaction proceeds, W189 moves toward the β-γ bridging oxygen atom of GTP and away from the Q61 side chain, which can then flip to interact directly with the γ-phosphate in a manner analogous to that observed in the Ras/RasGAP structure [43]. Indeed the ordered structure of switch II in our Ras- GppNHp model is not so different from that observed in the complex with GAP, and a flip of the Q61 side chain would put it within reach of the γ-phosphate. This process would release the allosteric switch, resulting in a disordered switch II, ultimately leaving more room for the release of the inorganic phosphate product at the end of the reaction. The allosteric switch that we propose leads to enhanced catalysis is consistent with recently published molecular dynamics simulations where correlated motions are observed between switch II

Figure 2.5: Proposed mechanism of intrinsic hydrolysis in Ras. (A) Ras-GppNHp active site showing Y32, Q61, the catalytic (W175) and bridging (W189) water molecules near the nucleotide. Electron density (gray) is from a 2Fo-Fc map contoured at the 1σ level. H-bonding interactions involving these residues in the ground state are shown in red dashed lines. The orange dashed line indicates the H bond between W189 and the β-γ oxygen of GTP that we propose helps stabilize the transition state of the reaction. (B) Schematics of the reaction mechanism leading to the transition state. Hydrogen atoms are not shown except those for W175 and W189 and hydrogen atoms that interact directly with them. The flexibility in directionality of H bonds may be important. residues 66–74 and helix 3/loop 7 residues 93–110 in Ras-GTP, but not in Ras-GDP [91]. Correlated or “breathing” motions involving switch II and helix 3 have also been observed experimentally by NMR [92].

Reconsidering the Relevance of Intrinsic Hydrolysis in Ras

Ras is a protein that has long been considered to undergo conformational changes associated with regulation strictly due to the state of the bound nucleotide, where it can interact with effectors in the GTP-bound but not in the GDP-bound state. We have uncovered an additional level of modulation of switch II (and along with it catalytic residue Q61) for Ras in the GTP-bound state that may influence its ability to hydrolyze GTP and

therefore the timing of interaction with Raf. We propose that the allosteric switch is not general to all effectors, but is activated by Raf, which interacts with Ras through switch I [88, 93]. In addition to leaving a free switch II in contrast to RasGAP and other effectors that bind both switches, Raf is unique in that its 3.5nM affinity for Ras [20] is 3 orders of magnitude stronger than the micromolar affinity observed for Ras/RasGAP [94] and for Ras interaction with effectors such as PI3K [14] and RalGDS [21]. Given that GAPs and effectors have overlapping binding sites [95], GAPs must displace the effectors in order to turn off the GTPase signaling and directly control the timing of the Ras/effector interaction. Since RasGAP, PI3K and RalGDS have similar affinities to Ras, it is likely that GAP-catalyzed hydrolysis is the primary mechanism of negative regulation of pathways involving these effectors. However, it is unlikely that GAPs can outcompete Raf at the concentrations found in cells1 [22]. It has been suggested that the role of RasGAP in regulating the Ras/Raf pathway could be to deplete the pool of available free Ras and that Raf could somehow increase the GTP hydrolysis rate [95, 96]. However this has not been part of the discourse in the literature for the last 15 years, given the slow intrinsic hydrolysis rate of Ras and the lack of rate enhancement observed in vitro in the presence of Raf. Given our discovery of the allosteric switch, this possibility must be seriously reconsidered and investigated further. We propose a model in which the binding of Raf is controlled by the state of the bound nucleotide through interaction of the RBD, but the timing of interaction is modulated allosterically at the remote site by Ca2+ in conjunction with another cellular factor containing a negatively charged group, mimicked by the acetate in our structure.

The discovery of an allosteric switch in Ras provides insight into the mystery associated with the function of Q61 in intrinsic catalysis and the slow rate of hydrolysis observed for Ras in the absence of GAPs. It suggests a unique mechanism by which the binding of Raf and simultaneous association of a negatively charged ligand at the remote allosteric site could increase hydrolysis rates to biologically relevant levels in the presence of Ca2+, promoting

1 The concentration range is 0.1–1.6 μM for Ras and 3.0–500 nM for Raf.

attenuation of the signaling in Ras/Raf-associated pathways. It appears that in order to have a fully formed active site for intrinsic hydrolysis of GTP, Ras needs to have both switch I and switch II in highly ordered conformations. Full stabilization of switch I can be achieved with the binding of Raf. Both in free Ras and in the complex with Raf, however, switch II is likely disordered in the absence of the allosteric switch [92, 93]. In this situation the correct placement of Q61 for catalysis would be expected to be a slow, rate-determining step in the hydrolysis reaction, leading to the previously observed hydrolysis rates, where the binding of Raf alone would not be expected to have an effect. However, the simultaneous binding of Raf at switch I, placing the hydroxyl group of Y32 in position to interact with the bridging water molecule and enable the allosteric switch, and a natural ligand at the remote allosteric site (perhaps a membrane phospholipid or carboxylate group) in the presence of Ca2+ could stabilize switch II through the mechanism we described above involving H- bonding networks 1 and 2. The completion of the active site would then no longer be a rate- determining step in the reaction, leading to an increase in intrinsic hydrolysis rate. Calcium has been shown to be one of the activators of the Ras/Raf/MEK/ERK pathway, promoting increases in Ras-GTP levels leading to the recruitment of Raf to the membrane [97]. Furthermore, we show here that the allosteric site binds Ca2+, but not Mg2+, supporting a specific role for calcium as we propose. It is conceivable that the timing of the Ras/Raf interaction is affected by the higher Ca2+ levels with binding of a membrane negatively charged group to the allosteric site once the complex with Raf is in place. An increase in Ca2+ concentration has already been shown to inactivate the Ras/Raf/MEK/ERK pathway in at least two different instances [98, 99]. Although the balance between the activating and inactivating roles of Ca2+ with respect to the Ras/Raf pathway is still not well understood, it is possible that the allosteric switch may be one of the means through which this balance is achieved by direct action of Ca2+ on Raf-bound Ras at the membrane.

The idea that a molecule at or near the membrane may act in conjunction with Ca2+ to activate the allosteric switch is supported by molecular dynamics simulations of full length

lipid-modified Ras in a 1,2-dimyristoylglycero-3-phosphocholine bilayer, showing that in Ras-GTP the catalytic domain makes more extensive interactions with the membrane phospholipids than in Ras-GDP, and is positioned such that the allosteric site near loop 7 is adjacent to the membrane [100]. The computational model shows that Ras-GTP interacts with phospholipid head groups at residues R128, R135, and Q165, anchoring helix 4 to the membrane. This is supported experimentally in cells by mutation of the two arginine residues to alanine, which results in a decrease in neurite outgrowth due to impaired signal transduction [100]. In our crystal structures of Ras-GppNHp, helix 4 is nestled against extensive crystal contacts, with R135 making a salt bridge with the terminal carboxylate group of a symmetry-related molecule. Thus, the stabilization of helix 4 by the membrane appears to be mimicked to some extent in crystals with symmetry R32. This may be an important element of the allosteric switch, as helix 4 contains Y137 (only two residues away from R135) that coordinates to the Ca2+ ion through its carbonyl group. Lack of this stabilizing interaction in solution may prevent the activation of the allosteric switch, accounting for the fact that we have been unable to measure an increase in the intrinsic hydrolysis rate by adding calcium acetate to the solutions in which the hydrolysis experiments are performed in vitro. If our assessment is correct that the crystal contacts serve a similar function to that of the membrane as far as stabilizing Y137 on helix 4, it appears that the structural components that lead to a highly ordered switch II and placement of Q61 in the active site are sensitive to both placement of Ras against the membrane in a manner that happens only in the GTP-bound form [100] and to the binding of Raf, which is necessary for the stabilization of Y32. In this situation Y32 could serve as a venue through which the binding of Raf is imposed as a requirement in the activation of Ras in lieu of activation by GAPs, ensuring that the membrane-bound Ras-GTP does not engage in futile hydrolysis of GTP to GDP before it has a chance to recruit Raf from the cytoplasm and activate the signal transduction cascade.

Contribution:

This article was published in Jan of 2010. I optimized crystallization of H-Ras that gave a high resolution 1.3Å that was deposited with a PDB code 3k8y. This structure was an improvement over previous data sets of WT Ras grown in CaAcetate based upon which the analysis for this part of the paper was made. As in previous structure, this structure clearly showed electron density for the allosteric on state including the bound CaAcetate and the side chain of Q61, but to a higher resolution which showed some of the details more clearly. I also performed a set of soaks designed to probe the specificity of the allosteric site for Ca2+. These soaks demonstrated the specificity of the allosteric site.

CHAPTER THREE

Small Molecules Selectively Shift the Equilibrium Between On and Off Allosteric States in Ras-GppNHp Crystals.

Genevieve Holzapfel, Greg Buhrman and Carla Mattos

Summary:

We have recently described an allosteric binding site in Ras where binding of calcium acetate in this site resulted in a shift in helix 3 and loop 7. The resulting shift allowed for a disorder to order transition in the active site of Ras GTPase to what we term the catalytically active conformation. Earlier we also reported that the highly and moderately transforming mutants stabilize a conformation of Ras in a catalytically inactive conformation. Organic solvent soaks of Ras using the multiple solvent crystal soaks method established several cluster sites in the interlobal region. Here we examine the binding of DTT or DTE to a site in the interlobal region referred to as the switch II/helix 3 pocket. Binding of DTT or DTE is associated with a disorder to order transition of switch II to the catalytically inactive conformation seen in the Ras oncogenic mutants. We use the small molecules acetate and DTT or DTE to selectively shift the equilibrium between different Ras conformations in the crystal environment.

Introduction:

Ras is a small monomeric GTPase that functions as a molecular switch in signal transduction [101, 102]. It is involved in cell proliferation, apoptosis, and multiple cellular functions that play critical roles in the tumorigenesis of a variety of human cancers [103, 104]. It is therefore not surprising that Ras and its mutants have been the focus of numerous

biochemical and cell biology studies [44, 50, 78, 79, 105, 106] as well as structural biology experiments primarily using the GTP analog GppNHp to obtain the activated state [5, 80, 82, 83, 85, 107, 108]. Ras is anchored to the membrane via an isoprenyl group as well as other posttranslational modifications at the C-terminus [59, 109] and is normally activated through cell surface receptors [110]. When bound to GTP, Ras propagates its signal by interacting with effector proteins such as Raf [93], phosphoinositide-3-kinase (PI3K) [14], Ral guanine nucleotide Dissociation Stimulator (RalGDS) [21], NOR1A [16] and many others [59]. Once GTP is hydrolyzed to GDP on Ras, interaction with effectors is no longer favored and signaling is turned off. The levels of Ras-GTP are kept in check by the opposing actions of guanine nucleotide exchange factors (GEFs) that catalyze the loading of GTP [77], and of GTPase activating proteins (GAPs) that increase the intrinsically slow GTPase activity of Ras for timely depletion of Ras-GTP [43]. The active site residues are situated primarily in the so- called switch I, switch II and the phosphate binding loop (P-loop) comprised of residues 30- 40, 60-76 and 10-17 respectively [111]. Oncogenic mutations interfere with the ability of Ras to hydrolyze GTP, resulting in a prolonged signal that promotes uncontrolled cell growth [104]. RasG12V and RasQ61L have received the greatest attention for being two of the most frequent point mutants commonly found in human cancers [66, 112, 113].

The accepted view of the Ras cycle implies the absolute necessity of GAPs to increase hydrolysis rates in vivo, as the intrinsic hydrolysis rates measured in vitro are too slow to be biologically relevant [78]. However, it has been known for many years that the RasG12P mutant is insensitive to GAPs, yet has a normal intrinsic hydrolysis rate and a nontransforming phenotype [80]. Furthermore, the effector protein Raf binds to Ras with an affinity that is a thousand fold greater than the affinities of GAPs for Ras, and GAP would have to displace Raf for binding [20, 94, 114]. This is not the case for effectors such as PI3K and RalGDS that have affinities comparable to that of GAP [14, 115]. We have recently discovered an allosteric switch in Ras where binding of calcium acetate in crystals with symmetry R32 promotes a shift of helix3/loop7 (residues 87-104/105-109) toward helix4

(residues 126-137) and an extensive H-bonding network that results in a disorder to order transition in the active site with placement of catalytic residue Q61 for catalysis (“on” state of the allosteric switch) [107]. This is in contrast to the structure from similar crystals grown in calcium chloride where switch II is disordered, resulting in an incomplete active site (“off” state of the allosteric switch) [50]. In our model intrinsic hydrolysis is activated by ligand binding at the allosteric site in the presence of Raf and plays a role in the control of signaling through the Ras/Raf/MEK/ERK pathway, alleviating the need for GAPs to compete with Raf for attenuation of the signal. In this context RasG12P is expected to function normally in the Ras/Raf/MEK/ERK pathway despite its insensitivity to GAPs, consistent with its normal phenotype.

Our model provides a new venue through which to understand the complexities of the structural biology of Ras and its associated function. In the canonical crystal form with symmetry P3221 [47] the highly conserved switch I residue Y32 is turned away from the nucleotide as in the Ras/RasGAP complex, in what has been determined by 31P NMR to be “state 1” [38, 115]. In the crystal form with symmetry R32, switch I is stabilized by crystal contacts with Y32 near the nucleotide as observed in the Raps/Raf-RBD complex [36] and determined by NMR to be “state 2” [115]. This is the switch I conformation associated with intrinsic hydrolysis [38]. We have therefore based our proposed mechanism for the reaction on the Ras structure obtained from crystals with symmetry R32 [107]. In the context of this switch I conformation the allosteric switch may be either “on” or “off” to modulate the conformation of switch II and we capture either one of these substrates of Ras-GppNHp by growing the crystals in calcium acetate (“on”) or calcium chloride (“off”) [105]. Since switch II is unhindered by crystal contacts in the R32 crystal form we have an excellent mimic of the Ras active site in the Ras/Raf complex, where switch II is not part of the binding interface [93].

Although clearly not a physiological ligand, we propose that the calcium acetate at the allosteric site in our crystals mimics a functional group associated with the head group of a membrane lipid, perhaps in the presence of calcium, as this site on Ras has been shown to interact with the membrane in the GTP, but not GDP-bound state [100, 116]. We have found that in the “on” state of the allosteric switch with a fully ordered active site, Y32 is closed over the nucleotide partially overlapping the position of the GAP arginine finger in the Ras/RasGAP complex [43]. In our crystals, as in the complex with Raf-RBD, Y32 interacts with a water molecule (WAT189) that bridges it to the ɣ-phosphate of GTP. Q61 is positioned by the allosteric switch to interact with this same water molecule which could receive a proton and develop a partial positive charge near the β-ɣ bridging oxygen atom of GTP to stabilize the dissociative transition state of the reaction [117, 118]. We propose that WAT189, aided by Y32 and Q61, is therefore a critical element in Ras catalysis in the absence of GAP when the allosteric switch is “on” due to the binding of Raf at switch I and a membrane-associated ligand at the allosteric site. Although we have observed these structural changes associated with ordering of the active site in our crystals, we have been unable to measure the expected increase in hydrolysis rate for wild type Ras in the presence of calcium acetate and Raf-RBD in solution [107]. This is most likely due to the fact that a salt bridge involving R135 on helix4 and a symmetry-related molecule in the crystal may be mimicking in vivo stabilization of the allosteric binding site by a salt bridge between R135 and a membrane phospholipid [100, 116]. A destabilized allosteric site in solution would limit intrinsic hydrolysis rates measurements in vitro to a state in which the allosteric switch is “off” for wild type Ras. The idea that without interaction at the allosteric site the allosteric switch is normally “off” is consistent with the fact that disorder of the switch regions, and thus an incomplete active site, is seen in solution by NMR spectroscopy [42].

Ras mutants commonly found in human cancers occur most frequently at positions 12, 13 or 61 [113]. Interestingly, rather than having a disordered switch II in the “off” state, the highly transforming Q61 mutants, including RasQ61L, have a well ordered switch II structure

stabilized in an anti-catalytic conformation by a hydrophobic cluster of switch I and switch II residues that isolate the nucleotide from bulk solvent [50]. We call this the “ordered off” state. In contrast to the “off” state where catalysis is slow, the “ordered off” state has no measurable catalytic activity [50, 105]. Interestingly, the G12V mutant does not adopt this conformation and switch II is disordered in the “off” state, just as it is in the wild type structure [105]. This structural difference between RasG12V and RasQ61L corresponds to differences in both intrinsic hydrolysis rates in the presence of Raf kinase and to differences in MEK and ERK phosphorylation levels in NIH-3T3 cells, particularly in the presence of a PI3K inhibitor that eliminates crosstalk effects to ERK due to Akt phosphorylation [105]. Taken together, these results suggest that modulation of the switch II conformation affects signaling through the Ras/Raf/MEK/ERK pathway. It is therefore of great interest to be able to affect this modulation with small molecules.

Here we show that either dithioerythritol (DTE) or dithiothrietol (DTT) binds between helix 3 and switch II in crystals, stabilizing the “ordered off” anti-catalytic conformation of switch II in wild type. We show that the allosteric switch can be predictably modulated by soaking crystals of wild type Ras-GppNHp in solutions containing either calcium acetate, which stabilizes the “on state”, or DTE/DTT, which stabilizes the “ordered off” state. Having previously found a binding site where ligands stabilize the “on” state [107], we identify one here where DTE/DTT stabilizes the “off” state and show that when both sites are “empty” we can capture a mix of the “on” and “off” states of the allosteric switch in our crystals. Furthermore, the equilibrium between the two states is reversible, dependent on the presence of the opposing molecules in the soaking solutions.

Results:

All of the experiments in this work were done with a C-terminal truncated construct of H- Ras that includes residues 1-166 (the catalytic domain), referred to as Ras throughout this

paper. In order to establish the reversibility of the “on” and “off” states in our crystals we did a series of soaking experiments as an extension of the soaks we published previously showing the specificity of the allosteric site for calcium versus magnesium [107]. In those experiments crystals were grown in mother liquor containing calcium acetate with the Ras allosteric switch in the “on” state, calcium acetate bound at the allosteric site and the active site ordered in what we propose is the catalytic conformation. Soaking in magnesium acetate resulted in an “empty” allosteric site and disruption of the H-bonding networks of the allosteric switch, but the extent of reversibility of the allosteric switch was not fully established. Here we present a systematic series of soaking experiments, clearly demonstrating that whether we start with the “on” state or “off” state of the allosteric switch we are able to induce full transition in both directions in our crystals.

DTE and DTT bind in a pocket between switch II and helix 3

When initially working out stabilization conditions for the soaks we hit upon conditions in which the crystals were very stable and diffracted to a resolution of about 1.4 Å, higher than the 1.6-1.8 Å we usually get when we take crystals directly from the mother liquor. The condition contained 30% PEG400 in addition to 30% PEG3350 (increased from the 25% PEG3350 in the mother liquor), as well as buffer and either calcium chloride or calcium acetate. As expected, we found that crystals originally grown in the presence of calcium chloride and 5mM DTE and then soaked in the stabilization conditions containing 100mM calcium chloride and no DTE had the allosteric switch in the “off” state (structure refined at a resolution so 1.39 Å – structure ID 888) (Table 3.1). Surprisingly, however, rather than a disordered switch II, we observed the “ordered off” conformation previously seen for the RasQ61L mutant [50]. Furthermore, this structure showed clear positive difference density in a Fo-Fc electron density map, consistent with binding of DTE in a cleft that forms between helix 3 and the “ordered off” conformation of switch II in a position where we previously had located a hot spot for protein/ligand interaction using MSCS experiments [5]. Soaking

Table 3.1: Data refinement and collection statistics for Chapter 3.

Structure ID 885 888 1062 1162 1187 Data collection Space group R32 R32 R32 R32 R32 Collection Temperature 100K 100K 100K 100K 100K Cell dimensions 87.414 88.521 89.029 88.704 88.822 a , b , c (Å) 87.414 88.521 89.029 88.704 88.822 133.468 134.06 135.207 134.36 134.779 α, β, ɣ (°) 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 Resolution (Å) 35-1.42 35-1.39 50-1.82 50-1.70 50-1.60 (1.44-1.42) (1.41-1.39) (1.85-1.82) (1.76-1.70) (1.66-1.60)

R sym or R merge 0.059 0.063 0.058 0.065 0.075 (0.335) (0.796) (0.793) (0.865) (0.681) I / σ 65.1 55.6 49.1 21.0 19.7 (9.1) (2.3) (2.9) (1.5) (2.2) Completeness (%) 100 99.9 99.9 99.5 99.9 (100) (98.4) (100.0) (95.0) (99.3) Redundancy 10.9 10.1 10.9 7.9 6.6 (10.7) (5.0) (9.2) (4.5) (3.8) Refinement Resolution (Å) 1.42 1.39 1.80 1.7 1.6 No. reflections 46257 39055 17736 20843 25192

R work / R free 17.07/18.88 18.07/19.63 15.92/19.41 17.77/20.05 17.89/20.26 No. atoms 1561 1494 2720 1502 1552 Protein 1345 1328 2575 1316 1317 GppNHp 1 1 1 1 1 Acetate 0 0 0 1 1 DTT/DTE 1 1 0 0 0 Calcium/Mg 4,3 2,2 2,2 2,2 2,2 Water 159 161 109 146 195 B-factors Protein 14.5 19.4 28.75 29.91 24.71 GppNHp 8.98 12.19 23.9 22.38 18.63 Acetate 37.28 28.67 DTT 35.54 43.51 Water 26.03 31 40.08 38.66 34.1 R.m.s. deviations Bond lengths (Å) 0.007 0.008 0.008 0.008 0.008 Bond angles (°) 1.154 1.258 1.060 1.117 1.129

Table 3.1: Continued Structure ID 1201 1204 1512 1515 1521 Data collection Space group R32 R32 R32 R32 R32 Collection Temperature 100K 100K 100K 100K 100K Cell dimensions 88.822 88.395 88.085 88.207 88.261 a , b , c (Å) 88.822 88.395 88.085 88.207 88.261 134.053 133.923 133.9 134.142 134.403 α, β, ɣ (°) 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 Resolution (Å) 30-1.57 50-1.72 50-1.73 50-1.57 50-1.66 (1.63-1.57) (1.75-1.72) (1.76-1.73) (1.60-1.57) (1.69-1.66)

R sym or R merge 0.067 0.054 0.100 0.064 0.082 (0.827) (0.666) (0.737) (0.295) (0.654) I / σ 37.3 49.3 33.2 52.7 39.4 (1.2) (3.0) (2.6) (7.3) (3.0) Completeness (%) 97.4 100 100 99.6 100 (79.8) (100) (100) (92.9) (100) Redundancy 9.6 10.4 10.8 10.9 11 (3.7) (8.9) (8.5) (9.0) (9.3) Refinement Resolution (Å) 1.63 1.72 1.73 1.57 1.66 No. reflections 23884 20777 21703 27768 23146

R work / R free 19.48/21.74 17.41/20.24 16.94/19.76 15.77/17.90 16.17/18.42 No. atoms 1540 1529 1495 1561 1547 Protein 1320 1318 1328 1324 1378 GppNHp 1 1 1 1 Acetate 0 1 0 0 0 DTT/DTE 1 0 1 2 2 Calcium/Mg 3,1 2,2 1,3 3,2 2,3 Water 176 171 165 184 161 B-factors Protein 23.22 26.83 20.34 15.7 19.4 GppNHp 16.18 19.84 13.6 10.55 13.4 Acetate 35.56 31.2 DTT 38.53 53.59 36.41 46.35 Water 31.78 34.89 31.96 27.43 32.3 R.m.s. deviations Bond lengths (Å) 0.008 0.007 0.007 0.008 0.007 Bond angles (°) 1.182 1.110 1.173 1.375 1.152

of crystals in the high PEG stabilization condition containing 100mM calcium acetate also gave an unexpected result. Rather than the “on” state of the allosteric switch, this structure showed the allosteric switch in the “off” state with switch II in the “ordered off” conformation (structure refined at a resolution of 1.42 Å – structure ID 885) (Table 3.1). This structure too contained a bound DTE at the helix3/switch II interface. It is clear that under the high PEG conditions with bound DTE near the active site, the presence of calcium acetate is not sufficient to promote the transition of the allosteric switch to the “on” state.

The DTE molecule in both structures binds in a pocket lined by Y96 from helix 3, with one of its two hydroxyl groups making a good H-bonding interaction with Wat365, which in turn H- bonds to the hydroxyl group of the tyrosine residue and to the side chain of D92 in helix 3 (Figure 3.1A). Wat365 is part of a network involving Wat308 and the carbonyl group of G60, the residue immediately preceding the catalytic switch II residue Q61. Wat308 is highly conserved in the Ras structures and also makes H-bonding interactions to the mainchain amide group of G12 in the P-loop and another water molecule, Wat416, which bridges it to the mainchain amide of E62. This network of H-bonding interactions linking G60 to the DTE molecule results in a shift of the N-terminal portion of switch II in this structure relative to the “off” state in which switch II is disordered (PDB code 2RGE). The DTE molecule is linked to the middle of switch II though one of its sulfur atoms that receives an H-bond from R68, which in turn interacts with Wat345 (Figure 3.1A). Thus, through its interaction with R68 the DTE molecule is connected to both the hydroxyl group of Y96 in helix 3 and the carbonyl group of G60 in switch II. In essence the DTE molecule brings together elements of switch II, the P-loop and helix 3, contributing to stabilization of the “ordered off” conformation. Interestingly, R68 in the “on” state is central to the H-bonding interactions of the allosteric switch leading to an ordered conformation of switch II [107]. The presence of DTE does not allow the positioning of R68 critical for the “on” state and at the same time optimizes interactions found in the “ordered off” state, favoring the anti-catalytic conformation of the

Figure 3.1: Difference density for bound DTE and DTT in the switch II/ helix 3 pocket. The 2Fo-Fc density map was contoured to 1sigma. The Fo-Fc σ was contoured to 3.0σ. A: DTT (slate) or B: DTE (cyan) bind the switch II/helix 3 pocket. active site. The binding of DTT in this pocket has a very similar effect with only minor differences. There is a loss of H-bonding interaction between one of the DTT hydroxyl groups and Wat365 while its other hydroxyl group gains an H-bond with R68 (Figure 3.1B). The sulfur atom retains its interaction with R68 as in DTE.

The active and allosteric sites have unique conformations in the “ordered off” state

The binding of DTE or DTT with stabilization of switch II in the “ordered off” state results in an active site where residue Q61 participates in a hydrophobic pocket with Y32, P34, T64 and I36, similar to that we observed previously for the Q61L, I and V and K mutants [50]. However, unlike the mutant residues that made van der Waals interactions within the active site (including the polar K side chain with its amino group exposed to solvent), the polar portion of Q61 makes an excellent H-bonding interaction with the hydroxyl group of Y32 (2.8 Å in 888) which also H- -phosphate of the nucleotide (2.6 Å in 888) (Figure 3.2). This conformation leaves no room for the bridging water molecule,

-phosphate, and which appears to be key to the mechanism of intrinsic hydrolysis in Ras [107].

Figure 3.2: Allostreric off active site comparison. The active site of the DTE and DTT bound structures are ordered in an off conformation (cyan). This conformation overlays with the conformation seen in the Q61L Ras oncogenic mutant and is stabilized by hydrophobic interactions. Unlike the Leu mutant the wild type has a hydrogen bond form Q61 to Y32 with a distance of 2.9Å.

The allosteric site has fewer water molecules in crystals soaked in high PEG solution with calcium chloride compared with 2RGE, perhaps due to a decrease in protein hydration associated with the presence of 60% total PEG. In the structure soaked in high PEG stabilization solution in the presence of 100mM calcium acetate crystallographic water molecules become more prominent in the allosteric site, with no visible binding of calcium acetate. This could be due to preferential interaction of the acetate molecules with PEG, relieving the protein dehydration effect and at the same time making fewer acetate molecules available to interact at the allosteric site. In this structure, two water molecules (Wat1461 and Wat1464) interact with the carbonyl group of Y137. These water molecules

are too close to each other (1.9 Å) to be present simultaneously and are therefore not at full occupancy. Y137 interacts with Wat1464 in a manner reminiscent of its interaction with calcium ion in the “on” state. Wat1461 represents an alternate location for a similar interaction with Y137 (Figure 3.3). Loop 7 is shifted toward switch II along with helix 3 in a way that is typical for the “off” state. This makes room for an additional water molecule, Wat417, to bridge between Wat1464 and the carbonyl group of D107 in a position that

Figure 3.3: View of the allosteric site in the Ras High PEG CaAcetate soaked structure (885).

overlays well with the location of the acetate molecule present in the “on” state. Residue R97 is found in a conformation that would sterically clash with a bound acetate and it makes an H-bond to Wat1464. In this structure R97 is in the same conformation found in the structures in the “off” state. However, there is weak positive electron density to support

an alternate conformation of R97 similar to that found in the “on” state. In summary, an overall comparison between the “on” state and that of the “ordered off” state in the high PEG solution in the presence of calcium acetate shows that the calcium ion and its three coordinating water molecules are replaced by two alternate positions for water interaction with the carbonyl of Y137 and a water molecule that connects it to the carbonyl group D107, which is positioned toward switch II increasing the size of the allosteric site.

Equilibrium between “on” and “off” states is modulated by small molecules

Our discovery that DTE or DTT binds near the active site between switch II and helix 3 with the result of stabilizing the “ordered off” state of the allosteric switch led to the idea that it should be possible to deliberately modulate the equilibrium between the two states in Ras crystals using calcium acetate to stabilize the “on” state and either DTE or DTT to stabilize the “off” state. However, given the prominence of the “ordered off” state in soaking solutions containing high PEG (ie 30% PEG 400 and 30% PEG 3350) with accompanying insensitivity to calcium acetate, we returned to our previously published stabilization solution obtained for soaks designed to test specificity of the allosteric site for calcium versus magnesium acetate [107]. This includes 30% PEG 3550 but no PEG 400 (details in Materials and Methods section) and results in crystals that diffract to slightly lower resolution (1.6 – 1.8, rather than 1.4 Å).

We performed two sets of soaking experiments in order to study the countering effects of calcium acetate and DTE/DTT in the transition between the “on” and “off” allosteric states in Ras crystals (Figure 3.4). In the first set, Set 1 in Table 1, soaks were started from crystals containing Ras in the “off” state grown in the presence of calcium chloride and 1-5 mM DTE, which has a disordered switch II as exemplified by the structure with PDB code 2RGE [50]. These crystals were transferred to stabilization solutions containing 100 mM calcium chloride and no DTE, DTT or calcium acetate, before a second transfer to solutions

Figure 3.4: Soak Flow Chart. Flow chart of two sets of experiments examining the transition between “on” and “off”. Set 1 (CaCl2) experiments are shown on the left and Set 2 (Ca Acetate) are shown on the right. containing 100 mM calcium chloride and either 100 mM DTE or DTT, or 100 mM calcium acetate without the reducing agents. This set of soaks resulted in four structures with data collection and refinement statistics shown in Table 1, solved to resolutions of 1.82 Å (structure ID 1062), 1.57 Å (structure ID 1512), 1.73 Å (structure ID 1515), and 1.60 Å (structure ID 1187) respectively. The structure from crystals transferred to stabilization solution with calcium chloride but no DTE/DTT shows clear electron density for both the “on” and “off” states of the allosteric switch, which were therefore both included in the refinement (Figure 3.5). This is clear evidence that in the absence of both calcium acetate and DTE/DTT, Ras is in equilibrium between the two states, sampled at detectable levels in our crystals. There is electron density for the entire switch II in the “on” state conformation as in our structure with PDB code 3K8Y, but no electron density for the N-terminal half of switch II in the “off” state, indicating that it is disordered as we previously observed in our structure with PDB code 2RGE. Starting at switch II residue M67 there is electron density to support both the “on” and “off” states of the switch.

The three other soaks in Set 1 were started from crystals of Ras that first soaked in calcium chloride to attain this “mixed” structure. Addition of either 100mM DTE or 100mM DTT shifted the equilibrium towards the “ordered off” structure with clear electron density for a DTE/DTT in the switch II/helix 3 pocket. There is no visible electron density for the “on” state in these structures. Conversely, soaking in the presence of 100mM calcium acetate

Figure 3.5: Mixed conformation electron density. Crystals soaked in the absence of acetate and DTT or DTE adopted a mixed conformation. Highlighted are the important residues V103 ad M67. In the allosteric off state V103 would clash with the on conformation.

resulted in a structure with an equilibrium shift to the “on” state with no electron density to support the “off” state of the allosteric switch. As expected, this structure has a bound calcium acetate at the allosteric site. From this first set of structures it is clear that starting from the “off” state with a disordered switch II, it is possible to attain a shift to a “mixed” state as well as to the “ordered off” with DTE/DTT bound near the active site and to the “on” state with calcium acetate bound in the allosteric site.

A second set of soaks, Set 2 in Table 3.1, was performed starting with crystals containing Ras in the “on” state grown in the presence of calcium acetate. These crystals were first transferred to a common solution containing 100mM calcium acetate and no DTE or DTT. This structure was solved to a resolution of 1.70Å (structure ID 1162) (Table 3.1). Not

surprisingly, it shows Ras with the allosteric switch in the “on” state and calcium acetate bound in the allosteric site. Transfer from this first soak to a solution containing 100 mM calcium chloride and 100 mM DTT resulted in a structure solved at 1.57 Å (DTT) (structure ID 1201) (Table 3.1) that transitioned to the “ordered off” state with bound DTT near the active site. This set of experiments shows that starting from the “on” state, Ras is able to fully transition to the “ordered off” state in our crystals. Two additional soaks in this set were designed to explore the competition between binding of calcium acetate to promote the “on” state and DTE/DTT to promote the “ordered off” state. Ras crystals grown in the presence of calcium acetate followed by a soak in stabilization solution with calcium acetate were then transferred to stabilization solutions containing either 100 mM calcium acetate and 100 mM DTE or 100 mM calcium acetate and 100 mM DTT. The two resulting structures were solved to 1.66Å (structure ID 1521) and 1.72Å (structure ID 1204) respectively (Table 3.1). Surprisingly, while the combination of calcium acetate and DTE resulted in a shift to the “ordered off” state with no bound calcium acetate in the allosteric site and DTE bound in the pocket between switch II and helix 3, the structure soaked in calcium acetate and DTT remained in the “on” state, with calcium acetate clearly bound in the allosteric site, the active site conformation ordered as in the structure with PDB code 3K8Y and no electron density for DTT. It is clear that there is a competition between opposing effects of calcium acetate and either DTE or DTT present at equal concentrations in solutions containing Ras crystals and that DTE has a stronger effect in stabilizing the “ordered off” state than DTT, presumably due to more optimized interactions within the binding pocket near the active site.

Discussion:

Allosteric mechanisms were originally described for oligomeric or multidomain proteins where allosteric changes induced by ligand binding involve changes in quaternary structure or interdomain interactions [119]. More recently, the definition of allostery has been

expanded to include ligand induced changes in tertiary structure and the distribution of conformational ensembles within a monomeric protein [120, 121]. It has become increasingly clear by our work and those of others that Ras-GTP (as modeled by Ras- GppNHp) is in the class of monomeric proteins that are regulated by shifts in conformational ensembles [38, 105, 107, 122], dynamically sampled in solution as shown for H-Ras-GppNHp [37, 38, 92] and K-Ras-GppNHp [5, 123]. Here we have explored the shifts in equilibrium between conformational states modulated by the binding of small molecules in Ras-GppNHp crystals. We showed that calcium acetate and DTE/DTT can effectively promote conformational shifts to the “on” and “off” states of the allosteric switch respectively.

We had previously identified the binding of calcium acetate at the allosteric site as promoting a network of H-bonding interactions associated with the “on” state that culminate in the ordering of switch II with placement of Q61 in the catalytic center [107]. Here we have identified DTE/DTT as binding near the active site to promote the “ordered off” state where switch II in the wild type Ras-GppNHp adopts the anticatalytic conformation we previously observed for the RasQ61L-GppNHp mutant. This site coincides with the most prominent binding site hot spot identified through our multiple solvent crystal structures (MSCS) experiments and predicted by FT-Map calculations to be a druggable site [5]. This is an indication that the site could be a binding pocket for a natural ligand of Ras. Significantly, it is a site of interaction between the GTPase Ran and importin- consequence of preventing GTP hydrolysis on Ran while cargo is being trafficked across the nuclear membrane [70]. In this structure, as in our structure of wild type Ras-GppNHp in the “ordered off” conformation, both the γ-phosphate and switch II residue Q69 (Q61 in Ras) make direct hydrogen bonds with the hydroxyl group of Y32. For Ras there have yet been no natural binding partners observed to stabilize this “ordered off” conformation.

Bulk solvent composition affects the binding of DTE/DTT to Ras-GppNHp crystals

A recently published structure of Ras-GppNHp from crystals of R32 symmetry soaked in the Ras pathway inhibitor zinc cyclen (PDB code 3L8Y) [7] has a switch II backbone conformation very similar to the “ordered off” conformation stabilized in our structure with bound DTE. Unlike our structures, though, the electron density for several switch II side chains is absent, indicating greater disorder in the region. The structure was solved at 2Å resolution and has a zinc cyclen molecule bound at a site near the C-terminal, contributed by the end of helix 5 and loop 7. We identified this site as the Loop 7 site in our analysis of hot spots in Ras- GppNHp [5] and it was labeled the p3 site in a similar study [124]. It was not obvious from our analysis of this structure, however, how the binding of zinc cyclen at this site could promote the “ordered off” conformation. Inspection of the active site with the associated electron density map downloaded from the PDB showed a model with an alternate conformation of the Q95 residue in helix 3, with its side chain nitrogen atom in electron density that extends significantly beyond it and superimposes well with the DTE binding position in our structure. Removal of the alternate Q95 side chain conformation, addition of DTE, and replacement of switch II with the “ordered off” conformation obtained from our 1.39 Å data set of Ras-GppNHp in high PEG, followed by refinement against the deposited data, resulted in an electron density map with greater continuity for switch II and a bound DTE molecule in the position observed in our structures (Figure 3.6). Although hard to discern a priori, the hind sight provided by our high resolution structures suggests that a bound DTE molecule contributes to the “ordered off” conformation in the zinc cyclen bound Ras structure.

This exercise is important because it provides an example where DTE, which is routinely used as a reducing agent in the crystallization mother liquor for Ras, has an unintended effect on its structure. We show here that either DTE or DTT can affect the structure of Ras in the crystalline environment and depending on the composition of the bulk solvent this

Figure 3.6: Zinc cyclen soaked switch II/helix 3 pocket. Electron density for the switch II/helix 3 pocket for the zinc cyclen soaked structure refined from the deposited structure [7] (PDB code 3L8Y). effect can be seen at concentration of less than 5 mM. As with the structure of Ras soaked in zinc cyclen [7] this was also the case for our structures soaked in high PEG stabilization solutions in the absence of reducing agents. The original mother liquor where Ras crystals were grown had only 1-5 mM DTE or DTT, but when transferred to a high PEG solution with a much higher concentration of 100 mM calcium acetate and no DTE/DTT, the allosteric site is “empty” and the DTE or DTT is still bound. Interestingly, crystals taken directly from the mother liquor or soaked in the lower PEG stabilization conditions were less sensitive to the presence of DTE or DTT. Under these conditions, soaking in 100 mM calcium acetate clearly stabilizes the “on” state as we show in the present work. The presence of 1 mM DTT in crystals used to generate the “off” state in mother liquor containing calcium chloride (PDB code 2RGE) has no effect as far as ordering switch II [50]. Since in the soaking experiments presented here we used 100 mM DTE or DTT we do not know the minimum concentration

necessary to observe binding with stabilization of the “ordered off” conformation under these conditions. We do know, however, that at 10 mM DTE, which was used to obtain the structure with the allosteric switch in the “on” state, an ordered switch II and calcium acetate in the allosteric site (PDB code 3K8Y), the DTE is not observed. Although the sensitivity to low concentrations of DTE/DTT seems to be dependent on the constitution of the bulk solvent, these reducing agents should be used with caution, particularly in crystallization soaks geared toward understanding the nuances of the switch regions in Ras or binding of small molecules in the search for Ras inhibitors. The soaking conditions for obtaining the zinc cyclen-bound Ras-GppNHp structure, for example, contains 17% PEG 6000 and 25 mM zinc cyclen [7]. It is possible that the higher molecular weight PEG may have a similar net effect to our high PEG solutions, providing a more crowded environment in which switch II is stabilized in the “ordered off” conformation conducive to binding DTE or DTT. It is important to note that high PEG solutions are not unique in this respect. In our multiple solvent crystal structures (MSCS) experiments soaks in four organic solvents at high concentrations were seen to have the same effect with a bound solvent molecule in place of the DTE/DTT: neat hexane, 60% 1,6-hexanediol, 55% dimethylformamide and 50% 2,2,2- trifluoroethanol [5]. As is the case with high PEG, it is likely that these solvents have a tendency to stabilize the “ordered off” conformation, which then becomes more conducive to binding either DTE/DTT or other molecules.

The active site conformation in the “on” state is associated with tight binding effectors

Our discovery of the allosteric switch in Ras-GppNHp crystals with symmetry R32 suggested a relevance to the interaction with Raf because this crystal form stabilizes a conformation of switch I identical to that seen in the Ras/Raf complex [107]. Furthermore, as in our crystals where switch II is free of crystal contacts, it is not part of the Ras/Raf interface. We subsequently established a connection between the allosteric switch and activation of the Ras/Raf/MEK/ERK signaling pathway in NIH-3T3 cells [105]. The major consequence of

calcium acetate binding at the allosteric site in our crystals is that it promotes an active site conformation where the entire switch II, including catalytic residue Q61, becomes highly ordered. This structure is consistent with an intrinsic hydrolysis mechanism that stabilizes developing charges in the transition state through a bridging water molecule that interacts simultane -phosphate of the nucleotide. A slight increase in intrinsic hydrolysis rate in the presence of Raf compared to uncomplexed Ras has been previously reported [38], presumably due to ordering of switch I. We propose that this effect could be greatly enhanced by binding of membrane components at the allosteric site that promote the order of switch II through the allosteric switch mechanism in Raf-bound Ras [5, 107]. Raf has an unusually high affinity for Ras compared to the effectors PI3K and

RalGDS, with the Ras/Raf-RBD interaction having a kD of 3.5nM [20]. This would be tough competition for RasGAP, which has affinity for Ras in the micromolar range, similar to those of PI3K and RalGDS, and would have to displace Raf to enhance the hydrolysis reaction. Intriguingly, a more recently discovered effector, NOR1A, also has an affinity for Ras in the nanomolar range and the crystal structure of its complex with Ras-GppNHp is available in the literature (PDB code 3DDC) [16]. Remarkably, the structure of the Ras-GppNHp/NOR1A complex has an identical active site to the one observed in the “on” state of the allosteric switch in our crystals bound to calcium acetate (Figure 3.7). The switch I conformation is as in the Ras/Raf-RBD complex and switch II makes interactions with an N-terminal extension of the NOR1A-RBD, through residues Y64 and M67, that stabilize the entire switch II in what we call the catalytic conformation for intrinsic hydrolysis in Ras. NOR1A is a tumor suppressor protein that also interacts with the pro-apoptotic kinases MST1/2 [125, 126] and has been shown to co-localize with microtubules [127, 128]. Not all of the functional consequences of its interaction with Ras are currently understood, but it has been shown that Ras binding is required for NOR1A’s growth suppressor activities and that it leads to the inhibition of the ERK pathway [129]. Given the similarity of the Ras active site in the NOR1A complex with that in our structures with the allosteric switch in the “on” state, we propose that one mechanism through which NOR1A may exert its tumor suppressor properties is

Figure 3.7: Allosteric “on” active site comparison. The structure of the complex of Ras-NORE1A (pink) reveals an active site that is consistent with the active seen in the allosteric on state (green). through enhancing hydrolysis of GTP on Ras, thus turning off Ras mediated signaling through various pathways known to promote growth.

Conclusions

We have identified small molecules that have opposing effects with respect to stabilization of substrates in Ras-GppNHp and propose that they mimic effects that may be functional in the cell. This is supported by the proximity of the allosteric site to the membrane as detailed previously [5, 100, 107, 116] and by the functional properties associated with binding at the helix 3/switch II pocket in the homologous GTPase Ran [70]. We propose that the “mixed” state structure obtained for crystals grown in calcium chloride with a disordered switch II is representative of the Ras-GTP/Raf complex in the absence of modulators, since in our

crystals switch I is ordered as seen in the complex. It can be thought of as the “off” state which is catalytically slow due to a disordered active site with occasional access to the catalytic conformation. Binding at the allosteric site to activate the allosteric switch would promote a shift in equilibrium to the “on” state, hydrolysis of GTP to GDP and termination of signaling through the Ras/Raf interaction. Alternatively an effector such as NOR1A could have a similar effect with stabilization of both switches through a direct interaction rather than through binding at the allosteric site. Both effectors have much higher affinities for Ras does RasGAP, so promotion of intrinsic hydrolysis is a good alternative.

Binding in the pocket between helix3 and switch II shifts the equilibrium to the “ordered off” state where the active site is in a non-catalytic conformation. Interaction of a protein or ligand at this site could render Ras incapable of hydrolyzing GTP with the consequence of maintaining active signaling in the cell. This type of modulation would add an additional level of complexity and fine-tuning in the regulatory mechanisms that affect Ras signaling. Interestingly, a high resolution “mixed” structure has previously been reported for crystals of Y32C/C118S H-Ras bound to R-cagged GTP [130], with structures representing “on” and “off” states simultaneously. These structures showed an ordering of switch II consistent with the ordered on and off conformations that we observe for the upper portion of switch II. However, the lower portion of switch II adopts different conformations especially the conformation of Q61 which in one conformation is swung way from the active site (PDB code 2CL7). The reported structure was not presented in the context of the allosteric switch. Correlated dynamics of the structural features associated with the allosteric switch has been observed by NMR for both H-Ras and K-Ras isoforms [5, 92]. The activation of the switch through binding at the allosteric site may be particular to the Ras/Raf interaction, but the same resulting shift can be achieved just as well by the binding of RasGAP as pointed out in our analysis of binding site hot spots in Ras-GppNHP [5] or other effectors such as NOR1A that promote a conformation with an enhanced rate of GTP hydrolysis.

Materials and Methods:

Protein expression, purification and crystallization:

H-Ras 1-166 was expressed and purified as previously described [85]. GDP was exchange to the GTP analog, GppNHp using the previously published procedure [107]. The protein was put in a final solution buffer (20mM HEPES pH 7.5, 50mM NaCl, 20mM MgCl2 and 1-10mM DTE). Sitting drop crystallization trays were set with a protein concentration of 10-15 mg/mL. Crystals were grown at 18°C for at least 1 week. Initial sitting drops were 5 uL protein solution and 5 uL reservoir solution. A 0.05% n-Octyl-β-D-glucopyranoside solution was made by adding n-Octyl-β-D-glucopyranoside to either: 0.2 M Calcium acetate hydrate and 20% w/v PEG 3350 (PEG Ion Screen 28 from Hampton Research) or 0.2M CaCl2 and 20% w/v PEG 3350 (PEG Ion Screen 7 from Hampton Research). The reservoir solution was comprised of 500uL of one the above solutions diluted with 100-200uL of final solution buffer and 0-150uL 50% w/v PEG 3350.

Soaks:

Soaks of calcium acetate grown crystals were transferred into a calcium acetate stabilization solution which was 10mM HEPES pH 7.5, 20mM NaCl2, 10mM MgCl2, 100mM calcium acetate, and 30%PEG 3350 for 60min. Crystals grown in CaCl2 were transferred to a CaCl2 stabilization solution which was 10mM HEPES pH 7.5, 20mM NaCl2, 10mM MgCl2, 100mM

CaCl2, and 30%PEG 3350 for 60min. Soaking in the stabilization condition was used to equilibrate crystals grown in different wells to the same starting condition. After soaks in the stabilization solutions, crystals were transferred to their experimental conditions. All experimental soaks had 10mM HEPES pH 7.5, 20mM NaCl2, 10mM MgCl2 and 30%PEG 3350.

Varied conditions were: calcium acetate 100mM, CaCl2 100mM, 100mM DTT and 100mM

DTE. Soaks in experimental conditions were for 1-2 hours. After soaking, crystals were flash frozen with liquid N2 with cryo conditions of 30% v/v glycerol with soak condition.

Data Processing:

Data was collected at 100K at the SER-CAT ID-22 and BM-22 lines at APS (Argonne, IL). The x-ray wavelength was 1.0Å with a crystal to detector distance was 150mm. Exposure time varied from 1-10 seconds with an oscillation of 1˚ per frame. Data processing was performed using HKL2000. The coordinates from pdb code 3K8Y [107] were used for phasing of all allosteric on structures. The model used for phasing in the high peg DTE structure was pdb 2RGE [50]. Subsequent allosteric off structures used the high PEG DTE structure for the phasing. Rigid-body refinement was performed using PHENIX refine [8] to 2.5Å. Simulated annealing was performed to 2000K after rigid refinement. Model building was performed using COOT [10]. PHENIX was used for refinement and generation of maps.

CHAPTER FOUR

Neutron Diffraction of Ras.

Summary:

Neutron crystallography allows visualization of hydrogen in a protein at reasonable resolutions, which can be used to look at hydrogen positions, protonation states and enhance understanding of catalytic mechanisms. We have proposed a mechanism of intrinsic Ras hydrolysis involving hydrogen bonding in the active site. Here we use deuteration and perdeuteration (techniques replacing hydrogen with deuterium) to enhance hydrogen visualization. We obtained large crystals (>1mm in at least one dimension) of both hydrogenated and perdeuterated proteins that are suitable for neutron diffraction. Hydrogenated Ras crystals soaked in D2O diffracted at the neutron beam line at LADI-III at the Institut Laue-Langevin (ILL) (Grenoble, France). Perdeuterated crystals have been mounted for neutron analysis. Feasibility of the neutron diffraction of Ras has been established and we are awaiting beam time to collect a full data set.

Introduction:

X-ray crystallography is a powerful tool for determining atomic level structural information. However, hydrogen atoms, which make up ~50% of protein atoms, are rarely visible by X- ray diffraction and generally require a resolution of less than 1.0Å to visualize [131, 132]. Even at sub-angstrom resolution, hydrogen can be difficult to resolve due to thermal atomic motion. It is possible to hypothesize hydrogen positions based on stereochemistry, but due to rotational freedom and titratable functional groups some positions are more difficult to predict [133]. Distinguishing hydrogen positions can answer questions of catalytic mechanism, hydrogen orientation, and protonation state. Neutron crystallography, unlike

X-ray, can resolve hydrogen positions at resolutions of 1.5-2.5Å [134-136] making it a powerful tool in examining enzymatic mechanisms, including GTP hydrolysis in Ras. The goal of the work presented here is to use neutron crystallography to determine hydrogen positions associated with our proposed mechanism of intrinsic hydrolysis in Ras, discussed in detail in chapter 2. That mechanism is centered around two catalytic water molecules and the side chains of residues Y32 and Q61 to promote hydrolysis of GTP with GDP and Pi as products. We distinguish between the two catalytic water molecules by calling one the nucleophilic water molecule and the other the bridging water molecule in the discussion that follows.

Ras Dynamic Regions Switch I and Switch II and GTP Hydrolysis Mechanisms:

Ras contains two highly dynamic regions, switch I (30-40) and switch II (60-76) whose conformations vary with the nucleotide binding state [34]. In switch I, NMR data suggests the presence of at least two conformations [37, 38]. In state 2 (the active conformation), Y32 is closed over the nucleotide and is poised to interact with downstream effector molecules. In state 1 (the inactive conformation), switch I can adopt different conformations where Y32 is swung away from the nucleotide. It was shown that state 2 is predominant in H-Ras (64±2%) whereas in M-Ras state 1 predominates (93±2%) [39]. M-Ras mutation of P40D and D41E were shown to affect the equilibrium between state 1 and state 2 [137]. The mutation, D41E, in M-Ras changed the equilibrium between state 2 and state 1 (79±2% for state1) and resulted in crystals with two different switch I conformations represented in the electron density maps, corresponding to each of the two states [1]. Along with the change in switch I conformation it was also observed that the pre-switch I region (defined as residues 39-41 (29-31 in H-Ras numbering)) also had two different conformations. V39 (V29) moves 1.83Å away from the ribose ring of GppNHp in state 1 and V39 (V29) and P40 (D30) loses interactions with the ribose ring. It was postulated that a water molecule would bridge the V29 to the ribose ring, however the resolution limitation

of the structures prevented definitive placement of water molecules. H-Ras mutants have been solved in state 1 conformations including T35S [137] and G60A [69]. These structures had a V29 conformation that was shifted significantly further (>10Å) from the nucleotide binding site than seen in the M-Ras state 1 structure (Figure 4.1).

Figure 4.1: Cartoon of Ras state 1 and state 2. Comparison of the crystal structures of M-RasD41E- GppNHp types 1 and 2 with those of M-Ras-GppNHp and H-Ras-GppNHp with a special emphasis on switch I, switch II, and the α3-helix. Superimposition of the backbone structures of M-Ras-GppNHp, M- RasD41E-GppNHp type 1, M-RasD41E-GppNHp type 2, and H-Ras-GppNHp. Only switch I, switch II, and the α3-helix are colored as indicated. The models were generated with PyMOL, based on the least square fittings of the Cα atoms of the residues excluding those of the two switch regions (From Matsumoto et al. 2011 [1].

H-Ras crystallizes in several space groups including P3221 and R32 and this has given us the opportunity to analyze various conformations accessed by the switch regions stabilized by crystal contacts. In the canonical P3221 space group (PDB code: 1ctq [4]), Ras switch I is found in a conformation similar to the one observed in the Ras-RasGAP structure (PDB code: 1wq1 [43]). In this conformation Y32 is swung away from the active site and is interacting with the ɣ-phosphate of a symmetry related Ras molecule. In this crystal form Y32 from a symmetry-related molecule is inserted in the active site in a position that coincides with the RasGAP Arg finger (R789) in the Ras-RasGAP complex (Figure 1.4). In the course of GTP hydrolysis it had been proposed that Q61 may activate the nucleophilic water molecule by abstracting a proton [47]. But due to glutamine being a weak base, it is now thought that GTP itself abstracts a proton from the nucleophilic water molecule in a substrate assisted mechanism [48]. Then nucleophilic attack by the activated water molecule proceeds on the ɣ-phosphate. The Arg finger is proposed to stabilize the negative charge on the phosphates that develop during the course of the hydrolysis reaction [46]. Though it is now agreed that the role of Q61 is not to activate the nucleophilic water molecule its function is still not fully understood. In the Ras-RasGAP structure the hydroxyl group of Q61 interacts with the nucleophilic water. From this interaction it was proposed that Q61 stabilizes and orients that water for nucleophilic attack [43]. Subsequent kinetic isotope effect (KIE) experiments revealed that mutation of Q61 to His resulted in a dramatic change in the KIEs for the leaving group oxygen atoms, suggesting a change in electrostatic potential in the ɣ-phosphate. Given this, a new role for Q61 as an organizer of active site features involved in oxygen stabilization was proposed [46]. This was supported by the fact that in the structure of the complex it was observed that the NH2 group of Q61 interacts with the carbonyl group of the Arg finger [43]. In the P3221 structure Q61 interacts with the nucleophilic water molecule in a conformation similar to that seen in the Ras-RasGAP structure. Given the active site conformations of switch I and switch II, Ras crystallized in the P3221 space group mimics the Ras-RasGAP complex and can give an insight into the mechanism of GAP stimulated hydrolysis in Ras.

Ras in the R32 space group is in a different conformation where switch I is stabilized in the state 2 conformation by crystal contacts. Switch II is found free of crystal contacts. We have found that two conformations of Ras are seen in this space group [50, 107] (Figure 2.1). In what we call the catalytic off state, helix 3 and loop 7 are positioned farther from helix 4 towards switch II. In this substrate of Ras-GTP switch II can be either disordered, stabilized by small molecule binding or stabilized in a catalytically inactive conformation by mutation of position 61 [50] [Chapter3]. In the off state where switch II is disordered, a bridging water molecule was observed between Y32 and the ɣ-P whereas in the catalytically inactive conformation Y32 is making a direct interaction with the ɣ-phosphate. Residue 61 stabilizes the catalytically inactive conformation by hydrophobic interactions with residues Y32, P34, Y64, and I36 [Chapter 3]. In the wild type structure Q61 is also within hydrogen bonding distance of the hydroxyl group of Y32. This conformation is nearly identical to the conformation seen in the Ran-Importin-β complex. Ran, another small monomeric GTPase, when in complex with Importin-β does not hydrolyze GTP and this conformation is therefore said to be catalytically inactive [70]. Binding of DTT/DTE was shown to limit the sampling of Ras to the off state and to help stabilize switch II [Chapter 3].

Like the off state, the on state can be stabilized by small molecule binding or by protein- protein interactions. The on state is adopted in the Ras-RasGAP structure [43], Ras solved in the P3221 space group, and in Ras effector complexes such as Raps-Raf [17], Ras-NORE1A [16] and Ras-PI3K [14]. We found that CaAcetate could also stabilize the on state by binding a remote allosteric site [Chapter 2]. The binding stabilizes a shift in helix 3 and loop 7 toward helix 4. The resulting shift allows switch II to adopt a different conformation. In this conformation Y32 moves away from the ɣ-phosphate and a bridging water molecule is observed between the hydroxyl group of Y32 and one of the oxygen atoms of the ɣ- phosphate. Q61 also interacts with the bridging water molecule. We proposed a mechanism of intrinsic hydrolysis for Ras based on the on state [107] (Figure 2.5). In this mechanism GTP acts as a general base, similar to the GAP stimulated hydrolysis, and

abstracts a proton from the nucleophilic water molecule. In the proposed mechanism this proton is shuttled to the bridging water molecule where a hydronium ion is created. The bridging water molecule therefore serves as the second catalytic water molecule in this mechanism. Q61 and Y32 orient and accept H-bonds from the bridging water molecule so that it becomes more prone to accept the proton shuttled from the ɣ-phosphate. The hydronium ion may serve to stabilize the developing negative charge in the transition state as the hydrolysis reaction proceeds. The proton would then be transferred to the β- phosphate of GDP and inorganic phosphate would be released. This proposed mechanism not only reveals a role for Q61 as a residue that organizes features in the active site but one that is important in catalysis.

Since both the intrinsic and GAP stimulated hydrolysis mechanisms involve hydrogen bonding to stabilize the transition state, the hydrogen positions would be valuable in evaluating and refining hydrolysis mechanisms. Here we seek to use neutron crystallography to resolve the hydrogen positions in Ras-GppNHp. From this information we can garner the protonation state and the orientation of hydrogen in Ras: specifically in the active site. This will help dissect the mechanisms of intrinsic hydrolysis catalyzed by Ras. Given that mutations of Ras affecting the GTP/GDP bound ratio are oncogenic and that we have uncovered a mechanism through which intrinsic hydrolysis could be important in modulating signaling pathways [105], understanding the Ras hydrolysis mechanism is significant in elucidating the fundamental mechanisms of how Ras works and in providing further information in drug design strategies aimed at affecting the allosteric switch.

Neutron Crystallography:

Neutron crystallography is a powerful technique for hydrogen position determination; it also has some challenges and limitations. The result of these difficulties is a low number of structures solved by neutron crystallography and deposited into the Protein Data Bank as

compared to X-ray crystallography. There are currently forty-eight deposited structures in the Protein Data Bank which were solved with the experimental method of neutron diffraction [138]. This is compared to 65,674 X-ray structures [138]. The true number of novel proteins solved by neutron diffraction, however, is less than forty-eight as some proteins have multiple structures. Examples of proteins with solved neutron structures include D-xylose isomerase [139, 140], rubredoxin [141, 142], perdeuterated aldose reductase [143], dihydrofolate reductase [144], lysozyme [145, 146], TOHO-1 [147, 148], and ribonuclease A [149]. Obtaining the neutron structure of Ras would represent an advancement in the technique. Most of the structures obtained are of “classical” proteins such as lysozyme and RNAse A or bacterial proteins such as TOHO-1. Obtaining the neutron structure of Ras would provide the first GTPase structure to be solved by neutron crystallography. To appreciate the challenges presented in neutron crystallography, an understanding of the technique from neutron generation to data processing is necessary.

From neutron to structure: How does neutron crystallography work?

Neutrons can be generated in two ways: fission and spallation. A nuclear reactor is used to generate neutrons by fission. Fission is where large atomic nuclei (such as U235) absorb a neutron, creating a high-energy, unstable nucleus. The return to a stable nucleus results in the splitting of the atom, release of gamma radiation, kinetic energy and neutrons. This can set off a sustained chain reaction to generate neutrons. Examples of available beam lines that are reactor based are the LADI-III at the Institut Laue-Langevin (ILL) (Grenoble, France) and, in the future, IMAGINE at High Flux Isotope Reactor (HFIR) (Oakridge, TN USA). A spallation neutron source generates neutrons by collision of high molecular weight atoms with protons accelerated to near the speed of light. In brief, a proton accelerator accelerates protons which then smash into a mercury, or other high molecular weight atom, target. This collision generates neutrons of different wavelengths. The protons are generated in pulses subsequently generating neutron in pulses. This adds a third dimension

to the diffraction data from a spallation source: time of flight (TOF). This is in contrast with a reactor generated beam source where neutrons are continuously generated and a third dimension is not possible. A Spallation source is available for biological crystallography at the Protein Crystallography Station (PCS) at the Los Alamos Neutron Science Center (LANSCE) (Los Alamos National Laboratory, USA) and at the future Macromolecular Diffractometer (MaNDi) at Spallation Neutron Source (SNS) (Oak Ridge National Laboratory, USA).

In neutron crystallography it is difficult to get a strong signal to obtain data to build a model. This is mainly caused by weak flux of neutron beams and weak scattering of neutrons. The currently available neutron sources (either reactor or spallation) have a relatively low flux especially when compared to X-ray sources. The best neutron sources in the world have a flux of 10^8 neutrons/cm-2/s-1. For comparison, a modern rotating anode X-ray generator has a flux of about 10^11 photons/cm-2/s-1 and a third generation synchrotron has an even greater flux of X-rays at 10^16 photons/cm-2/s-1. To solve this problem, advances in beam line technology are being developed to increase flux. A second reason for weak signal is that neutrons scatter weakly and thus most are not diffracted. Since these two problems are not manageable by the end-user, solutions to these problems on a smaller, more individual scale include optimizing crystal volume and increasing the signal to noise ratio by using deuteration [150]. Increasing the volume of the crystal increases the intensity of the spots, and the amount of data collected, with respect to the following equation: 2 2 I=Io*F *V*A/(vo) where I is diffracted intensity, Io is the incident neutron intensity, F is the structure factor, V is the crystal volume, A is the area subtended by the sample and vo is the volume of the unit cell [151]. Using deuteration, where exchangeable hydrogen atoms are exchanged by soaking in deuterium, neutron diffraction has been successful with a crystal volume as low as 0.3mm3 [144]. Using perdeuteration, where protein is expressed in deuterium and therefore all molecules are fully deuterated, this volume can be reduced to ~0.1mm3 [143], and is used successfully in this study. The use of a polychromatic beam also

increases the amount of data collected [152]. The use of multiple wavelength neutrons is referred to as Laue diffraction. In neutron data collection the quasi-Laue diffraction is the method employed; quasi-Laue diffraction uses a range of wavelengths.

The quasi-Laue diffraction method which utilizes a multiple wavelength beam is different from X-ray diffraction, which traditionally uses a monochromatic beam. The quasi-Laue diffraction method is used primarily to collect as many neutrons as possible to increase the signal, and to decrease the collection time to a realistic level [152]. Typically, a range of wavelengths from 2-5Å is used. With current neutron technology exposure times are long; one frame can be collected for over 24 hours. Given this, when designing neutron collection strategies it is important to collect as few frames as possible and use as many of the generated neutrons as possible. The benefit of using multiple wavelengths is that there are more reflections. Using the Ewald Sphere to define spots that will give diffraction, two circles are drawn instead of just one. One circle has a radius of 1/the shorter wavelength and the other circle has a radius of 1/the longer wavelength. Any reflections that fall between these two spheres satisfy Bragg’s law and gives rise to diffraction. An obvious additional attempt to increase the amount of data collected over time is to collect as many diffracted neutrons as possible. The use of a spherical detector allows for significant increase in the amount of diffracted neutrons collected since neutrons diffract in all directions. This unfortunately creates a fixed detector distance and therefore limits the size of the unit cell that can be collected and analyzed [152].

The Laue method does have drawbacks: problems with harmonic and spatial overlap and mosaicity. Spatial overlap is when a diffraction spot encroaches on its neighbor. This can make resolving the intensities more difficult as the intensity of one spot interferes with its neighbor. A large unit cell (>100Å) which gives diffraction spots that are closer together would therefore result in greater problems with spatial overlap. Space groups with higher symmetry reduce this problem by negative interference of diffracted neutrons resulting in

systematic absences and fewer spots on the detector. Advances in detector technology and use of TOF have also increased the unit cell dimensions possible for data collection. For example, MaNDi will be optimized to collect data on crystals with unit cells lengths of 150Å and beyond. This increase opens the door to solve more protein structures by neutron crystallography, including those of Ras crystallized in the P3221 form with a longest unit cell length of ~160Å and in R32 with a unit cell edge of ~135Å. Mosaicity also increases potential spatial overlap in the Laue method and since neutron diffraction data processing is particularly sensitive to mosaicity, crystals with low mosaicity have less issue with spatial overlap. Another cause of spatial overlap is harmonic overlaps. This arises from the ambiguity in Bragg’s Law which states diffractions are possible where nλ=2dsinθ where λ is the wavelength of incident wave, d is the spacing between the atomic lattice and θ is the angle between incident ray and the scattering plane. What holds true for a given (λ,d) also holds true for (λ/2,d/2), (λ/3,d/3), and so forth.

Cryo crystallography was a significant advancement in X-ray crystallography reducing the damage to crystals from the radiation given off from the X-ray beam. Cryo freezing also reduced B-factors and trapped ligands [133]. Neutrons used in neutron crystallography do not observably damage biological crystals; an advantage given the long exposure times. Neutron crystallography has also typically been performed at room temperature. Studies have shown that decreasing the temperature to 15K has improved the B-factors and allowed a greater number of water molecules to be modeled in a neutron crystallography structure [153]. There is great interest in advancing cryo technology in neutron crystallography to better be able to trap ligands or highly mobile areas. One problem with cryo neutron crystallography is that mosaicity can be increased in the freezing process. Large crystals, needed for these studies, are sometimes even more sensitive to the freezing process as it is difficult to freeze the entire crystal simultaneously. As described above, the increased mosaicity causes problems with special overlap and the ability to process the data.

Despite the list of limitations for neutron crystallography, the main benefit of the technology is the ability to see hydrogen at a reasonable resolution. The visualization of hydrogen is possible because the scattering length density of hydrogen with respect to neutrons is significantly greater than with respect to X-rays. This difference is due to neutrons diffracting by interaction with atomic nuclei rather than the electron cloud as in X- ray diffraction. This means that the diffraction capability of a given atom is generally consistent across the periodic chart, though isotopes can vary. In X-ray diffraction, hydrogen has a low coherent scatter length due to its small number of electrons compared with atoms such as carbon or oxygen making it difficult to visualize. In neutron diffraction, hydrogen has a coherent scatter length of -0.374 (10-12 cm) compared to nitrogen, carbon and oxygen of 0.937, 0.665 and 0.580 respectively. The negative scatter length density of hydrogen makes hydrogen atoms appear as negative density in the Fo-Fc map (Figure 4.2). This differentiates hydrogen from deuterium as deuterium, has a positive scatter length density of 0.667 and appears as positive density in the data. The greater absolute value of deuterium’s scattering length density and the fact that it is positive makes it beneficial to replace hydrogen with deuterium for visualization of hydrogen atoms in protein molecules by neutron crystallography. Another consideration for using deuterium is that hydrogen has a large incoherent scatter length of 80.27 barns. This produces a significant amount of noise in the data. Deuterium has an incoherent scatter length of 2.05 barns which can improve the signal to noise ratio 40 fold [154], thus improving the data and resulting maps of samples that use deuterium versus hydrogen [152].

Figure 4.2: Density map difference between deuteration levels. (F calc, ϕcalc) Fourier syntheses computed at 1.0Å and corresponding to three different cases: neutron fully deuterated, neutron fully hydrogenated and neutron partially deuterated, in which the hydroxyl H (HH) of tyrosine shares its site with deuterium (DH) with an occupancy ratio of 0.6:0.4 (Modified from Afonine et al. 2010) [6]. Deuterium is present in electron density map whereas hydrogen is present as negative density in the difference density map.

There are two methods of deuteration, or replacing hydrogen with deuterium, in protein crystallography. In deuteration, crystals are soaked in D2O resulting in the replacement of exchangeable hydrogen atoms (~50% of hydrogen) associated with the protein, water molecules, and other solvent molecules. This greatly improves the signal to noise ratio and thus results in better quality data. Perdeuteration is a technique that takes deuteration one step further. Perdeuterated proteins are expressed in media containing only D2O and fully deuterated carbon sources, which results in all hydrogen positions being replaced with deuterium, not just exchangeable hydrogen atoms. This yields further improvement to the data by reducing the signal to noise ratio. In some cases perdeuteration has been observed to significantly lower the limit of minimum crystal volume to 0.1mm3 [143]. In general, the advances in deuteration and perdeuteration has decreased the crystal volume necessary for data collection, opening the door for a greater number of proteins to be studied by netutron diffraction. It is no longer necessary to obtain crystals of sizes approaching 1.0mm3, a great limiting factor in protein neutron crystallography until recently. There are, however, considerations to be kept in mind in substituting hydrogen for deuterium. One of the most important is that the bond length is longer for deuterium than hydrogen and therefore care must be taken to ensure that deuteration or perdeuteration does not alter the structure. Although studies have suggested that perdeuteration does not notably alter

protein structure [154-157], it is not uncommon to collect X-ray data on crystals of deuterated (or perdeuterated) proteins for comparison with the structures solved from crystals of hydrogenated proteins.

Because D2O is expensive as compared to the ubiquitous H2O, the use of bioreactors to create a small volume, high concentration of protein expressing bacteria is ideal for neutron crystallography. Since these cells tend to grow significantly slower when adapted to D2O media, the use of Ampicillin (Amp) resistance as a selective pressure is not ideal for perdeuteration expression. The protein giving rise to Amp resistance, β-lactamase, is found in the periplasmic space between the inner and outer membranes of the bacteria. The outer membrane is leaky, releasing β-lactamase into the media. Ampicillin can then be degraded and it is possible that over long growth times enough β-lactamase can build up and dividing cells lose the pressure to keep the PET21-Ras plasmid. This can result in lower protein yields as the cells can eject the plasmid during expression. Kanamycin, unlike ampicillin, is cytosolic and therefore has a reduced likelihood to leak into the media keeping selective pressure to retain the plasmid over the long period of time necessary for protein expression in D2O [158].

Neutron diffraction and Ras hydrolysis:

Here we seek to advance both the technique of neutron crystallography and the understanding of the structure and mechanism of hydrolysis mechanism of Ras. Neutron diffraction provides a way to examine the hydrogenation state of Ras especially with respect to the active site. Knowledge of the protonation state and positions of active site hydrogen atoms will help elucidate the mechanism of Ras hydrolysis of GTP. Currently there is controversy as to the mechanism of intrinsic hydrolysis for Ras. Using neutron diffraction we will examine the proposed mechanisms with regards to the hydrogen atoms present in the active site and further refine it based on neutron data.

Methods:

Deuteration: Crystal optimization and soaking of hydrogenated protein:

H-Ras (1-166) was purified as in Chapters 2 and 3. An exchange reaction was performed to exchange the bound nucleotide to GppNHp, a non-hydrolysable GTP analog. Crystal screening was performed using sitting drop CombiClover Crystallization Plates (Emerald BioSystems). The drop consisted of a 1:1 ratio of protein solution to well solution. The drop size was 10-20µL. The largest crystals were obtained using a protein concentration of 12- 18mg/mL and were grown to a length of greater than 1mm in the longest dimension.

Crystals were transferred to a D2O solution of 10mM HEPES pH 8.0, 25mM NaCl, 10mM

MgCl2, 10mM DTT, 100-200mM CaAcetate, 30% PEG-3350 and soaked for greater than 6 weeks in hanging drop trays. The soaking solution was exchanged periodically for fresh D2O solution. Soaked P3221 and R32 crystals appeared undamaged and were sent to LADI-III at the ILL (Grenoble, France).

Cells, plasmid and D2O adaptation for perdeuteration:

Expression of perdeuterated Ras was adapted from the protocol in Meilleur et al. 2009 [158]and was performed at Oak Ridge National Lab. Kanamycin resistance was introduced into H-Ras (1-166) in the PET21 vector using theTn5 Kan insertion kit (Epicentre an Illumina company) and is referred to as PET21-Ras-Kan. BL-21 cells were transformed with the modified PET21-Ras-Kan vector then plated on LB plates. The sequence of Ras was checked to ensure no mutations occurred in the process. Cells grown on LB plates were streaked on plates made with Enfors minimal media (minimal media) made with H2O. Once cells are adapted to growth on minimal media plates made with H2O, they are transferred to minimal media plates made with D2O. Reagents used to make solutions for growth in D2O underwent an exchange from H to D. In this process the reagents were dissolved in D2O

and the liquid was evaporated using a rotovap. This process was repeated to ensure full exchange of H for D and thus ensuring the greatest incorporation of D into expressed protein. Once cells are adapted to D2O growth they are transferred to liquid minimal media made with D2O. Cells adaption continues over several days keeping cells in log phase by diluting the cells when they reach OD600 of ~1.0.

Adapted cells were used to inoculate a bio-fermentor. A volume of 100mL of adapted cells was used to inoculate 900mL of deuterated minimal media. The dissolved O2 levels were monitored to assess cellular growth. As cells consume the available deuterated glycerol the dissolved O2 levels decrease. When the dissolved O2 leveled a feed solution (10%w/v glycerol and 0.2%w/v MgSO4) was added. During growth pH is monitored and maintained by addition of NaOD. Cells were induced by addition of IPTG at an OD600 of 9.0). Cells were harvested ~24hrs after inductions by centrifugation. For initial rounds adapted cells are flash frozen (without glycerol) to act as a stock for future inoculations.

Perdeuterated Protein Purification

Expressed perdeuterated protein was purified using the non-perdeuterated Ras protocol

(Chapter 2 and 3) and all purification buffers were made with H2O. Briefly, cell paste was resuspended in 50mL of buffer A (20mM TRIS pH 8.0, 50mM NaCl, 5mM MgCl2, 1mM DTT, 5% v/v glycerol, and 5µM GDP). Cells were lysed by sonication and debris pelleted by centrifugation. DNA was removed by addition of PEI and centrifugation. The supernatant was run over a QFF anion exchange column at a flow rate of 2.0mL/min. When UV wavelength 280 returned to base line a gradient of 0-30% buffer B (buffer A with the addition of 1M NaCl) was run over 200min with a flow rate of 3.0mL/min. Fractions of 2.5mL were collected over the gradient. Ras elutes at around 15%B. Fractions containing Ras were concentrated to less than 5mL and applied to a S100 gel filtration column at 1.3mL/min. Ras eluted at around 130mL. Fractions were combined and concentrated.

When necessary, purity was improved by running an additional anion exchange column. QHP was run with a gradient of 0-25% with buffer A and buffer B over 200mL. Ras eluted at around 10% B. After purification a nucleotide exchange of GDP to GppNHp was performed as in non-perdeuterated Ras.

In different perdeuterated Ras preparations D2O or H2O was used in the Ras exchange buffer (32mM TRIS pH 8.0, 200mM (NH4)2SO4, 10mM DTT and 0.1%w/v NOG) and the Ras crystal stabilization buffer (20mM HEPES pH 7.5, 50mM NaCl, 20mM MgCl2 and 1-10mM

DTE). The pH of Ras exchange buffer made with D2O was adjusted to 8.4 to adjust for the different properties of H versus D. Ras exchange buffer made with H2O was modified to include 10% glycerol and 0mM DTE. Performing the exchange reaction on perdeuterated protein in H2O exchange buffer resulted in a significantly higher protein loss which was reduced by the addition of glycerol. After the exchange reaction was completed, a buffer exchange to Ras crystal stabilization buffer was performed. Exchange from Ras exchange buffer made with D2O to Ras crystal stabilization buffer made with D2O was performed using a NAP desalting column to reduce the amount of deuterated crystal stabilization buffer required. The exchange of the solutions made with H2O was performed using concentrators rather than a NAP desalting column to reduce protein loss. The pH of the Ras crystal stabilization buffer made with D2O was also adjusted to pH 8.0 to account for the different properties of hydrogen and deuterium. Glycerol at 5%v/v was also added for protein stability. Purified and exchanged protein was concentrated to greater than

20mg/mL, flash frozen in liquid N2, and stored at -80˚C for crystallization.

Perdeuterated Crystallization:

Crystallization of protein exchanged to a D2O solution was initially performed by the hanging drop method using a 1:1 ratio of protein solution and well solution. In this case, 3µL of a protein solution at a concentration of 14-18mg/mL was used with 3uL of well

solution. The well solution was comprised of 400µL of PEG Ion Screen #28 (200mM CaAcetate and 20% PEG 3350) with 0.1%w/v NOG added with varying amounts of additional PEG 3350 (50µL to 150µL of a 50% PEG 3350 solution) and crystal stabilization buffer (50µL to 175µL of 10mM HEPES pH 8.0, 25mM NaCl, 10mM MgCl2 and 0-10mM DTE). All solutions were made with D2O. A crystal size of 300-400µm in the longest dimension was obtained.

These crystals were obtained in the P3221 space group. Data sets on perdeuterated Ras crystals were collected using X-ray diffraction at room temperature and at 100K.

In crystallization trials with hydrogenated protein it was shown that the addition of NOG produced a higher quantity of the R32 crystal form compared with P3221 crystal form.

Crystallization trials were set up using protein in D2O crystallized in conditions similar to above with the addition of 0.1% NOG added to the PEG Ion Screen #28 solution. Unlike hydrogenated protein set in hydrogenated conditions, the resulting crystals were still in the

P3221 space group. It is sometimes necessary to crystallize perdeuterated protein in H2O and then soak in D2O because crystallization in D2O can vary from H2O crystallization. It was necessary to switch from hanging to sitting drops to increase the crystal volume. Sitting drop trays were set with perdeuterated protein in a hydrogenated solution and resulted in R32 crystals. After optimizing crystal drop sizes of 5µL by 5µL and 10µL by 10µL in clover leaf sitting drop trays (Emerald Biosystems) the best performing wells were scaled up and set using 9-well glass plates and a drop size of 100µL to 120µL. Crystals grew to a maximum 3 size of approximately 1mm . Crystals were transferred to a D2O soak solution (10mM

HEPES pH 8.0, 25mM NaCl, 10mM MgCl2, 100mM CaAcetate, 30% PEG 3350) and soaked for over 2 months. Crystals were transferred into a new drop and the well solution was changed every few weeks throughout the soak process. After soaking crystals were frozen in liquid N2 with cryo protectant (30%v/v glycerol in well solution). Some crystals were transferred to a higher CaAcetate soak solution (150mM rather than 100mM) to improve occupancy of the allosteric site. These crystals soaked for 1hr and then were either mounted in capillaries for room temperature or cryo studies as above. In initial crystal

optimization the story of the effect of presence of DTT/DTE was evolving. Subsequently, in early crystallization trials the protein stabilization buffer contained 10mM DTE. The crystal stabilization buffer used in the well solution and in the soak solution also contained DTE. Once it was clear that DTT/DTE was able to shift the equilibrium between the on and off states of Ras in the R32 space group, the concentration of DTE was dropped to 1mM in the protein stabilization buffer. The crystal stabilization buffer used for the well solution and soak solutions was made with 0mM DTE.

Data Collection and structural refinement:

Data collection was performed at room temperature on our home MAR345 area detector mounted on a Rigaku RuH3R rotating anode generator. Structures were also obtained at 120K on the same source. Synchrotron data were collected on a MAR CCD detector on the SER-CAT 22-ID beamline at the Advanced Photon Source (Argonne National Lab) at 100K. Data collected on the Rigaku generator was processed using HKL or HKL2000 [3]. Data collected at SER-CAT was processed using HKL2000. Molecular replacement was performed using PHENIX [8] with previously solved Ras-GppNHp structures with PDB codes 1ctq, 3k8y or 2rge used for phasing. A round of rigid body refinement was then performed with a high resolutions limit of 2-2.5Å. This was followed by a round of simulated annealing to 2000K and refinement using phenix.refine. Subsequent rounds of refinement were also performed using phenix.refine. COOT [10] was used for model rebuilding.

Results:

Deuteration results:

Optimized crystals of hydrogenated Ras were grown in H2O to a length of 1.0mm or greater in the largest dimension for both the P3221 and the R32 space group (Figure 4.3Aand B). Crystals were then soaked in deuterium to exchange the exchangeable hydrogen atoms. The soak conditions did not visibly damage crystals. Crystals were shot on the LADI-III at the ILL (Grenoble, France), test shots showed neutron diffraction spots were visible after 2hrs for both the deuterated P3221 and the R32 space group (Figure 4.4A and B). After 18hrs of exposure the P3221 crystal revealed higher resolution diffraction (Figure 4.4C).

Perdeuteration results:

Expression of perdeuterated Ras was successful and Ras was found in the soluble fraction. In the highest yielding prep the yield of protein was 50mg Ras after purification and exchange starting with 18.9g of cell paste. Purification of perdeuterated Ras was identical to the purification of hydrogenated Ras and high purity was obtained for crystallization.

Ras P3221 crystallization and structure:

Perdeuterated crystals were obtained in both the P3221 and R32 space group.

Perdeuterated Ras crystals grown in D2O conditions with or without NOG were in the P3221 crystal form. A crystal length of 400-500µm was obtained in the P3221 crystal form.

Crystals obtained were fairly chunky (Figure 4.3C and D). Crystal optimization of the P3221 crystals was only performed using hanging drop trays. This limited the size of the drop and

subsequently the total protein and crystal size. The largest crystals were mounted in capillaries and test shots were performed at LANSCE (Los Alamos National Lab, USA). No visible diffractions were detected after 12hrs of exposure.

Figure 4.3: Optical photographs of Ras crystals. A: P3221 crystal of hydrogenated Ras soaked in D2O grew to an approximate size of 1.0X0.5X0.5mm. B: R32 crystal of hydrogenated Ras soaked in D2O grew to an approximate size of 1.0X0.5X0.5mm. C and D: P3221 crystals of perdeuterated Ras grown in D2O. E: R32 crystal of perdeuterated Ras grown in H2O. F: Crystal from E transferred to a D2O soak solution shows some crystal damage. G and H: Perdeuterated Ras grown in H2O R32 crystals. G: Crystal size was about 1.0X0.8X0.4mm. H: Crystal size was about 1.3X1.0X0.6mm.

Figure 4.4: Neutron Laue diffraction patterns for Ras at different exposure lengths. Collection was performed at the LADI-III at the ILL (Grenoble, France). A 2hr exposure of a P3221 crystal (pictured in FigureA) resulted in diffraction spots (A). After 18hr of exposure of the same crystal higher resolution spots are detected (B). Diffractions were also seen for the R32 crystal (pictured in FigureB) after 2hr of exposure (C). No 18hr exposure for the R32 crystal was performed due to beam line time restrictions.

Room temperature data (Structure ID: 713-2) was collected on our home generator to 1.86Å. Several structures were collected under cryo conditions on our home source at 120K (Structure ID: 713, 714, and 714-2) with a resolution of 1.91Å, 1.86Å and 1.78Å respectively. Data was also collected on the ID-22 line at SERCAT at 100K (Structure ID: 849 and 858) to

1.69Å and 1.78Å respectively (Table 4.1). The high resolution (1.3Å) P3221 structure (1ctq) [4] was used as the comparison model. Overall the perdeuterated structures are consistent with the hydrogenated structures.

Figure 4.5: SDS-PAGE separation of perdeuterated Ras. The column labeled A corresponds to concentrated Ras protein used for crystallization. Column B represents a molecular weight ladder. Lanes in between represent fractions from the Ras peak from QHP separation.

Though the dynamic switch regions in Ras are partially stabilized by crystals in the P3221 space group there is still varying degrees of order amongst the data. Given that neutron data is generally collected at room temperature it is important to examine the conformations seen at room temperature. Ras in the P3221 space group was previously solved to high resolution (1.31Å) at room temperature (PDB code: 3tgp) [9]. A comparison between the cryo structure of hydrogenated Ras in the P3221 crystal form (1ctq) and room temperature data (3tgp) reveal structures that are consistent (Figure4.6A) with an average backbone root mean square deviation (RMSD) of 0.222Å. In the room temperature structure, Q61 interacts with the catalytic water molecule as in the Ras-RasGAP complex [9].

Table 4.1: Data collection and refinement statistics for perdeuterated Ras crystals grown in D2O conditions in the P3221 space group. Structure IDs are assigned numbers and correspond to the data storage number. Structure ID 713 714 714-2 849 858 713-2

Data collection

Space group P3221 P3221 P3221 P3221 P3221 P3221 Beamline Rigaku Rigaku Rigaku ID-22 ID-22 Rigaku Collection 120K 120K 120K 100K 100k RT Temperature Cell dimensions 39.134 40.147 40.116 39.142 39.253 40.107 a , b , c (Å) 39.134 40.147 40.116 39.142 39.253 40.107 158.215 160.947 160.594 158.625 158.862 160.861 a, b, g (°) 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 Resolution (Å) 35-1.91 35-1.86 35-1.78 35-1.78 35-1.69 35-1.86 (1.98-1.91) (1.93-1.86) (1.84-1.78) (1.84-1.78) (1.75-1.69) (1.93-1.86)

R sym or R merge 0.104 0.109 0.136 0.091 0.076 0.317 (0.857) (0.628) (0.757) (0.184) (0.420) (0.000) I / σ 22.77 38.68 23.58 61.52 54.88 12.56 (2.204) (5.731) (2.851) (29.29) (12.36) (2.138) Completeness (%) 96.3 99.2 90.1 96.6 99.8 84.4 (91.1) (96.9) (91.3) (97.8) (100) (94.2) Redundancy 4.4 9.4 6.5 8 9.6 4.6 (3.1) (6.5) (4.5) (8.5) (10.4) (5.4) Refinement Resolution (Å) 1.91 1.86 1.78 1.78 1.69 1.86 No. reflections 11260 13335 13763 13721 16606 11321

R work / R free 22.07/29.73 17.26/21.10 20.48/24.40 21.56/27.54 22.55/29.45 19.90/25.85 No. atoms Protein 1314 1297 1286 1322 1297 1322 GppNHp 31 31 31 31 31 31 Mg 1 1 1 1 1 1 Ca 0 0 0 0 1 0 Water 82 88 70 109 138 61 B -factors Protein 30.37 21.80 23.03 24.55 16.63 22.22 GppNHp 26.26 14.10 16.30 16.06 10.77 14.20 Water 34.17 30.48 31.40 31.98 24.03 30.30 R.m.s. deviations Bond lengths (Å) 0.008 0.008 0.008 0.008 0.008 0.008 Bond angles (°) 1.201 1.186 1.218 1.244 1.245 1.132 Backbone RMSD (Å) 0.486 0.403 0.317 0.539 0.557 0.403

In 1ctq Q61 was away from the catalytic water in a non-catalytic conformation. The catalytic water also was absent. The room temperature data set reveals a fairly ordered Ras active site Figure 4.6B). In the perdeuterated room temperature structure switch II is only partially ordered with little density for residues 61-67. There is electron density to support a nucleophilic water molecule in the active site (Figure 4.7).

Figure 4.6: Room temperature data set of Ras in P3221 crystal form. A: Ribbon diagram showing a high resolution (1.26Å) P3221 Ras cryo structure (PDB code: 1ctq [4], warm pink) superimposed on the bound GppNHp molecule with a high resolution (1.31Å) room temperature structure (PDB code: 3tgp [9], blue). B: The active site of room temperature P3221 Ras (blue). Y32 is swung away from the active site (Y32-A) and Y32 of a symmetry related molecule (Y32-B, light blue) is interacting with the GppNHp. Electron density maps are shown in grey and contoured to 1.0σ.

The perdeuterated structures collected at 100K and 120K all overlaid well with the high resolution P3221 structure (Figure 4.8A). The RMSD between the hydrogenated structure (1ctq) and perdeuterated structure collected at cryo temperature was varied from 0.317- 0.557Å. The largest variation is in the pre-switch I region and in switch II. The degree of order in switch II varied in the different structures with residues E62-M67 having weak or no electron density. When the side chain of Q61 was ordered it was away from the active site

in a similar conformation to the 1ctq structure where it is not thought to be active. There was electron density to support the nucleophilic water molecule in some of the structures including 714-2, 849 and 858.

Figure 4.7: The active site of the perdeuterated Ras grown in the P3221 crystal form collected at room temperature (cyan). The symmetry Y32 (Y32-B) is inserted into the active site is shown in grey. Electron density maps are shown in grey and contoured to 1.0σ.

In two data sets (849 and 858) residues N26-E31 (pre-switch I region) and D107-V109 (loop 7) adopted an alternate conformation. In these structures V29 moves 2.8Å relative to its position in 1ctq from the core of the protein (Figure 4.8B). The resulting shift in F28 would result in steric hindrance between the carbonyl of F28 and the side chain O of D109 in a symmetry related molecule therefore correlating the conformational changes seen in the pre-switch region and loop 7 (1.78Å). The shift in the pre-switch I region is similar to the shift seen between state 2 and state 1 in M-Ras. The high resolution P3221 structure shows similar interactions between Ras and the bound nucleotide (Figure 4.9A). In M-Ras water molecules were proposed to bridge the interactions lost upon movement of V29 [1]. The

structures obtained here support the proposed water molecules in state 1 (Figure 4.9B). Wat29 bridges the interaction between the carbonyl of V29 and the ribose sugar which would be lost. Wat182 also bridges the backbone amide of V29 to Wat1008. In the state 2 structure Wat1008 bridges the backbone amide of Val29 to the carbonyl of A18 (Figure 4.9A).

A B

Figure 4.8: Pre-switch I variation. A: Cartoon of a superposition of the various P3221 perdeuterated Ras structures collected on Ras in the P3221 space group (1ctq). Overall little backbone variation is seen except for small variations in the pre-switch I region, switch II, and loop 7. The colors are as follows: 1ctq (warm pink) 713 (orange), 714 (yellow orange), 714-2 (pale yellow), 849 (light blue) and 858 (purple). B: A closer view of the variation in the pre-switch I region reveals two positions for V29. In 1ctq, 713, 714, and 714-2, V29 is found in a conformation toward the nucleotide. In 849 and 858 V29 is flipped away from the nucleotide.

Ras R32 Crystal Optimization and D2O soaking:

Perdeuterated Ras crystals in the R32 space group were obtained by using hydrogenated crystal growth conditions with NOG. Crystals were grown in sitting drop trays with a 1:1 protein to well solution drop with a final drop size of 10-20µL. The concentration of DTE originally used for the protein stabilization buffer and the crystal stabilization buffer was 10mM. This was dropped to 1mM in the protein stabilization buffer and 0mM in the crystal

Figure 4.9: Pre-switch I hydrogen bonding networks. A. The pre-switch region in 1ctq (warm pink) forms interactions with the bound nucleotide. Wat 1008 bridges between H27 and V29 with A18. B: In the open pre-switch I (858 purple) water molecules 29 and 182 bridge interactions that would be lost by the movement of V29. stabilization buffer. This change in both hydrogenated or perdeuterated crystallization did not seem to alter the quality of crystal obtained. The concentration of CaAcetate was also varied from 100mM to 400mM in the well solution to determine the concentration necessary to occupy the allosteric site. The largest crystals obtained through optimization in a 20µL volume were around 300-500µm in the longest dimension. To improve crystal size larger volumes were needed and to accomplish this, 9-well plates were used. After

optimization in the smaller sitting drop trays the best performing wells and protein concentrations were used in the 9-well sitting drop plates with a total volume of 100-120µL. The largest resulting crystals were around 1mm3 (Figure 4.3E-H).

The first crystals obtained in the R32 space group were soaked in a D2O exchange solution that also contained 10mM DTE. Crystals under 300µm visibly appeared undamaged and diffracted well by X-ray diffraction. A batch of 300—500µm crystals was transferred to a the D2O exchange solution for over 2 months that contained 0mM DTE with the well solution being exchanged every few weeks. Unlike the previous crystals the largest of these crystals showed some cracking after soaking (Compare crystal in Figure 4.6E to Figure 4.6F). The smaller crystals appeared undamaged. Before mounting these crystals in capillaries or loops, crystals were transferred from the 100mM CaAcetate D2O exchange buffer to a higher CaAcetate (150mM or 200mM) containing drop for 1hr (contained 0mM DTE). This was meant to ensure occupation of the allosteric site. Data was collected at SER-CAT for soaked crystals at both 150mM and 200mM CaAcetate (Structure ID: 1507 and 1508 respectively). After observing the damage of the crystals in the soak condition, D20 exchange for the largest crystals obtained was performed by vapor diffusion. The hydrogenated well solution used to grow the crystals was exchanged for well solution made with D2O. This would provide a gentler soak. It also negates the necessity of extra PEG3350. Well solution was exchanged about every two weeks. After the first exchange mold was observed in some trays. NaAzide was added to the well solution to inhibit mold growth. Crystals that were soaked for over seven weeks (4-5 with NaAzide) visually appeared unaffected. After eight weeks of soaking and an earthquake, crystals were cracked.

Several data sets were collected of perdeuterated Ras crystals grown in H2O detailed in Table 4.2. All resulting crystals were in the R32 space group. Data sets were collected at room temperature and at cryo temperatures (100K-120K).

Table 4.2: Data collection and refinement statistics are presented for several structures of perdeuterated Ras in the R32 space group

Structure ID 1608 1614 1736 745 747 756 Data collection Space group R32 R32 R32 R32 R32 R32 Beamline Rigaku Rigaku Rigaku Collection RT RT RT 100K 100K Temperature Cell dimensions 90.262 90.474 90.527 88.500 88.448 88.367 a , b , c (Å) 90.262 90.474 90.527 88.500 88.448 88.367 136.909 135.253 134.928 134.965 134.872 134.562 α, β, ɣ (°) 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 Resolution (Å) 50-2.25 50-2.20 35-2.27 35-1.92 50-1.60 50-1.54 (2.29-2.25) (2.24-2.20) (2.31-2.27) (1.99-1.92) (1.66-1.60) (1.60-1.54)

R sym or R merge 0.191 0.134 0.11 0.385 0.060 0.056 (0.435) (0.697) (0.825) (0.717) (0.436) (0.520) I / σ 17.04 15.20 20.39 19.83 35.58 42.33 (2.25) (.556) (1.09) (3.96) (3.88) (4.07) Completeness (%) 98.9 84.2 93.7 100 97.6 100.0 (91.4) (15.7) (85.4) (100) (98.2) (100.0) Redundancy 6.2 7.0 5.7 9.5 7.4 9.8 (3.0) (1.1) (2.3) (7.9) (7.1) (8.1) Refinement Replecement Model 3k8y 3k8y 2k8y 2rgd 2rgd 3k8y Resolution (Å) 2.25 2.20 2.27 1.92 1.60 1.54 No. reflections 10299 9343 9422 15789 26459 30342

R work / R free 16.39/20.63 18.85/23.93 18.18/21.62 17.85/22.31 17.46/19.81 18.43/19.52 No. atoms Protein 1303 1266 1243 1304 1289 1331 GppNHp 32 32 32 32 32 32 Mg 2 2 2 2 2 2 Ca 1 1 1 2 2 2 Acetate 0 0 0 0 0 1 Water 60 36 41 145 146 154 B -factors Protein 36.80 50.83 51.41 25.05 21.03 22.96 GppNHp 26.24 43.54 44.09 17.96 14.99 16.20 Water 37.53 52.62 52.03 33.01 31.15 31.93 R.m.s. deviations Bond lengths (Å) 0.008 0.008 0.010 0.008 0.008 0.007 Bond angles (°) 1.080 1.091 1.159 1.137 1.140 1.132 Experimental: Conformation ON ON ON OFF OFF ON DTE conc (mM) 5 0 0 5 7 2 No Yes Yes No No Yes D2O Soaked

Table 4.2: Continued.

Structure ID 912 916 918 936 938 1507 Data collection Space group R32 R32 R32 R32 R32 R32 Beamline ID ID ID ID BM Collection 100K 100K 100K 100K 100K 100K Temperature Cell dimensions 88.050 89.249 89.047 88.359 88.703 88.918 a , b , c (Å) 88.050 89.249 89.047 88.359 88.703 88.918 134.459 136.138 135.509 134.971 135.449 135.749 α, β, ɣ (°) 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 90,90,120 Resolution (Å) 50-1.62 50-1.50 50-1.70 35-1.62 35-1.83 50-1.54 (1.65-1.62) (1.53-1.50) (1.73-1.70) (1.65-1.62) (1.86-1.83) (1.57-1.54)

R sym or R merge 0.077 0.047 0.064 0.067 0.134 0.064 (0.553) (0.672) (0.599) (0.540) (0.805) (0.414) I / σ 47.80 61.27 56.42 35.29 23.67 55.80 (3.43) (2.81) (3.49) (3.72) (2.5) (6.37) Completeness (%) 99.9 99.9 99.9 100 100 100 (100.0) (99.7) (99.8) (100) (99.9) (100) Redundancy 9.8 10.5 10.3 10.7 8.8 10.9 (7.8) (7.0) (8.3) (9.0) (5.7) (10.9) Refinement Replecement Model 3k8y 3k8y 3k8y 2rgd 2rgd 3k8y

Resolution (Å) 1.62 1.50 1.70 1.62 1.83 1.54 No. reflections 25700 33593 22894 25970 18278 30698

R work / R free 16.62/18.85 18.61/20.88 17.93/20.59 17.06/19.59 18.19/21.63 17.93/19.88 No. atoms Protein 1309 1311 1308 1319 1279 1316 GppNHp 32 32 32 32 32 32 Mg 2 2 2 2 2 2 Ca 2 2 2 2 2 2 Acetate 1 1 1 0 0 1 Water 208 218 172 155 139 218 B -factors Protein 20.27 23.05 24.47 17.77 25.84 18.36 GppNHp 15.81 16.82 17.61 10.95 18.79 12.86 Water 30.98 34.01 34.95 28.88 34.93 29.01 R.m.s. deviations Bond lengths (Å) 0.007 0.008 0.007 0.008 0.008 0.008 Bond angles (°) 1.163 1.151 1.111 1.164 1.099 1.149 Experimental: Conformation ON ON ON OFF OFF ON DTE conc (mM) 6 2 2 5 5 0 No Yes Yes No No Yes D2O Soaked

Table 4.2: Continued.

Structure ID 1508 1606 1611 Data collection Space group R32 R32 R32 Beamline BM Rigaku Rigaku Collection 100K RT 120K Temperature Cell dimensions 89.080 88.485 88.499 a , b , c (Å) 89.080 88.485 88.499 134.972 135.144 134.967 α, β, ɣ (°) 90,90,120 90,90,120 90,90,120 Resolution (Å) 50-1.90 50-2.00 50-1.95 (1.93-1.90) (2.03-2.00) (1.98-1.95)

R sym or R merge 0.099 0.105 0.106 (0.625) (0.683) (0.638) I / σ 42.37 23.69 22.91 (7.31) 1.77 (1.80) Completeness (%) 100 99.7 99.6 (100) (97.2) (95.2) Redundancy 10.9 9.9 9.5 (10.5) (4.6) (4.1) Refinement Replecement Model 3k8y 3k8y 2rgd

Resolution (Å) 1.90 2.00 1.95 No. reflections 16471 13963 15023

R work / R free 17.13/20.63 16.65/21.23 17.38/21.82 No. atoms Protein 1313 1318 1285 GppNHp 32 32 32 Mg 2 2 2 Ca 2 2 1 Acetate 1 1 0 Water 169 159 149 B -factors Protein 23.36 27.75 24.85 GppNHp 15.16 21.87 18.30 Water 31.46 35.33 33.64 R.m.s. deviations Bond lengths (Å) 0.008 0.008 0.008 Bond angles (°) 1.098 1.107 1.149 Experimental: Conformation ON ON OFF DTE conc (mM) 0 0 5 Yes No No D2O Soaked

Room temperature data of hydrogenated protein:

The switch regions in Ras are highly mobile and can be disordered unless stabilized by a protein binding partner or crystal contacts. To examine the effects that cryo freezing had on the stabilization room temperature data were collected for WT Ras grown in CaAcetate (Table 4.2: Structure ID 1608). The resulting structure overlayed well with the WT cryo data set grown in CaAcetate (3k8y) (Figure 4.10A) with an average backbone RMSDs of 0.29Å. Helix 3, helix 4 and loop 7 were shifted from the allosteric off conformation similar to the allosteric on conformation (Figure 4.10B). The backbone of switch II was ordered except for a density break at S65 (Figure 4.11A). As in the cryo structure not all side chains in switch II are ordered including E62, E63 and S65 with residues Y64, M67 and R68 being only partially ordered. Q61 comprising part of the active site is disordered past the CG (Figure 4.11B). The bridging water molecule is absent and Y32 is making a direct interaction with the nucleotide (2.47Å). Surprisingly, given the structure adopts the allosteric on conformation of loop 7, helix 3 and switch II, in the RT data set there is no density to support a bound CaAcetate (Figure 4.11C). R97 is found in two conformations one similar to that seen in the allosteric on state and one that would sterically clash with the bound acetate. Despite the “empty” allosteric site, loop 7 residues V103-V109 along with the backbone of helix 3 and switch II overlay with the on state (Figure 4.10B).

Figure 4.10: Room temperature comparison of Ras in the R32 crystal form. Cartoon of the room temperature structure of Ras grown in CaAcetate (violet) superimposed on Ras in the allosteric on state (PDB code:3k8y, green). Ribbon diagram shows that helix 3, loop 7 and switch II are in the allosteric on state. The allosteric off state represented by the Q61L mutant (2rgd) is shown in cyan. Residues M67, R68, Q99, V103 and Y137 represent important residues in the coordinated movement between helix 3/loop 7 and switch II and are shown as sticks. Superposition was on the bound GppNHp.

Perdeuterated R32 Ras Structure Results:

Data were collected for perdeuterated Ras grown in H2O before and after back soaking into

D2O conditions. The resulting structures adopted both the allosteric off and on states.

Soaking in D2O did not observably alter the structure. Not surprisingly, there was correlation with the presence of DTE either in the growth condition or in the soak condition and the structure adopting the off conformation (Table 4.2). The trend is not clear as crystals in the presence of >5mM DTE (Structure ID: 745, 912, 936, 938, and 1611) adopted both the on (912) and off (745, 936, 938, and 1611) conformations. Once the DTT/DTE effect was elucidated, the DTE concentration in the growth condition was dropped to 0.5mM and the DTE concentration in soak conditions was dropped to 0mM. All structure with 0mM DTE are found in the allosteric on state. Despite the effect of DTE there is not strong enough density to model a DTE.

B C

Figure 4.11: Room temperature electron density. A: Ras collected at room temperature reveals an ordered switch II (violet) in a conformation similar to the allosteric on state (3k8y; grey). B: Despite and helix 3, helix 4, loop 7 and switch II conformation consistent with the allosteric on state, the allosteric site at room temperature shows no density to support a bound CaAcetate. C: The active site in the room temperature data set has Y32 making a direct interaction with the nucleotide.

CaAcetate levels were varied in the perdeuterated structures to ensure better occupancy of the allosteric site. In 916 and 918 CaAcetate levels were 200mM in the growth condition. These structure, however, had higher levels of DTE and were subsequently in the allosteric off conformation. Crystals soaked at 100mM CaAcetate for over 2 months then transferred to 150mM (1507) were in the allosteric on conformation with a bound CaAcetate in the allosteric site (Figure 4.12A). Crystals transferred to 200mM CaAcetate (1508) were similar to the crystals in 150mM (1507). The average backbone RMSD was 0.24Å and 0.32Å respectively. In 150mM CaAcetate there is weak density to place Q61, however, there is density to support the bridging water molecule (Figure 4.12B). In the 200mM CaAcetate structure there is also weak density for Q61. Surprisingly, there is no density to support a bridging water molecule (Figure 4.13C).

Room temperature was also collected for perdeuterated Ras soaked in D2O. The data sets collected were from crystals undergoing the same growth and soak procedure at 150mM CaAcetate as 1507. Residue Q61 was partially ordered in this structure. Y32 was making a direct interaction with the nucleotide (Figure 4.13A). There was density to support a nucleophilic water molecule. Like the hydrogenated room temperature structure (1608), the protein was in the allosteric on state but there is no electron density to support a bound CaAcetate (Figure 4.13B).

Discussion:

Here we sought to examine the feasibility of using neutron crystallography to determine the H/D positions in Ras. We also examined the effect that perdeuteration has on the structure of Ras in the P3221 and R32 space group. Through crystal optimization I obtained large (>1mm in at least one dimension) Ras crystals in both space groups. The resulting crystals were soaked in D2O conditions for improvement in the signal to noise ratio. After two hours of exposure at the LANDI-III at the ILL (Grenoble France) diffraction spots were visible in

Figure 4.12: Perdeuterated active and allosteric site conformations. A: The allosteric site of 1507 (teal), a cryo structure of perdeuterated R32 Ras soaked in D2O, reveals an occupied allosteric site. B: The active site shows the presence of a bridging water molecule, Wat 189. Electron density of 2fo-fc map is shown in grey and contoured to 1.0σ.

both space groups (Figure 4.4A and C). The P3221 crystal was shot for an additional 16hr (18hr total) (Figure 4.4B). Spots were estimated to be diffracting to around 2.5Å, within the resolution for visualization of H/D. This shows clear feasibility for collecting neutron data on Ras.

Figure 4.13: Room temperature perdeuterated conformations. A: Similar to the hydrogenated room temperature structure (1608) the perdeuterated R32 room temperature structure (Structure ID: 713-2, violet purple) reveals no density to support a bound CaAcetate to the allosteric site. 3k8y is shown in grey to show placement in an occupied allosteric site. B: The active site reveals Y32 in direct contact with the ɣ-phosphate. There is density to support the catalytic water, Wat 175. Electron density of the 2fo-fc map is shown in grey and contoured to 1σ.

Though the soaked crystals diffracted well, we looked to improve of resolution by using the technique of perdeuteration. We especially want to improve the R32 which we had not the opportunity to test for longer data collection times. Perdeuterated Ras was expressed in a bio-fermentor and purified to high purity (Figure 4.3). Crystals were obtained in D2O growth conditions. Surprisingly, even with the addition of NOG these crystals were in the P3221

space group. This prompted crystallization trials of perdeuterated protein grown in hydrogenated crystallization conditions. The crystals grown in hydrogenated conditions were of the R32 space group. The hydrogenated grown crystals required soaking into D2O to exchange all exchangeable hydrogen atoms. Large crystals (>500um) did not perform well being directly transferred into a D2O soak condition (Figure 4.3F). Whether this was related to the D2O or the changes from the well conditions to the soak condition is unclear.

To address this, a gentle method was used where the well solution was exchanged to D2O and the drop was exchanged by vapor diffusion. After seven weeks of soaking, crystals appeared undamaged. A couple days later big cracks were seen which was perhaps due to an earthquake.

The structures resulting from the perdeuterated protein yielded some surprising results.

The crystals in the P3221 space group showed that a shift in the pre-switch I region and a related shift in loop 7 (Figure 4.8A and B). The change in the pre-switch I regions is similar to the conformational change seen in the M-Ras transition from state 1 to state 2 [1]. The distribution of H-Ras in solution favors the state 2 conformation where Y32 of switch I is closed over the nucleotide. This observation supports the idea that perdeuteration may change the dynamics of switch I and change the distribution of state 1 versus state 2. This would provide an explanation why in hydrogenated crystallization conditions containing NOG crystals grew in the R32 morphology while in perdeuterated conditions crystals were

P3221. The conformation of V29 in the open conformation would sterically clash with the Ca ion that coordinates the backbone of F28 and the hydroxyl group of D30, a symmetry related E31 and a symmetry related D33. This Ca ion overlays the position of an inserted Lys from Raf [17]or NORE1A [16] and presumably is involved in the stabilization of switch I in the effector bound conformation [50].

The results from the R32 space group showed there was also a change in the conditions that resulted in the off state where helix 3 and loop 7 are away from helix 4. Lower PEG soaks

(30% PEG 3350) with DTT/DTE concentrations of less than 10mM resulted in the allosteric off state. This may also represent a change in the equilibrium between hydrogenated and perdeuterated Ras. While in most structures there is little density to support a DTT/DTE in the switch II/helix 3 pocket, removal resulted in structures in the on conformation. Structures in the on state were consistent with non-perdeuterated structures. In 1507 the bridging water molecule was present and Q61 is partially ordered (Figure 4.12B). This shows that though perdeuteration may alter the equilibrium between switch conformations in Ras that the conformations remain similar.

The P3221 space group has Y32 in a conformation away from the active site as in the Ras- RasGAP complex. Y32 from a symmetry related molecule is inserted into the active site in a similar fashion to the Arg finger. Q61 in this space group is found in at least two conformations. One interacts with the catalytic water as in the GAP structure. One is swung away in an inactive conformation. A possible explanation for the multiple Q61 conformations is Q61 loses the interaction with the NH2 groups in the Arg finger. Though it would be ideal to solve the complex of Ras bound to RasGAP to examine GAP stimulated hydrolysis, this is currently not possible. The unit cell for the complex is ~210Å by ~210Å by

~108Å, to large for neutron crystallography. Though not an exact mimic, the P3221 space group would still provide information to further refine the understanding of GAP stimulated hydrolysis.

Neutron crystallography is typically performed at room temperature. It is a confirmation of feasibility that high resolution hydrogenated room temperature data of Ras structures in both the P3221 and R32 space groups reveal active sites that have most of the pertinent features in an ordered conformation (Figure 4.6B and 4.11B). Though Q61 is prone to disorder valuable insight can still be gained by learning the hydrogen positions in the rest of the active site. The perdeuterated structures showed weaker density for active site components such as Q61, however these structures were at lower resolution. Like the

room temperature hydrogenated structure there was minor density to support the catalytic conformation of Q61 (Figure 4.13B).

Here we show that we have made great progress in our efforts to solve the neutron crystal structure of Ras-GppNHp. We have proven feasibility and are currently in the process of producing more of the large Ras perdeuterated crystals for neutron data collection in both crystal forms. This information will strengthen proposed mechanisms for both GAP stimulated (P3221) and intrinsic (R32) hydrolysis. Ras will represent the first GTPase to have its structure solved by neutron crystallography.

CHAPTER 5

Structural Analysis of Uncomplexed Rap1A.

Summary:

Rap1a, a small monomeric GTPases, has emerged as an important signal transduction protein in varied cellular functions including cell-cell junction formation and cell adhesion. Rap1 is the closest homolog to the GTPase, Ras, which is mutated in 30% of human cancers. Understanding the structural nuances of Rap1 will aid in the understanding of Ras. Here we present uncomplexed structures of Rap1A. Together with a previously solved structure, the structures presented show that Rap1 can adopt five different active site conformations which when compared to Ras show interesting parallels. The data presented also shows that global conformational changes seen in Ras are regulated differently in Rap1.

Introduction:

Rap1 is a small monomeric GTPase belonging to the Ras subfamily. Rap1 acts as a signal transduction molecule involved in many cellular processes including cell polarity, secretion, cell-cell adhesion, differentiation among others. Rap1 has two isoforms that share over 90% sequence identity, Rap1a and Rap1b, where all but two of the substitutions are at the C-terminal hyper variable region (HVR) (Figure 1.2). Rap1 is activated by exchanging GDP for GTP, facilitated by guanine nucleotide exchange factors (GEFs). GEFs such as Epac bind Rap-GDP, loosen nucleotide binding and release the GDP allowing for the more abundant GTP to bind [159]. The binding of GTP organizes the dynamic regions, switch I (residues 30- 40) and switch II (residues 60-76), allowing binding of downstream effectors. The hydrolysis of GTP to GDP on Rap results in the inactive form. GTPase activating proteins (GAPs) such as Rap1GAP bind to switch I and switch II, helping to increase the rate of intrinsic hydrolysis

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from .00035 to 7.2min-1 [53, 54]. The cycling between GTP and GDP in GTPases is critical for maintaining proper cellular function. Many mutations in GTPases, GAPs or GEFs disrupt this cycling and result in a disease state. It is therefore important to understand how this balance is maintained.

Rap1 is the closest family member to Ras, sharing over 50% sequence identity, and was first identified for its ability to rescue K-Ras transformed cells [24]. Rap1 shares a high sequence homology with Ras in the effector binding regions and not surprisingly it was shown in vitro that Rap1 was able to bind the Ras effectors, c-Raf, PI3K and RalGDS [23]. Rap1 binds c-Raf in a manner similar to Ras through the Ras binding domain (RBD) and the cystine rich domain (CRD) [160], but the interaction with Rap1 results in very different biological outcomes from those of Ras. Where Ras binding to c-Raf was able to stimulate cell proliferation through the MEK/ERK pathway ([161]), Rap1 binding to c-Raf does not. This interaction, observed with C-terminal truncated Rap1 (residues 1-167), has not been shown in vivo with full length Rap1 and thus remains controversial. Rap1 also has a high affinity for the Ras effector RalGDS [23]. Interestingly, Rap1 but not Rap2 can stimulate RalGDS activity. Rap1 also interacts with PI3K (p110α) although it inhibits the pathway whereas Ras is an activator [162]. The ability for Rap1 to bind the Ras effector Raf may not be all that surprising given the high degree of similarity in their effector binding regions. These interactions highlight the need to understand the specificity between GTPases and their binding partners.

Ras has been intensively studied for years as it plays a role in oncogenesis. The result has been a wealth of structural and biochemical information that has provided insight into the nuances of Ras. This wealth of knowledge of Ras provides a point of comparison for all small GTPases. Rap especially benefits from comparison due to its high sequence similarity to Ras and a propensity for similar effectors. As a point of comparison, most GTPases in the Ras superfamily have a Gln at position 61 that has been determined to play a key role in the

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hydrolysis rate of Ras. Mutation to any residue other than Gln not only decreases the intrinsic hydrolysis rate by 10 fold but can also be oncogenic [44]. Though there is consensus on the crucial role of Q61 in Ras hydrolysis, there is still controversy on the mechanism through which it is oncogenic and what role it plays in the cell. In the canonical form of Ras within the P3221 space group, Ras adopts a conformation similar to when it is bound to RasGAP [9]. In this structure, Q61 interacts with a water molecule (wat175), the catalytic water molecule. It was proposed that Gln functions to place wat175, the nucleophilic water, for nucleophilic attack on the nucleotide. Ras was more recently solved in a different space group, R32, where the structure is more similar to an effector bound complex of Ras such as NORE1a and Raf (Chapters 2 and 3) [50]. This crystal form resulted in two different conformations termed allosteric on and allosteric off.

In the allosteric off conformation of Ras, helix 3 is away from helix 4, toward the active site. Switch II in the original growth conditions was disordered but introducing moderately or highly transforming Ras mutations (Q61L, Q61I, etc) or binding of a pocket between helix 3 and switch II resulted in an ordering of switch II where Q61 interacted with Y32 closing it on the nucleotide and preventing GTP hydrolysis [50]. In the allosteric on conformation, CaAcetate is bound to a remote site near R97 of helix 4. This binding accompanied a shift in loop 7 and helix 3 away from switch II, resulting in a highly ordered switch II conformation where the active site appears poised for catalysis. A second water, seen in the effector complexes Ras-NORE1A and Ras-Raf, is located between Y32 and the γ-phosphate and corresponds to the bridging water molecule seen in our wild type structure of Ras. While in the on state associated with the wild type, Q61 interacts with the bridging water molecule, it is not present in the highly or moderately transforming Ras mutants such as Q61L, but is present in the weakly transforming mutants. We have suggested that wat 189 is involved in catalysis and its absence may be related to the decrease in intrinsic hydrolysis of the different Q61 Ras mutants [50].

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Rap1 and Rap2 have a Thr instead of a Gln at position 61 and in this respect they both differ from Ras (Figure 1.2). This substitution results in a 10 fold decrease in hydrolysis rate similar to mutations of Ras at position 61. Mutation of Thr to Gln in Rap1 results in a hydrolysis rate that is similar to that of Ras [53, 163]. Several Rap structures have been solved including Rap2-GTP, Rap2-GTPγS, Rap2-GDP [13, 164], Rap1-Raf-RBD (residues 51- 131) [17], Rap1-RapGAP [41], and Rap1-Epac [165]. A structure of a Rap1 mutant E30D/K31E which makes switch I of Rap1 identical to Ras (named Raps) was also solved in complex with Raf-RBD [17]. The structures of Rap2-GTP, Rap1-Raf and Raps-Raf overlay well. Differences were observed in both switch regions that affect the active site. Switch I residue, Y32, of Rap1 in complex with Raf-RBD makes a direct hydrogen bond to the γ- phosphate where the Y32 interaction with the γ-phosphate in Raps is mediated by a bridging water molecule as in Ras. Switch II in the Rap1-Raf-RBD complex takes a conformation where T61 interacts with a water equivalent to the nucleophilic water molecule in Ras (Figure 5.1). This interaction was similar to the one seen between Q61 and the nucleophilic water in Ras solved from crystals with symmetry of the P3221 space group (Figure 1.4). The effector complex Rap1-Raf has switch I and switch II involved in crystal contacts. This complex is also controversial as to its biological relevance. Following the appearance of this complex in the literature, the structure of Rap1’s closest family member Rap2 (60% sequence identity) was solved bound to GDP, GTP and the GTP analog GTPγS [13]. This structure overlaid well with the Rap1-Raf structure. Switch I was in a similar conformation in the GTP structure with Y32 making a direct hydrogen bond with the γ- phosphate. In the Rap2-GTP structure the distance between Y32 and the nucleotide was shorter at 3.0Å compared to 4.0Å in the complex structure. This interaction was lost in the GTPγS structure perhaps due to the different hydrogen bonding properties of the S versus the O [13]. One difference in the Rap2-GTP structure is that the nucleophilic water molecule is not present. Subsequently, the structure of Rap2 was obtained from crystals with P21 symmetry, where switch II adopted an alternate conformation [164]. The nucleophilic water was present and was near T61. The backbone of T61 was ordered but

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the orientation of the side chain was not well defined. In this crystal form switch II was disordered from 62-64 or from 62-66 depending on the molecule in the asymmetric unit. This is consistent with a disordered switch II in Rap2 as observed for Ras. There are currently no published structures of uncomplexed Rap1.

Here we present the structure of Rap1a bound to both its natural ligand GTP and to the GTP analog GppNHp. The resulting crystals had two molecules in the asymmetric unit. In both molecules switch I was stabilized in a closed conformation by crystal contacts. Switch II was modulated by crystal contacts in one molecule but not in the other. The resulting structures showed novel active site conformations which may shed light on the functional mechanism of Rap1.

Figure 5.1: The active site of Raps in complex with Raf. T61 interacts with the bridging water in a similar manner to the interaction between Q61 and the bridging water molecule in Ras. T32 is shifted away from the ɣ-phosphate with a distance of 3.4Å.

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Experimental Procedures:

Cloning and Expression:

Truncated Rap1a (1-169) was mutated to prevent aggregation in expression and purification (C40S, C118S, and C139S). These residues were selected by comparing the sequences of Rap1, Rap2, Ras, and Ral and using the Rap1-Raf structure (1c1y [17]). The resulting clone was transformed into BL-21 DE3 cells (Novagen). Cells were grown in LB overnight at 37°C while shaking at 225rpm. Flasks were inoculated with the overnight culture and grown at

37°C. Cells were induced at an OD600 of 0.6-1.0 with 0.5 mM IPTG and then collected through centrifugation after 5-6 hours of induction. The resulting cell pellet was stored at - 80°C.

Purification:

The pellet was resuspended in 50mL Buffer A (20mM HEPES (pH 8.0), 40mM NaCl, 5mM

MgCl2, 1mM DTT, 5%w/v glycerol, and 5µM GDP) with protease inhibitors added (2µg/mL antipain, 5mM Benzamidine, 1mM pefabloc, 1µg/mL leupeptin, 1µg/mL pepstatin A, and 1µg/mL E64). The resuspended pellet was sonicated and cell debris was removed by centrifugation at 18,000 rpm for 20 min. PEI (0.5%) was added slowly on ice and stirred for 20 min. Precipitated DNA was removed by centrifugation at 18,000 rpm for 20 min. The sample was passed through a 0.45 µm filter then applied to a Q Sepharose Fast Flow column. Rap1a was eluted off the anion exchange column with a 150mL gradient of 0-16% Buffer B (Buffer A except 1 M NaCl) in Buffer A.Fractions containing Rap1a were located on a 12% SDS-PAGE gel. These fractions were concentrated to less than 2mL and run on a HiPrep 26/60 Sephacryl S-200 HR gel filtration column in gel filtration buffer (Buffer A except 150mM NaCl). Fractions were concentrated and the salt concentration was brought down by dilution with Buffer A. Samples were run on a Q Sepharose High Performance

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(QHP) anion exchange column and eluted with a 200mL 0-8% Buffer B in Buffer A. QHP separation was able to separate Rap1a-GDP and Rap1a-GTP (Figure 5.2). Rap1 bound to GTP was ready for crystallization. The GDP peak contained more contaminants; better crystals were obtained when only the GTP peak was used in further experiments.

Figure 5.2: Chromatogram of Rap1 run on a QHP column. Peak A corresponds to the GDP bound Rap1 protein. Peak B corresponds to the GTP bound peak and was used for crystallization.

Exchange Reaction:

GppNHp: Purified protein from QHP was concentrated to less than 2mL. Using a NAP desalting column the buffer was exchanged to Exchange buffer (20mM HEPES, pH 8.0, 200mM Ammonium sulfate, 10mM DTT, 0.1% n-octyl-glucopyranoside, 10% glycerol). GppNHp was added in three times molar excess with alkaline phosphatase conjugated to agarose beads and reacted for 45 min at 37°C. Following incubation, agarose bead were centrifuged and discarded. The resulting supernatant was exchanged to Crystallization

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buffer (Buffer A except 1µM GppNHp instead of GDP) through concentrations. Protein was then ready for crystallization.

Crystallization and Data Collection:

For Rap1a-GTP and Rap1a-GppNHp crystallization, hanging drop trays were set with a protein concentration of 5mg/mL. Crystals were obtained at concentrations from 3mg/mL to 15mg/mL but lower concentrations yielded fewer more singular crystals. The crystallization was Crystal Screen 20 (100mM Na Acetate Trihydrate, pH 4.6, 200mM Ammonium Sulfate, and 25% w/v PEG 4000) from Hampton Research or variations on precipitant or buffer concentrations. Crystals were also grown in 100mM Na Acetate, 100mM MES pH 5.0 or 5.5 with 25% PEG 4000). Crystals grew in less than 12hrs at 18°C. Resulting crystals were cryo protected in Crystal Screen 20 with 30%v/v PEG 400 and frozen in liquid nitrogen. Diffraction data were collected at 100K at the SER-CAT ID-20 line at APS (Argonne, Il). An X-ray wavelength of 1.0Å with an oscillation angle of 1.0° per frame was used. The detector distance was 120mm. Data were processed with HKL2000 [3].

Soak:

Rap1-GppNHp crystals were soaked in 50mM Na Acetate Trihydrate, 50mM MES, pH 6.0,

5mM MgCl2, 100mM Li Sulfate, and 30% PEG 4000 for 1-2 hours. Crystals were cryo protected and frozen as above and data was collected at 100K on the ID-22 beam line at SER-CAT (Argonne National Lab).

Phasing and Refinement:

The initial phasing for all the structures was done by molecular replacement. The initial molecular replacement of Rap1-GTP was performed with the Rap1 structure taken from the

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Rap1-Raf-RBD complex (PDB code 1c1y) [17] with Raf and all non-protein atoms removed from the model for molecular replacement. Initial fit was generated with CNS [166]. Molecular replacement for subsequent structures was done using Phasar with either the solved Rap1-GTP or Rap1-GppNHp models after all non-protein atoms were removed. A round of rigid body refinement was followed by simulated annealing using Phenix.refine. Further refinement was performed using Phenix.refine [8]. COOT was used for the manual rebuilding of the model [10].

Results:

A C-terminally truncated form of Rap1a (1-169) containing the entire catalytic domain was used for this study and is subsequently referred to as Rap1. For better expression and purification three Cys to Ser mutations were introduced; C40S, C118S, and C139S. These residues were selected by examining the Rap1-Raf, Rap2-GTP and Ras structures. They also overlay well with the Rap1-Raf structure and do not seem to affect the global structure of the protein. Both the C40S and the C118S mutations make no interactions in the obtained structures. In two molecules the hydroxyl group of C139S is within hydrogen bonding distance of N108 but it does not seem to stabilize any particular conformation. Crystals of Rap1 bound to both GTP and GppNHp were obtained with symmetries of the space group

P21212 with two molecules in the asymmetric unit. The data refinement statistics are shown in Table 5.1. Rap1-GppNHp crystals grew in a solution of Na Acetate Trihydrate (pH 4.6). To examine if Rap1 has a similar allosteric site as Ras or if the site is Ras specific, soaks were performed in a solution containing 50mM Na Acetate or CaAcetate buffered with MES at pH 6.0. Crystals were not stable above pH 6.5 in the conditions tried. Unfortunately, soaks in CaAcetate resulted in a precipitate forming and destroyed the crystals. The soak in NaAcetate was gentler and the resulting structure, referred to here as Rap1-NaAcetate, has data collection and refinement statistics as shown in Table 5.1.

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Table 5.1: Data collection and refinement statistics for Rap1A bound to GTP or GppNHp Structure ID 334 337 612 LiSO4_soak_1.5A Structure Name Rap1-BW Rap1-GTP Rap1-Thr Rap1-NaAcetate Mol B Conf. Bridging Wat Disordered Thr Disordered Data collection

Space group P21212 P21212 P21212 P21212 Beamline 22-ID 22-ID 22-ID 22-ID Collection 100K 100K 100K 100K Temperature Cell dimensions 111.869 112.628 113.634 112.999 a , b , c (Å) 42.181 41.821 42.338 41.993 66.178 66.134 66.750 68.471 α, β, ɣ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 500-1.80 500-1.55 500-1.58 50-1.50 (1.86-1.80) (1.61-1.55) (1.64-1.58) (1.55-1.50)

R sym or R merge 0.048 0.060 0.093 0.076 0.497 0.549 (0.648) (0.509) I / σ 37.3 38.3 25.3 25.9 (2.19) (2.57) (1.93) (2.17) Completeness (%) 97.5 97.5 98.3 99.7 (82.1) (95.0) (90.3) (98.0) Redundancy 6.9 7.0 5.9 6.7 (4.9) (5.0) (3.4) (4.7) Refinement Resolution (Å) 1.8 1.55 1.58 1.5 No. reflections 28015 43014 41329 49090

R work / R free 21.29/26.01 18.71/21.29 19.53/22.60 19.48/22.88 No. atoms Protein 2678 2628 2671 2669 GTP/GppNHp 64 64 64 64 Mg 2 2 2 2 Water 129 316 312 323 Sulfate 0 1 1 2 B Factors Protein 33.06 21.78 23.72 21.10 GTP/GppNHp 27.47 15.85 16.65 15.78 Water 38.52 28.92 30.29 26.33 R.m.s. deviations Bond lengths (Å) 0.007 0.007 0.007 0.007 Bond angles (°) 1.171 1.170 1.150 1.114

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Table 5.1: Continued

Structure ID 1000 1003 1024 1030 Structure Name 40uL 35uL pH 5.0 pH 5.5 Mol B Conf. Thr Thr Thr Thr Data collection

Space group P21212 P21212 P21212 P21212 Beamline 22-ID 22-ID 22-ID 22-ID Collection 100K 100K 100K 100K Temperature Cell dimensions 112.561 112.833 112.938 112.908 a , b , c (Å) 42.226 42.353 42.967 42.065 66.778 66.672 66.896 66.183 α, β, ɣ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 50-2.08 50-1.93 50-2.05 50-2.00 (2.15-2.08) (2.00-1.93) (2.12-2.05) (2.07-2.00)

R sym or R merge 0.085 0.106 0.098 0.138 (0.394) (0.515) (0.537) (0.400) I / σ 16.4 12.5 12.1 13.8 (3.55) (2.34) (2.30) (2.73) Completeness (%) 99.8 99.2 95.0 99.3 (99.0) (97.2) (84.8) (95.0) Redundancy 4.6 5.6 4.7 5.9 (4.0) (4.3) (3.5) (4.7) Refinement Resolution (Å) 2.08 1.93 2.05 2.00 No. reflections 18649 23069 18516 20709

R work / R free 18.08/23.82 18.04/23.26 17.86/23.48 18.55/24.39 No. atoms Protein 2668 2672 2670 2664 GTP/GppNHp 64 64 64 64 Mg 2 2 2 2 Water 142 180 188 173 Sulfate 1 1 1 1 B Factors Protein 20.95 14.81 23.81 17.75 GTP/GppNHp 16.54 10.62 18.88 13.21 Water 24.21 19.58 28.67 20.52 R.m.s. deviations Bond lengths (Å) 0.008 0.008 0.008 0.007 Bond angles (°) 1.135 1.146 1.130 1.148

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The structures obtained were globally very similar, with variations primarily in the active site, switch II, and the loop regions (Figure 5.3). These differences were seen when comparing molecule A to molecule B in the asymmetric unit, but were also seen when comparing molecule B from the different structures. Among the resulting structures there were three different active site conformations. The active site variations accompanied different switch II conformations. Other regions that showed variations amongst the solved structures were loop7, loop9, helix4 and the C-terminus. Examining these different conformations may give insight into how Rap1 utilizes flexible regions to interact with protein binding partners in the cell and provides a valuable point of comparison with Ras.

Figure 5.3: Global comparison of Rap1a. A: Molecule A from the Rap1-GTP, Rap1-BW, Rap1-Thr and Rap1-NaAcetate are structurally similar. Molecule A of Rap1-GTP is used a model of this conformation. B: Molecule B from Rap1-GTP, Rap1-BW, Rap1-Thr and Rap1-NaAcetate reveal more structural differences especially in Loop 7, Loop 9 and switch II. Rap1-GTP molecule A is included for comparison. The Rap1-BW molecule B conformation is consistent with the conformation seen in molecule A.

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T61 interacts with the nucleophilic water molecule:

In molecule B of the Rap1-GTP structure, Y32 is making a direct hydrogen bond with the nucleotide (2.57Å) and wat230 (2.59Å) as seen in the Rap2 and Rap1-Raf structures (Figure 5.4). A direct interaction is also seen in molecule B of the Rap1 structure solved in the presence of NaAcetate at a higher pH bound to GppNHp (2.54Å) (the Rap1-NaAcetate structure). In this structure however, wat230 is absent. The catalytic water molecule is coordinated by the ɣ-phosphate of GTP/GppNHp as well as the carbonyl of T35 as is common in GTPases. In the canonical form of Ras, with P3221 symmetry, an interaction between the side chain of Q61 and the nucleophilic water molecule is observed. Rap1-GTP and Rap1-NaAcetate are similar to the canonical form of Ras. The nucleophilic water molecule is within hydrogen bonding distance to the hydroxyl group of T61 at a distance of 2.77Å and 2.83Å respectively. The backbone amide of T61 is also brought within hydrogen bonding distance of the nucleophilic water with a distance of 3.11Å in the Rap-GTP structure and 3.15Å in the Rap1-NaAcetate structure. This conformation is similar to the one observed in the Rap2-GTP and was termed the non-catalytic conformation.

Figure 5.4: T61 interacts with the catalytic water molecule. The active site of the Rap1-GTP (teal) reveals an active site where the hydroxyl group of T61 interacts with the nucleophilic water with a distance of 2.8Å

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Pre-bridging water molecule:

A bridging water molecule can be observed in Ras-effector complexes or in structures of Ras solved from crystals with symmetry of the R32 space group, which mimics Ras bound to its downstream effectors. In molecule A of the solved Rap1 molecules switch I is stabilized by crystal contacts in a closed conformation. This closed conformation is similar to the state 2 conformation in Ras which is stabilized by effector binding of switch I. Y32 makes a direct interaction with the ɣ-phosphate of either GTP or GppNHp bound Rap1 structures with distances ranging from 2.6Å to 3.0Å. A water molecule is present (wat87) that is within 2.9Å of Y32 and within 3.5Å of the ɣ-phosphate in all molecule A structures (Figure 5.5A). The proximity of wat87 to both the nucleotide and Y32 is similar to the bridging water molecule seen in published Ras and Raps structures. However, Y32 makes a direct interaction with the nucleotide so w87 does not act as a bridging water molecule. Due to its close proximity it could be called a pre-bridging water molecule as it is positioned to become the bridging water molecule awaiting a shift of Y32 away from the ɣ-phosphate. Another difference seen in these structures is that the carbonyl group of G60 is oriented towards the active site rather than away. This change allows the carbonyl to be in proximity of wat87 with distances between 2.9Å to 3.7Å. In molecule A of the one structure in which we see the bridging water molecule as in Ras (referred to here as Rap1-BW), wat87 is the furthest from G60, and is closer to the bridging water molecule position. This is seen as a decrease in the distances between the w87 and Y32 and ɣ-phosphate, which is shortest in molecule A of the Rap1-BW structure.

Presence of a bridging water molecule in Rap1.

While in Ras both direct and indirect interactions between Y32 and the nucleotide have been observed, in previously published Rap1 and Rap2 structures the hydroxyl group of Y32 makes a direct interaction with the nucleotide. The exception to this is the Rap1-GTPɣS

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structure where the distance is 7Å. In molecule B of the Rap1-BW structure the distance between Y32 hydroxyl group and the ɣ-phosphate is 4.23Å. This distance is far for a direct interaction and similar to the distances in the indirect interaction seen in Ras and Raps. Like these structures, a water molecule is seen between Y32 and the nucleotide (wat87) at a distance of 2.47Å to Y32 and 2.48Å from the ɣ-phosphate (Figure 5.5B). In the Raps-Raf structure and in Ras structures the hydroxyl group of residue 61 interacts with the bridging water molecule. In our structures, T61 is facing away from the active site. This conformation for T61 is also seen in molecule A, however, in molecule B wat87 acts as a bridging water molecule whereas in molecule A Y32 is in a direct interaction.

T61 can replace the bridging water molecule between Y32 and the nucleotide

A third active site conformation is seen in molecule B of one of the Rap1-GppNHp structures (referred to here as the Rap1-Thr structure). This structure was also obtained when varying buffer concentration of pH of the growth condition. Like in the Rap1-BW structure Y32 of switch I is no longer in direct contact with the nucleotide, with a distance of 4.16Å between Y32 and the ɣ-phosphate of GppNHp. Previously this shift accompanied positioning of a bridging water molecule. In Rap1-Thr, the hydroxyl group of Thr61shifts ~3Å from the position found in the Rap1-GTP structure and replaces the bridging water molecule. As a result, the hydroxyl group of T61 is 2.5Å away from both Y32 and the ɣ-phosphate (Figure 5.6).

The shift in T61 also accompanies a different coordination of the nucleophilic water molecule. As in the previous structures, the backbone amide and carbonyl group of Thr35 and the ɣ-phosphate interacts with the nucleophilic water. In addition, switch II gains an interaction with the nucleophilic water in this structure as well as with the amide groups of Gly60, Thr61, and Gly62, with distances of 3.27Å, 3.12Å and 2.97Å respectively. This gain of a hydrogen bond may explain the observed ordering of switch II.

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Figure 5.5: Pre-bridging and bridging water molecule conformations. A: The Rap1-GTP structure acts as a representation of the active site seen in Molecule A (yellow). T61 is swung away from the active site. Y32 interacts is in a direct conformation with the nucleotide with a distance of 2.7Å. B: The active site of Rap1-BW molecule B structure reveals that Y32 adjusts to accommodate a bridging water molecule with a distance of 4.2Å between the hydroxyl group of Y32 and the ɣ-phosphate. T61 is swung away similar to the conformation seen in molecule A.

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Figure 5.6: T61 replaces the bridging water molecule. Rap1-Thr molecule B (cyan) adopts a novel conformation where T61 replaces the bridging water molecule seen in Ras and the Rap1-BW molecule B structures. Electron density is contoured at 1.0σ.

Switch II conformations:

The different active site conformations including differences in switch II residue 61 results in different switch II conformations. Switch II in molecule B is free of crystal contacts and has more freedom to sample different conformations than does molecule A. Thus in the Rap1- GTP structure, switch II becomes disordered between Thr61 and Met67. In the Rap1- NaAcetate structure switch II is disordered from residues 62-67.

Switch II of molecule A where a pre-bridging water molecule is seen also adopts a previously unseen conformation in Rap1. It is ordered and is involved in crystal contacts with switch I of a symmetry related molecule A. In switch II, T61 is oriented away from the active site as

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in the Rap1-Rap1GAP structure but the overall conformation of switch II is unlike that seen in complex with Rap1GAP. The hydroxyl group of T61 interacts with the backbone amide of Q63. The carbonyl is within hydrogen bonding distance of the NH groups of R68 (3.01Å and 3.08Å) tying the lower part of switch II to the upper half of switch II. There is also a water molecule that is 3.11Å away that interact with S11 and would thus connect switch II to the P-loop. The carbonyl of Q63 interacts with wat6, which through wat5 bridges to the hydroxyl of Y71 and the carbonyl of A59. The hydroxyl group of T65 interacts with the backbone amide of R68 and is within hydrogen bonding distance of wat6. Residue A59 begins alpha helix 2 in Rap1. In the molecule A conformation F64 is flipped towards helix-3 and is in a different conformation than previously seen. This conformation is unlike what was seen in the Rap1-Raf structure, where switch II is also stabilized by crystal contacts, and is a change from the location of Y64 in Ras in different Ras effector complexes, Ras-PI3K and Ras-NORE1a. The conformation of switch II seen with a pre-bridging (molecule A) and bridging water molecule (Rap1-BW) do not vary significantly. There is weaker density for molecule B of the Rap1-BW structure for residues 62 and 63 which may result from the decrease in crystal contacts

The Rap1-Thr structure has a different switch II conformation (Figure 5.7). This is not surprising given the movement in T61 to replace the bridging water molecule.

Global comparisons:

Two global conformations are seen in Ras. In the “on” state helix 3 and loop 7 are pulled towards helix 4 resulting in an ordering of switch II. The “off” state does not have a helix 3/loop 7 shift and there are steric clashes with the switch II conformation seen in the on state. This results in either a disordered switch II or switch II ordered in what we call the anti-catalytic conformation. The conformation seen in the Rap1 structures overlays with the “on” state in Ras where helix 3 is shifted towards helix 4. In fact, the shift is more

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Figure 5.7: A comparison of the conformations adopted by switch II in Rap1. The Rap1-BW molecule B (violet) shows a novel switch II conformation where F64 is swung towards helix 3, this conformation is also adopted in all Molecule A solved. The Rap1-Thr molecule B (cyan) structure reveals a switch II conformation where F64 is swung away from helix 3 which is similar to the placement of Y64 in Ras bound to effector molecules. pronounced in the Rap1 structures. In Ras this shift required either a binding partner or a CaAcetate bound at the allosteric site. In Rap1 there is no bound CaAcetate. The conformation of the allosteric site is also different. In Ras residue 101 is a Lys where in Rap1 it is a Leu. The L101 conformation overlaps the acetate binding site observed in Ras. In Ras R97 is involved in the binding of the Ca2+ in the allosteric site. In the Rap1 structures this residue is shifted and overlays where the Ca2+ would bind in Ras. In Ras it is seen that R97 stacks with Y137 and that this is shifted upon binding of CaAcetate in the allosteric site. In Rap1 Y137 is equivalent to W138 and R97 stacking is preserved.

The changes seen upon ligand binding at the allosteric site in Ras is a coordinated movement between helix 3, loop 7 and the active site. There are substitutions in Rap1 that may decouple this movement (Figure 5.8). Y96 is involved in the coupling in Ras whereas in Rap1 this residue is L96. In Rap1 residue 80 is a Leu rather than a Cys. These differences

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may help stabilize hydrophobic interactions with L93 and I100 in helix 3, leading to the on state conformation where it is shifted toward helix 4 and disfavoring a shift to the off state.

Figure 5.8: Comparison of Rap1 (violet) helix 3 and helix 4 residues with Ras (green) in the allosteric on state. The conformation of R97 in Rap1 overlaps the Ca2+ binding site in Ras. L101 in Rap1 overlaps the acetate binding site in Ras and would prohibit acetate binding in the allosteric site as in Ras.

Loop9 is a site of an insertion (N140) in Rap1 compared with Rap2 and Ras. The extra residue gives more flexibility to loop9 and results in two observed conformations (Figure 5.9A). In most structures (all molecule A structures, Rap1-NaAcetate molecule B, and Rap1- BW molecule B) S139 (one of the C to S mutants) N140 and C141 continue helix 4 (Figure 5.9B). In the Rap1-Thr and Rap1-GTP structures these residues aren’t part of helix 4 and are swung towards the C-terminus to form a β-turn from W138 to C141 (Figure 5.9C). The side chain of S139 is within hydrogen bonding distance of the side chain of D108 with a distance of 2.9Å in the Rap1-Thr structure and a distance of 3.36Å in the Rap1-GTP structure. N140 gains interactions with the C-terminus and loop 7. The hydroxyl group is about 3.4Å from

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D108 in both structures. The NH group is 3.32Å and 3.21Å from Q164. The carbonyl group interacts with the backbone amide of M111 with distances of 3.07Å and 3.01Å.

B C

Figure 5.9: Two different conformations of loop 7 are observed in the presented Rap1 structures. A: In all molecule A structures (yellow, orange, pink, light green) and in molecule B of the Rap1-BW (magenta) and Rap1-NaAcetate (plum) structures N140 is towards the solvent. In the Rap1-GTP (teal) and Rap1-Thr (cyan) structures N140 is shifted towards the C-terminus. B: The Rap1-BW molecule B structure is used as a representation showing the stabilization and continuation of helix 4. In this conformation S139 (Cys in wild type Rap1) is within hydrogen bonding distance of R97, however, this interaction does not seem to alter the overall structure. C: The Rap1-Thr shows a different loop7 conformation where loop9 adopts a β-turn. In this conformation loop9 forms interactions with loop7 and C-terminal residues.

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The C-terminus has been truncated at residue 169 and in most cases is ordered only to R167 or K168. The C-terminus of Rap1-GppNHp-BW is shifted from the other molecule A conformations. The backbone is shifted from R163 to K168 (T169 has weak to no density in all structures). In the Rap1-GTP, Rap1-NaAcetate, and the Rap1-Thr structures where the C- terminus overlays well there is density to support the entire side chain of both R167 and K168. In the Rap1- BW where the C-terminus is shifted, the side chains of R167 and K168 are disordered. Molecule A is consistent amongst all the structures obtained and as such we used molecule A from the Rap1-GTP structure for comparison.

Small molecule binding:

In Ras small molecule binding accompanied changes in global conformations. In Rap1 a - bound SO4 from the mother liquor and acetate in soak conditions bound Rap1 with - differing results. In unsoaked structures there is a SO4 , found in the crystallization condition, bound to molecule A. This sulfate makes interactions with the backbone amides of Q87 and S88 as well as with two water molecules. It is replaced in molecule A of Rap1- - LiSO4 and in molecule B with two water molecules. The sulfate binding does not seem to have any structural repercussions. In molecule B of the Rap1-NaAcetate structure residues 87 and 88 are less defined. There is weak electron density for a water molecule interacting with the backbone amide of Gln87 but no density is there to support a water molecule interacting with the backbone amide of Ser88.

The allosteric binding site in Ras involves binding of CaAcetate. This binding is not observed with MgAcetate or in CaCl2. To examine whether this is also seen in Rap1 we performed soaks of Rap1 crystals in the presence of CaAcetate and NaAcetate. NaAcetate is found in the mother liquor (100mM) as the buffer at pH 4.6. First a stabilization condition was determined where Rap1 crystals could be soaked for greater than 1hr. The stabilization condition substituted lithium sulfate for ammonium sulfate and raised the pH from 4.6 to

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6.0. Crystals soaked in stabilization condition were stable and resulted in a 1.55Å data set (Table 1). Unfortunately, substitution of CaAcetate for NaAcetate in the soak condition was not successful at similar concentrations. Substitution of CaAcetate caused a precipitation at pH 6.0. The Rap1-NaAceate structure soaked at pH 6.0 revealed two acetate binding sites not in unsoaked crystals.

The first bound acetate binds near where the allosteric binding site is seen in Ras (Figure 5.10A). This acetate molecule interacts with the NE of Arg97 (3.12Å), the same residue that acetate binds to in the allosteric switch of Ras. However, the binding of acetate to Arg97 in Rap is in a different manner to the acetate binding in Ras (Figure 5.10B). The effect on the hydrogen bonding network in Ras is not seen in Rap1 and switch II is disordered beyond residue Thr61. There are conformational changes in Rap1-NaAcetate compared to unsoaked structures. Loop8 and helix-4 have unclear density and helix 4 is shifted. The density becomes less defined at E121 where there is a break in the backbone electron density continuity. This continues through loop8 and to the end of helix4. Many side chains in helix4 become disordered. The backbone density for E129, G131 and W138 is also weak. The conformation of Loop 7 is also altered resulting the carbonyl of E107 being 2.99Å away from NH-R97.

The second acetate binding site is found interacting with the carboxyl group of residue Phe143 of molecule A and with three water molecules. In the soaks there is also a different SO4 binding site observed. This site is in crystal contact between location and helix 3 of symmetry related molecule As. The sulfate interacts with the backbone amide of E45. The binding of this acetate and sulfate do not seem to alter the structure as no conformation differences are seen in molecule A compared with the unsoaked structures.

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Figure 5.10: Calcium Acetate binding site. A: Rap1 crystals soaked in NaAcetate (plum) at pH 6.0 have a bound acetate molecule near R97. B: Both Ras (green) and Rap1 have an bound acetate near R97, however, the binding occurs differently.

Discussion:

While there is no correlation established between oncogenicity and Rap, it is an essential protein in the cell; this makes understanding how it functions important. Rap1 plays a role in the formation of cell-cell junctions an in cell-cell adhesion. Rap1 is the closest family member to Ras, an important cancer protein, and has been shown to rescue Ras transformed cells [24]. The ability of Rap1 to rescue Ras transformed cells was originally attributed to competitive binding of Raf, but a direct interaction between full length Rap and Raf in the cell has yet to be established [26]. It is now believed that the ability of excess Rap1 to revert Ras transformed cell is related to Rap1 function in cell-cell adhesion. Given that Ras is mutated in 30% of human cancers and is prominent in high mortality cancer such as pancreatic (90%), lung (30%) and colon (50%) cancer, a drug inhibiting Ras function in cancer is desirable [66]. Rap1 provides a protein with which to study specify determinants in Ras, as it is one of its close family members. This is important in the design of a drug that would be specific for Ras. To date there has been little structural information available for Rap, particularly for Rap1, since virtually all of the structural work to data has focused on

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Rap2. Many of the solved structures have a disordered switch II conformation or have switch II conformations stabilized by crystal contacts [13, 164]. Here we present structures of Rap1A solved bound to GTP and to the GTP analog, GppNHp. These structures adopt novel conformations and add to the complexity of Rap function.

We observed two conformations for Y32, where it makes either a direct and an indirect interaction with the ɣ-phosphate of GTP or its analogue GppNHp. A bridging water molecule between Y32 and the nucleotide was observed as seen in our Ras structures. The Rap1-BW is the first structure of wild type Rap1 where Y32 is making an indirect interaction with the nucleotide through a bridging water molecule (Figure 5.5B). The Rap2-GTP S structure had a distance of 7Å between Y32 and the ɣ-phosphate [13]. This distance is consistent with the distance observed in the Ras solved from crystals with symmetry of the

P3221 space group and the Ras-RasGAP structure as well [4, 41]. The Raps-Raf structure, which is Rap1 with two switch I residues mutated, has a bridging water molecule (Figure 5.1). The movement in Y32 was attributed to the tighter interactions between Raps and Raf compared with Rap-Raf. In the Raps-Raf structure, the switch II of Raps and the switch II conformation of Rap1 is similar. There is, however, a 2.5Å shift in the hydroxyl group of T61. T61 shifts to interact with the bridging water molecule [17]. This conformation is analogous to what is seen with Ras in the allosteric on state. There, Q61 forms an interaction with the bridging water molecule [107].

In molecule A in all of our Rap1A structures, we see a conformation where Y32 is within hydrogen bonding distance to the ɣ-Phosphate but a water molecule is seen in a similar position to the bridging water molecule (Figure 5.5A). We call this a pre-bridging water molecule. The Rap1-BW chain B takes an almost identical conformation where the pre- bridging water molecule now is shifted into position of the bridging water molecule. It is logical to suggest that the pre-bridging water molecule shifts upon Y32 movement to become the bridging water molecule as seen in Rap1-BW chain B.

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Hydrolysis experiments examined mutations of T61 in Rap1. Mutation to Q61 resulted in hydrolysis rates similar to that of Ras as shown previously. Mutation of T61 to Leu or to Ala did not significantly alter the hydrolysis rate with respect to intrinsic or GAP stimulated hydrolysis. The Rap1-RapGAP complex shows a T61 conformation where T61 is away from the active site and thus not involved in the hydrolysis mechanism. It may, however, help stabilize the formation of the GAP complex. We have proposed a mechanism for intrinsic hydrolysis in Ras that involves placement of a bridging water molecule. In Rap1 we saw a bridging water molecule with T61 swung away from the active site. This would be consistent with a mode of intrinsic hydrolysis in which T61 is not involved.

None of the Rap structures solved adopt the allosteric off conformation seen in Ras. In Ras the on state was stabilized by either effector or small molecule binding. In Rap the on state was observed in the absence of small molecule or effector binding. This could be due to an increase in the hydrophobic interaction stabilizing helix 3 toward helix 4 (Figure 5.8). The active site is stabilized in the off state of Ras by either small molecule binding or oncogenic mutation and is also seen in the Ran-Importin-β complex. The Q61T mutation in Ras is weakly transforming. Substitution of this residue may not be able to stabilize the off active site conformation as does the highly transforming mutations that are hydrophobic. This may explain why this conformation is not seen in Rap.

Little is known about the structures of Rap-effector complexes. While the Rap-Raf complex was solved over a decade ago little structural information has been gathered since then. This complex is also unclear as to its biological significance and does not involve interaction with switch II as the NORE1 and PI3K does in Ras and likely in Rap.

We also see a Rap1 conformation in which T61 replaces the bridging water molecule. It is unclear whether this would be a catalytically active conformation. The hydroxyl group on

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the Thr may be able to act as a shuttle for a proton between the ɣ-phosphate and Y32. The Thr, however, would be less flexible in position than a water molecule. Movement of the proton to the bridging O between the β and ɣ-phosphates might be more restricted than in the bridging water molecule due to constrains associated with switch II backbone conformation. However, this could be an advantage if the position of the Thr hydroxyl group is optimized for catalysis. The accompanying switch II conformation shows similar placement of F64 to Y64 in the Ras-NORE1a complex. This is important because Rap1 is able to bind NORE1a, forming interactions with switch I and switch II. The comparison and similar placement of residue 64 suggests it is an important residue for binding in Ras and Rap1. In other structures, the placement of F64 is shifted slightly but may be a result of crystal packing.

Ras has been solved in several different crystal forms and reveal different switch II conformations. Here we reveal several conformations accessible to switch II, and consequently the active site, in Rap1. The Raps and Rap complexes with Raf show slightly different active sites. In the Raps-Raf complex a bridging water molecule is observed whereas in both the Rap1-Raf and Rap1-RapGAP structures, as well as in all Rap2 structures, no bridging water molecule is observed. This lead to a conclusion that Rap does not utilize a bridging water molecule and that Y32 is closed over the nucleotide. We have now shown that both T61 and a bridging water molecule may be present, linking the hydroxyl group of the highly conserved Y32 to the ɣ-phosphate of GTP. The significance of these conformations to catalysis in Rap1A remains to be determined.

- There is an observed SO4 binding site in the molecule A in both the structures of Rap1A-GTP and Rap1A-GppNHp. This binding involves interactions with Q87 and S88. While there are no obvious structural changes due to binding, these residues are involved in Rap1 trafficking in the cell. Rap1 is gernylgernylated and contains a polybasic region at its C-terminus. Substitution of 85-89 with the sequence found in Ras (TAQST to NNTKS) resulted in a shift

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from a perinuclear localization to Rap1 localizing to the plasma membrane. The reverse substitution in Ras was enough to keep Ras localized perinuclearly despite having undergone prenylation. It was proposed that Rap1 localization to the perinuclear region may involve protein binging to residues 85-89 [61].

We examined whether the allosteric switch is similar in Rap1 as it is in Ras. All of our Rap1 structures were in the allosteric on state regardless of binding partners. We performed soaks at a higher pH with NaAcetate. Soaks with CaAcetate were unsuccessful. Despite this we were interested that acetate bound Rap1 in the absence of calcium. This was not observed in Ras (Chapter 2). The acetate binding site was near helix 4 as it was in Ras but was shifted with respect to the position found in Ras (Figure 5.10A and B). When examining the allosteric site, there are features in Rap1 that may prohibit CaAcetate binding. K101 is a Leu creating an environment that maybe intolerant to Ca2+ binding. Taken together it seems unlikely that Rap1, the closest Ras family member, has an allosteric site similar to Ras. This may make the allosteric site a desirable drug target reducing the chances of the drug being promiscuous. Interestingly, in Ras it has been shown that helix 4 makes direct interaction with the membrane. We showed that in Rap1 the conformation of this helix is sensitive to pH, where it becomes destabilized in going from pH 4.6 to pH 6.0. Whether Ras has a similar pH sensitivity remains to be determined.

We have shown that switch II and the active site in Rap can adopt various conformations in a manner that is highly sensitive to the surrounding conditions. This is consistent with what is found for Ras. The switch regions, particularly switch II, exhibit a high degree of disorder in the uncomplexed forms of both GTPases and can be modulated to a variety of conformation stabilized by binding partners. This is an important venue through which GTPases can interact with such a large variety of effectors and regulatory proteins such as GAPs. These interactions select conformations associated with the various functions

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performed by these proteins, through complex mechanisms that we are only now beginning to understand.

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CHAPTER 6

The Multiple Solvent Crystal Structures Method Performed on Rap1a.

Abstract:

The Multiple Solvent Crystal Structures (MSCS) method is designed to probe binding sites on protein surfaces. MSCS has been performed on the classical, well-studied, proteins such as elastase and RNaseA. A MSCS data set has also been collected for Ras. This information combined with FTMap results (a computational version of MSCS) provides insight into hotspots of protein-protein interaction in Ras. Rap1 is the closest homolog to Ras and will provide valuable comparison with Ras by which specificity determinants will be identified for each of the two closely related GTPases. Here we present the methods developed to crosslink Rap1A, transfer into organic solvent soaks and X-ray data collection of the Rap1A MSCS. There are 18 data sets with 11 unique solvents all collected to around 2Å resolution, which should provide a complete picture of the binding surface of Rap1A and a comparable MSCS set to that obtained for Ras.

Introduction:

Rap is a member of the Ras family of monomeric GTPases. GTPases act as molecular switches alternating between a GTP bound active form and a GDP inactive form. In the active form the dynamic regions of switch I (30-40) and switch II (60-76) change conformation and allow for effector binding. Rap functions in a variety of cellular process from cell differentiation to cell-cell adhesion. Rap consists of Rap1a, Rap1b, Rap2a, Rap2b and Rap2c. Rap1 and Rap2 share 60% sequence identity. Rap1 is the closest family member to Ras with about 50% sequence identity. Ras is an oncogene, mutated in 30% of human cancers and thus is an attractive target for drug therapy [66]. The MSCS of Rap1 will

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provide a good venue for comparing the location of hot spots with those of Ras and for determining the key interactions that may lead to high specificity towards Ras, but not its close homologue Rap1A.

The crystal structure of Ras was solved over 20 years ago. Since then many structural studies have begun to tease out the nuances of the structure-function relationship in Ras. The catalytic domain of Ras has been split into two lobes, an N-terminal (1-86) and C- terminal lobe (87-171). We termed the N-terminal lobe which includes switch I, switch II, the P-loop and most of the nucleotide binding pocket the effector lobe as it contains residues involved in protein-protein interaction. The C-terminal lobe contains areas known for membrane interaction as well as an allosteric site we identified (Chapter 3). This allosteric site located between helices 3 and 4 binds CaAcetate and results in a conformational shift with helix 3 and loop 7 moving toward helix 4, away from the active site. The shifted structure was termed the allosteric “on” structure as it is thought to be the conformation seen with GTP hydrolysis. This conformation can also be stabilized by protein binding partners such as GAP, NORE1A or PI3K [14, 16, 41]. In Ras crystals with switch II free of crystal contacts the allosteric on conformation was accompanied by the ordering of switch II.

The multiple solvents crystal structures (MSCS) technique uses small molecules as probes to the surface of proteins [71]. In the MSCS method crystals of a given protein are transferred to different organic solvents at high concentrations. As organic solvents are quite harsh to crystals, crystals are generally crosslinked with glutaraldehyde before transfer to organic solvent solutions. After crosslinking and soaking, X-ray data are collected. Structures are solved and superimposed for comparison. It has been shown that the organic solvents typically displace water molecules at sites that have evolved for interaction. Thus, MSCS has been shown to be able to identify hot spots, areas of protein-protein or protein-ligand interactions, on protein surfaces. The technique was established by examining classical

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proteins where there was abundant information on protein and ligand binding sites. These included Elastase (a peptide binding protein) [71], RNAse A [72] (an RNA binding protein) and Ras [5] (interacts with other proteins). In both cases MSCS was able to pick out known sites of protein-protein or protein-ligand interaction. Subsequently, the MSCS data sets have been obtained for tail spike (a sugar binding protein), trypsin (a peptide binding protein), chymotrypsin (a peptide binding protein), lysozyme (a sugar binding protein) and BAF (a DNA binding protein). Analyses from these structures are consistent with previously published MSCS results and support the ability of MSCS to identify hot spots in proteins [unpublished results].

FT-Map is a computational analog of MSCS where molecular probes are computationally placed on the surface of the protein in a dense grid. The program then finds favorable positions by utilizing empirical free energy functions. Once positions are identified, clusters are located and ranked based on the average free energy of the probes. FT-Map can probe organic solvents that are generally too harsh or dangerous to work with in an experimental setting. It also can probe areas that are found in crystal contacts or structures that are less stable in the organic soak environment.

MSCS and FT-Map are complimentary techniques that together can give a complete picture of hot spots in a protein. Recently, our lab performed analysis of the binding surface of Ras using both MSCS and FT-Map. The results from MSCS identified 9 clusters (2 were the same cluster located in vicinity of two symmetry related molecules where members of the cluster interacted with both molecules) (Figure 6.1). MSCS located two sites in the effector binding lobe, clusters 3 and 7. Both overlay the region of GAP binding. Cluster 3 also overlaps the region where the Ras Binding Domain (RBD) of Raf binds. Clusters 1, 2b, 4, 5, 6, and 8 were located in the inter-lobal region between the effector and allosteric lobe. Clusters 6 and 8 in the interlobal region also overlapped the region of GAP binding. Cluster 5 was located where Raf-Cystine Rich Domain (CRD) is thought to interact. Of the remaining clusters two,

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2b and 4, were not known interaction sites but were found in areas that undergo large conformational changes in Ras GDP. Solvents in cluster 4 were interacting with residue K147 and D30. K147 has been shown by NMR data to be involved in the global correlated dynamics seen in Ras. Binding of cluster 1 by the solvents HEX, YEG, ETF, and GOL accompanied a disorder to order transition in switch II to adopt an off state where Y32 is interacting with the ɣ-phosphate and Q61 interacts with the closed Y32 resulting in the catalytically inactive conformation seen for the RasQ61L mutant and for wild type bound to either DTT or DTE (Chapter 3).

Figure 6.1: MSCS results for H-Ras-GppNHp in the “off” state of the allosteric switch. The effector lobe is shown in green, and the allosteric lobe is shown in gray. MSCS clusters 1 through 8 are shown with red spheres superimposed on the organic solvent molecules, which are in stick representation within each cluster. (b) is rotated by 90° relative to (a) in order to show sites 3 and 7. (From Buhrman et al. 2011 [5]

Using the computational method, FT-Map, it was possible to probe conformations that weren’t available in the experiments performed. With MSCS it was only possible to probe the allosteric off state. With FT-Map the allosteric off state and on state were probed. In

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the allosteric on state two different conformation of switch I were tested. In state 1, Y32 is swung away from the nucleotide as in the Ras-RasGAP complex and the P3221 space group. In state 2 Y32 is positioned toward the nucleotide as in the Ras effector complexes and the observed in the Ras structure solved from crystals with symmetry of the R32 space group. The results were similar between the different structures tested. Cluster 1 shifted in K-Ras. This was due to the substitution of H95 for Q95 as in H and N Ras. Interestingly, FT-Map did not locate cluster 3, which was in the location of Raf-RBD binding. The solvents in this area were stabilized by water molecule interactions as well as interactions with the protein. FT- Map strips the waters so this maybe why this site wasn’t selected. The weaker MSCS clusters with only 2 solvents, 4, 5, 6, 7 and 8, were not seen in FT-Map. FT-Map, however, identified 4 cluster spots not seen in MSCS. The site of these clusters were between helices 4 and 5 an area in crystal contacts in the crystal form used for the analysis. This area is also thought to form interactions with the membrane and is near the allosteric site. Fewer sites were seen in the allosteric on conformations. Together the results show a more comprehensive view of the hot spots in Ras.

Relatively few structures of Rap can be found in the protein data bank (PDB). Rap2 has been solved bound to the ligands, GDP, GTP, and GTPɣS [13, 164]. Rap1 has been solved in complex with Raf-RBD [17], RapGAP [41], and with Epac (a Rap GEF) [165]. Recently, we solved the structure of uncomplexed Rap1 adding to the conformations seen in Rap1 [Chapter 5]. Four active site conformations were identified in Rap1. In two very similar conformations, the global conformation of the protein was nearly identical and overlayed with the allosteric on conformation of Ras. In the active site there were subtle differences. In one conformation Y32 was making a direct interaction with the nucleotide with a pre- bridging water molecule within 4Å of both Y32 and the ɣ-phosphate. In the second conformation Y32 moves away from the nucleotide and a bridging water molecule is observed. In both conformations switch II was ordered in a similar conformation with T61 pointed away from the active site. A different active site conformation was seen where T61

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replaced the bridging water molecule. Switch II also orders to a unique conformation associated with this structure. Finally, a third active site conformation was observed in the Rap1-GTP and a soaked structure. In this conformation T61 interacts with the nucleophilic water molecule. Globally the structures were similar outside of switch II. Differences were seen in loop 9 where D140 shifts closer toward the active site.

Rap1 soaks were performed at higher pH (pH 6.0 rather than 4.6) in Na Acetate and Ca Acetate to assess the allosteric site in Rap1. The Ca Acetate soak condition had solubility problems and Rap1 crystals did not perform well in this solution. In the Na Acetate structure a surprising conformational change occurred. The result was that a large area of the protein loop 8, loop9 and helix 4 had poor density and were shifted from the original structure solved at pH 4.6. Due to the fact that the crosslinking reaction required deprotonation of Lys side chains for the reaction with glutaraldehyde, the MSCS data collection was done on the structures at pH 6.0, the highest pH we could attain without destroying the crystals. The crosslinking reaction must also occurs in buffers that do not contain amines, and therefore crystals were transferred to a stabilization solution containing lithium sulfate rather than staying in the original crystallization mother liquor containing ammonium sulfate.

The MSCS technique provides a way to garner insight into Rap1 binding sites, for which little is known. Also, given the similarity between Rap1 and Ras, Rap1 provides a good comparison to Ras. In drug targeting it is important that the drug be specific. Understanding the binding site differences in Ras and Rap1 would provide information to better design a drug specific to Ras. Here we present the MSCS data sets collected for Rap1.

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Methods:

Crystal Optimization:

Rap1a 1-169 was expressed and purified as in Chapter 5. Initial Rap1 crystals were very thin plates which diffracted well but did not perform well after soaking in different conditions. To perform the MSCS technique on Rap1, crystals had to be improved. First, purity of the final protein was improved. Rap1 eluted from the final purification step, QHP, as two peaks, a GDP bound peak and a GTP bound peak. The GDP peak contained greater impurities than the GTP peak. For optimum crystallization, the GDP peak was discarded before exchanging Rap1 to GppNHp. Hanging drop crystal trays were used with a 2µL by 2µL drop. Other ratios were tried in an attempt to improve crystal quality, but changing the drop volume seemed to have no effect. Second, crystals were also improved by decreasing protein concentration to 3-5mg/mL. To further improve crystallization, screens were performed around Crystal Screen 20 (0.2M Ammonium Sulfate, 0.1M Sodium Acetate Trihydrate, and 25%w/v PEG4000) (Hampton Research). One screen used 300-350µL Crystal Screen 20 as the base solution. 60-100µL of 50%w/v PEG4000 was added and the solution brought up to a final volume of 500µL with H2O. The resulting screen varied (NH4)2SO4 concentration from 0.12-0.14M, NaAcetate concentration from 0.6-0.8M, and PEG4000 concentration from 21- 27.5%w/v. The second screen varied the concentrations of the individual components of

Crystal Screen 20 in a final volume of 500uL. In this screen (NH4)2SO4 concentration was 0.1M (14.25µL), NaAcetate concentration was varied from 0.5-0.9M (25-44µL), and PEG4000 concentration was varied from 14-27.5%w/v (140-220µL). We also discovered that Rap1 would crystallize in Li2SO4 rather than (NH4)2SO4, which aided in crosslinking the resulting crystals. Crystals grew in less than 12hrs and performed more poorly if left for more than 1-2 weeks in the crystallization condition. The result of these screens was fewer, thicker crystals that proved more robust for soaking in the organic solvent solutions.

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Crystal Stabilization and Solvent Soaking:

In the MSCS technique glutaraldehyde cross-linking is utilized to stabilize the crystal in the presence of high concentrations of organic solvents. Glutaraldehyde crosslinks free amines and it is therefore important to remove amines from the crystallization condition. The first step in the crosslinking procedure is to find a stabilization buffer that is ideal for crosslinking and in which crystals are stable for 1+hrs. (NH4)2SO4 was exchanged to Li2SO4 to eliminate free amines and the level of PEG4000 was increased to aid crystal stability. An initial stabilization condition at low pH (pH 4.6) was tested (0.2M Li2SO4, 0.1M Sodium Acetate Trihydrate, pH 4.6, 20% PEG 4000 and 20% PEG 6000). Crystals were directly transferred to the low pH stabilization condition and soaked for 1hr in 5-10µL volumes in sitting drop trays. These soaked crystals diffracted similarly well as unsoaked crystals. As glutaraldehyde crosslinks more efficiently at a higher pH the low pH stabilization buffer was set aside for a stabilization with a higher pH. A pH range of 5.0-7.0 was attempted using ~40% PEG 4000 (888µL of a 45%w/v PEG4000 solution) and ~0.1M buffer solution. The buffer solution varied with pH. In the end, the highest pH with stable crystals was found at pH6.0. The buffer solution was a mix of 0.1M Sodium acetate trihydrate and 0.4M MES pH 6.0.

With a stabilization buffer selected, crosslinking was performed. As stated, Rap1 crystals were very thin and diffusion of glutaraldehyde would presumable be fast, therefore, a lower level of glutaraldehyde was tried (0.4%). The final cross-linking was 950µL stabilization buffer and 50µL gluderaldehyde (8.26 % w/v in distilled water, pH 4.25, Electron Microscopy Sciences). Various crosslinking times were tested from 15min to 1 hour. Diffraction was decreased after 45min of cross-linking so the crosslinking time selected was 30min. The crosslinking reaction was stopped by the addition of TRIS-glycine buffer. After crosslinking, crystals were transferred to different organic solvents at different concentrations for 1 hr (see data refinement tab le for organic solvents and corresponding concentrations).

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Organic solvents were diluted with crystal stabilization buffer when necessary. Synchrotron data were collected on the ID-22 line at SER-CAT at APS (Argonne, Il). The detector distance was 120mm. Data refinement statistics are shown in Table 6.1.

Results and Discussion:

Data were collected for 18 data sets including the crosslinked structure (x-link) and 12 unique organic solvents to around 2.0Å (Table 6.1). The organic solvents included 75% hexanediol (HEZ), 50% R,S,R-bisfuranol (RSF), 70% isopropanol (IPR), 2M TMAO, 1M acetate (ACE), 50% S,R,S-bisfuranol, 75% acetonitrile (ACN), 50% DMSO, 70% acetone (ACT), neat hexane (HEX), 50% t-butanol (TBUT) and 70% glycerol (GOL). Organic solvents, in part, were chosen for comparison to Ras [5]. Several solvents overlap the two MSCS data sets including: GOL, IPR, RSF, RSG, HEZ, and HEX. Both dimethylformamide (DMF) and cyclopentanol (YEG) were attempted for Rap1 but did not diffract well enough for use in MSCS analysis. The organic solvent 2,2,2-trifluoroethanol (ETF) was also attempted but the crystals did not survive soaking.

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Table 6.1: Data collection and refinement statistics are presented for the Rap1A MSCS collected. Synchrotron data were collected at 100 K. Data were processed using Denzo HKL or HKL2000 [3] and refined with PHENIX [8] and Coot [10].

Structure ID 580 582 604 605 607 609 Experimental Condition Condition Hexanediol RSR Furanol Isopropanol TMAO Acetate X-Link Concentration 60% 50% 55% 1M 1M 30min

Data collection

Space group P21212 P21212 P21212 P21212 P21212 P21212 Beamline 22-ID 22-ID 22-ID 22-ID 22-ID 22-ID Collection 100K 100K 100K 100K 100K 100K CellTemperature dimensions 112.401 112.183 112.684 112.789 112.763 112.127 a , b , c (Å) 41.934 42.087 42.027 42.470 42.257 41.769 66.359 66.536 66.661 67.164 66.681 66.171 α, β, ɣ (°) 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 Resolution (Å) 35.00-1.96 35.00-2.14 35.00-1.85 35.00-2.02 35.00-1.90 35.00-1.96 (2.03-1.96) (2.22-2.14) (1.92-1.85) (2.09-2.02) (1.97-1.90) (2.03-1.96)

R sym or R merge 0.101 0.131 0.098 0.08 0.095 0.074 (0.660) (0.497) (0.731) (0.513) (0.748) (0.465) I / σ 16.8 15.6 33.3 40.4 25.9 20.7 (2.25) (0.18) (1.95) (3.45) (1.58) (2.16) Completeness (%) 84.5 97.4 98.5 97.9 98.8 88.8 (83.5) (82.0) (94.7) (94.0) (91.2) (74.9) Redundancy 4.6 5.1 5.6 5.2 6.3 2.7 (3.4) (2.4) (4.0) (3.7) (4.2) (2.3)

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Table 6.1 Continued

Structure ID 610 614 621 626 629 648 Experimental Condition Condition X-Link Isopropanol SRS Furanol Acetonitrile DMSO Acetone Concentration 30min 70% 50% 60% 50% 70%

Data collection

Space group P21212 P21212 P21212 P21212 P21212 P21212 Beamline 22-ID 22-ID 22-ID 22-ID 22-ID 22-ID Collection 100K 100K 100K 100K 100K 100K CellTemperature dimensions 112.597 113.594 112.722 112.169 112.188 111.944 a , b , c (Å) 41.681 41.704 42.432 41.921 41.919 41.532 66.168 66.075 66.760 66.459 66.682 66.267 α, β, ɣ (°) 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 Resolution (Å) 35.00-2.05 50.00-1.90 50.00-2.02 35.00-2.04 35.00-2.09 35.00-2.02 (2.12-2.05) (1.97-1.90) (2.09-2.02) (2.11-2.04) (2.16-2.09) (2.09-2.02)

R sym or R merge 0.099 0.112 0.103 0.095 0.106 0.119 (0.546) (0.738) (0.000) (0.000) (0.536) (0.000) I / σ 19.4 31.5 25.5 18.2 21.7 27.2 (1.95) (3.95) (2.31) (2.36) (1.72) (1.40) Completeness (%) 97.2 99.8 99.4 87.1 88.3 99.7 (84.2) (99.9) (99.7) (92.2) (50.9) (99.7) Redundancy 5 7 6 5.3 5.4 6.1 (2.6) (6.6) (5.6) (4.3) (3.4) (5.2)

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Table 6.1 Continued

Structure ID 649 650 664 665 666 Experimental Condition Condition Hexandiol Hexane T-butanol TMAO Acetonitrile Glycerol Concentration 75% neat 50% 2M 75% 70%

Data collection

Space group P21212 P21212 P21212 P21212 P21212 P21212 Beamline 22-ID 22-ID 22-ID 22-ID 22-ID 22-ID Collection 100K 100K 100K 100K 100K 100K CellTemperature dimensions 112.097 112.907 111.608 112.161 111.369 113.377 a , b , c (Å) 41.881 42.381 42.239 42.472 42.015 42.973 66.206 67.033 66.506 66.393 66.439 67.356 α, β, ɣ (°) 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 90,90,90 Resolution (Å) 35.00-2.02 35.00-1.97 35.00-2.04 35.00-2.02 35.00-1.90 35.00-2.04 (2.09-2.02) (2.04-1.97) (2.11-2.04) (2.09-2.02) (1.97-1.90) (2.11-2.04)

R sym or R merge 0.093 0.079 0.071 0.086 0.091 0.076 (0.586) (0.000) (0.774) (0.770) (0.827) (0.000) I / σ 20.1 34.8 25.5 25.0 38.6 37.6 (2.09) (1.77) (2.77) (1.70) (2.75) (2.00) Completeness (%) 91.3 97.8 87.3 90 98.9 99.8 (90.2) (100.0) (81.6) (84.9) (99.2) (99.9) Redundancy 5.1 5.7 4.7 4.7 5.2 6.3 (4.0) (4.7) (3.7) (2.9) (4.7) (6.4)

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The data collected for the MSCS analysis of Rap1 has been collected and processed. Molecular replacement has been performed on all data collected thus far. The x-link (1.96Å), RSR, glycerol, and 1M acetate structure have all been solved to a reasonable R- value. Molecule A in the MSCS set is consistent with the conformation previously seen (Chapter 5). Molecule B, on the other hand, has structural differences from the previously solved structures. Rap1 soaked in NaAcetate (Rap1-NaAcetate) at pH 6.0 showed a disordering of helix 4. This soak was performed under conditions that were similar to crosslinking conditions for the MSCS set. Both contained 50mM NaAcetate trihydrate and 50mM

MES pH 6.0. Both contained 30% PEG 4000. The NaAcetate soak contained LiSO4 and MgCl2 while these were not part of the organic solvent solutions. In Rap1 a LiSO4 binding site was observed around residues 85-89 which was shown to be important for cellular localization in Rap1. Absence of LiSO4 in the x-link structure does not alter the conformation of molecule A.

In the x-link structure large areas of the protein are disordered. Switch II residues 62-66 are disordered with weak density for residues 67-69. Switch II is commonly disordered in GTPases and this is seen in the Rap1-GTP and Rap1-NaAcetate structures (Chapter 4). Loop 8 is mostly disordered including residues 118-121 and 126-128. Residues 122-125 have some density to support their positions. Loop regions are prone to disorder so this may not seem surprising, however, residues 116-119 comprise part of the nucleotide binding pocket. These residues being disordered may explain the weaker than normal density for the bound GppNHp and high associated B-factors. One possibility is that this had to do with the removal of MgCl2 from the soak condition. Magnesium binding helps stabilize the bound GTP in the active site. It would be surprising that this would disorder residues associated with the ribose ring (118-121) rather than residues near the ion binding site. Further disordering is observed in helix 3 residues 96-105 and these residues were removed from the model. Helix 3 in Ras was shown to adopt different conformations related to small molecule binding an allosteric site. In previous Rap1 structures helix 3 only adopted the on

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conformation seen in Ras (Chapter 4). The disordering of helix 3 in the x-link structure indicates that Rap1 may have more flexibility in helix 3 than was previously seen. Helix 4, like in the NaAcetate structure, has density but the density is messy. Comparison of the RSR and x-link structure reveals two helix 4 conformations. The density seen may be a result of alternate conformation of helix 4 but at the resolutions obtained this cannot be said for certain. Helix 4 has been reported to interact with the membrane in Ras where it is stabilized by interactions with the membrane. The flexibility of helix 4 in the soaked Rap1 structure may indicate that helix 4 in Rap1 may also have conformational flexibility that can be stabilized by molecular interactions. The above results show that the P21212 crystal form of Rap1 is suitable for MSCS analysis. Rap1 may benefit from FT-Map analysis as a compliment to the MSCS data as in Ras. This would allow better probing of the conformation adapted in molecule B which differs from molecule A (Chapter 5).

Rap1 crystallizes in the P21212 space group with two molecules in the asymmetric unit. An important consideration in an MSCS project is whether the crystal packing blocks sites of interest. For example, in Ras a few sites determined by FT-Map were not able to be probed due to crystal contacts. An examination of the Rap1 structure reveals that most sites that were accessible in Ras are also accessible in Rap1. In molecule A of Rap1A switch II is in a different conformation than previously seen, this conformation blocks the cluster 1 binding site. The clusters 3 and 4 binding site are also blocked by crystal contacts with molecule B. The cluster 1 site is unhindered in molecule B, however, the residues forming interactions with the solvents in this cluster in Ras (R68, Q95, Y96, Q99, and D92) are part of a region that is partially disordered in Rap1 molecule B. It has been observed that organic solvents can order the disordered switch regions in Ras [85] so it may be possible that certain organic solvents would order areas destabilized in Rap1 but in the structures solved thus far this is not observed. Cluster 3, like in molecule A, is inhibited by crystal contacts and thus it would be impossible to probe this site in Rap1 in this crystal form. Cluster 4 is not hindered in molecule B. All other cluster sites determined in Ras are free of crystal contacts and

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could be accessed by solvent molecules. Cluster sites in FT-Map that were inaccessible in Ras may be accessible in Rap1.

The structures in the Rap1A MSCS set are at various stages of refinement with only a couple fully refined with added water molecules and organic solvents. To date, only the acetate and glycerol structures have bound solvent molecules. There were no solvents placed in the RSR structure, although this may reflect an early stage during the refinement process. Even so, it would not be surprising if there turned out to be fewer RSR molecules bound to Rap than the 11 placed in the Ras structure, since the Rap1 structure was soaked with 50% RSR compared to 90% in Ras. In the acetate soaked structure, there were 3 acetate molecules placed, however, none overlayed cluster sites in Ras or binding site in Rap1. In the glycerol structure three glycerol molecules were placed: GOL-H, GOL-I and GOL-J (Figure 6.2). Two bound GOL molecules were in crystal contacts and didn’t overlay cluster sites in Ras (GOL-H and GOL-J) (Figure 6.3). GOL-I, on the other hand, was in a similar position to cluster 4 making interactions with K147 (Figure 6.4). This site in Ras was associated with a recently discovered ubiquitination site [167]. Rap2 and Rap1 have also been shown to be regulated by ubiquitination [168, 169]. The preliminary results showing similar binding in Rap1 and Ras support the idea that the MSCS of Rap1 will be useful in determining Ras specificity.

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Figure 6.2: Electron density for GOL molecules placed in the Rap1 soaked structure. From left to right GOL-H, GOL-I and GOL-J are shown. Electron density was contoured at 1.0σ.

Figure 6.3: The binding sites seen in the Rap1-GOL structure (greencyan) are compared to the data obtained in the Ras MSCS set (light grey). Data sets include neat YEG (PDB code: 3rs0), 50% IPR (3rs7), neat HEX (3rs3), 70% GOL (3rrz) 55% DMF (3rs4), 60% HEZ (3rry), 50% ETF (3rs2), 20% RSG (3rso), and 90% RSF (3rsl). Bound GOL molecules from Rap1 are shown in hot pink while organic solvents from the Ras data set are shown in black.

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Figure 6.4: GOL-I (hot pink) from Rap1 GOL soaked structure (greencyan) is close to the cluster 4 site in Ras (comprised of RSG and RSF (black)). GOL-I is also near a GOL form the Ras set that wasn’t included as part of a cluster. Ras soaked in GOL is shown in light grey.

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