ABSTRACT

GAP ENGINEERING TO RESTORE GTP HYDROLYSIS TO ONCOGENIC KRAS MUTANTS

by Benjamin A. Fenton

The Kras gene encodes a small GTPase that is involved in pathways regulating cell growth and proliferation. Bound to GTP, Kras sends growth signals until the GTP is hydrolyzed. Hydrolysis is catalyzed by GTPase Activating Protein (GAP). Activating mutations in Kras are highly correlated to cancer development due to uncontrolled growth. Any mutation in the 12th amino acid of Kras (glycine) cause steric alterations in the catalytic site of GAP that prevent catalysis of GTP hydrolysis. Kras is a widely-studied signaling protein, and currently there are no clinical inhibitors. This project will attempt to redesign the catalytic site of GAP to circumvent the steric interference caused by G12 mutations, thus allowing hydrolysis to occur and signaling to be turned off. Recombinant proteins were purified from E. coli, and GAP mutants were screened for catalytic activity using a colorimetric GTPase assay.

GAP ENGINEERING TO RESTORE GTP HYDROLYSIS TO ONCOGENIC KRAS MUTANTS

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

Department of Chemistry and Biochemistry

by

Benjamin A. Fenton

Miami University

Oxford, Ohio

2014

Advisor______Dr. Michael A. Kennedy

Reader______Dr. Ann E. Hagerman

Reader______Dr. David L. Tierney

Reader______Dr. Michael W. Crowder Table of Contents Title Page i List of Tables v List of Figures vi Acknowledgements vii

Chapter 1: Introduction

1.1 The Kirsten Rat Sarcoma Viral Homolog 2 1.2 Mechanism of GTP Hydrolysis 5 1.3 Oncogenic Mutations in Kras 8 1.4 Project Goals 8 1.5 Chapter 1 References 10

Chapter 2: Gene Cloning and Site-Directed Mutagenesis of Kras and rasGAP

2.1 Introduction 14 2.2 Methods 15 2.2.1 Primer Design 15 2.2.2 Gene Amplification 16 2.2.3 Endonuclease Digestion 19 2.2.4 Ligation 20 2.2.5 Transformation and Plasmid Isolation 20 2.2.6 Colony Screening and Sequencing 21 2.3 Results and Discussion 22 2.3.1 Gene Amplification and Mutagenesis 22 2.3.2 Endonuclease Digestion of Insert and Vector 22 2.3.3 Colony Screening and Sequencing 22 2.4 Conclusions 23 2.5 Chapter 2 References 29

ii

Chapter 3: Expression, Purification, and Characterization of Kras and rasGAP 3.1 Introduction 32 3.2 Methods 34 3.2.1 Transformation, Expression, and Cell Lysis 34 3.2.2 Nickel Affinity Purification 35 3.2.3 Kras Expression in Minimal Media 35 3.2.4 Crystal Screening 36 3.2.5 Kras Nucleotide Binding State Determination 36 3.3 Results and Discussion 37 3.3.1 Protein Expression and Purification 37 3.3.2 Thrombin Cleavage 37 3.3.3 Binding State Determination by 1H-15N HSQC NMR 43 3.4 Conclusions 43 3.5 Chapter 3 References 48 Chapter 4: Measuring GAP-Mediated Catalysis of GTP Hydrolysis by Kras 4.1 Introduction 50 4.2 Methods 52 4.2.1 Kras GTP Loading and Desalting 52 4.2.2 Optimization of a Colorimetric GTPase Assay 54 4.3 Results 55 4.3.1 Kras GTP Loading and Desalting 55 4.3.2 Establishment of a Standard Curve 59 4.3.3 GTPase Activity of WT and G12A Kras with WT GAP 59 4.3.4 GAP Mutant Screening for Restoration of Rapid GTP Hydrolysis 63 4.4 Conclusions 68 4.5 References 69 Chapter 5: Conclusions and Future Directions

iii 5.1 General Remarks 72 5.2 Conclusions 72 5.2.1 Cloning and Mutagenesis of Kras and GAP 72 5.2.2 Expression and Purification of Kras and GAP 72 5.2.3 Optimizing a Phosphate Assay to Monitor GTP Hydrolysis 73 5.4.4 Screening Engineered GAP Mutants for Restoration of GTPase Catalysis 73 5.3 Future Directions 73 5.4 Closing Remarks 75 5.5 References 75

iv

List of Tables

Table 2-1: Successfully cloned genes and mutants 28 Table 3-1: Successfully expressed proteins 44

v

List of Figures Figure 1-1: Diagram of Kras signaling 3 Figure 1-2: Cycle of activation and deactivation of Kras 4 Figure 1-3: Crystal structure of Kras 6 Figure 1-4: Diagram of the active site of Kras in complex with GAP 7 Figure 2-1: Primers used for cloning and mutagenesis 17 Figure 2-2: Amplification of target genes 24 Figure 2-3: Amplification of genes for overlapping PCR mutagenesis 25 Figure 2-4: Digestion of pET28 vector plasmid 26 Figure 2-5: Restriction enzyme screening for successful ligation and transformation 26 Figure 2-6: Amino acid sequences of Kras and GAP gene products 27 Figure 3-1: Wild-type Kras purified from E. coli forms a dimer 38 Figure 3-2: Elution profile of wild-type Kras 39 Figure 3-3: Wild-type GAP purification 40 Figure 3-4: Purification of Kras mutants 41 Figure 3-5: Purification of GAP mutants 42 Figure 3-6: 1H-15N HSQC spectrum of wild-type Kras 45 Figure 3-7: 1H-15N HSQC spectrum showing single conformational state 46 Figure 3-8: Rapid conversion to GDP state of Kras upon addition of GAP 47 Figure 4-1: Mechanism of the GTPase colorimetric assay 53 Figure 4-2: Elution traces from PD-10 Desalting Columns 56 Figure 4-3: 1H-15N HSQC confirmation of GTP loading 57 Figure 4-4: 1H-15N HSQC representation of reaction time course 58 Figure 4-5: Standard curve for the PiColorLock Gold phosphate assay 60 Figure 4-6: GTPase activity of wild-type Kras with and without GAP 61 Figure 4-7: GTPase activity of G12A mutant Kras with and without GAP 62 Figure 4-8: GTP hydrolysis of Kras after two minutes 64 Figure 4-9: Activity of GAP mutants on wild-type Kras 66 Figure 4-10: Activity of GAP mutants on G12A Kras 67

vi Acknowledgements

I would like to thank my advisor Dr. Kennedy for his assistance and guidance throughout my career at Miami, starting from my first month here as a freshman. My time in his lab has been filled with incredible opportunities that I doubt I would have found elsewhere. I would also like to thank my graduate committee for their valuable insights and ideas, and assistance with my thesis. Next, I want to thank Dr. Ni, my mentor in the lab who taught me all of the procedures and instrumentation used in the lab, and who was always available to help troubleshoot and talk things through. I want to thank Dr. Ramelot as well, for her expertise and training on all of my NMR experiments. I also want to thank the graduate members of the Kennedy research group, for giving me a supportive and enjoyable lab environment. The undergraduate team that I started out a part of and ended up leading has been instrumental in this project. I want to thank the current team, Jackie Ehrman, Amielia Adams, Aaron Keck, Peter McCandless, Katy McGill, Colleen O’Neil, Nathan Burns, and Brett VanCauwenbergh, as well as past members of the team for their time and effort. I would like to thank my friends and family for their support, especially my parents, who have helped make all of this possible. Finally, I want to dedicate this thesis in memory of my grandmother, Marilyn Fenton, who we lost to cancer during my sophomore year of college.

vii

Chapter 1:

Introduction

1 1.1 The Kirsten Rat Sarcoma Viral Oncogene Homolog

The Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) protein is member of the of small . Characterized by a catalytic G domain, the Ras superfamily includes Ras, Rap, Rho, , and many others. Kras, and more specifically the Kras4B variant that is highly expressed in human cells, is one of the most well-studied members of the family. Kras plays a role in and acts as an on/off switch depending on its nucleotide binding state. When bound to GTP, Kras signaling is switched on, and Kras can interact with downstream effectors such as PI3K, MAPK, and Raf, ultimately resulting in the production of transcription factors that lead to cell growth and proliferation (1,2) (Figure 1-2). Kras has a degree of intrinsic GTPase activity and slowly hydrolyzes the bound GTP into GDP. When the GTP is hydrolyzed and the resulting phosphate is released, Kras undergoes a conformational change that stops interaction with effector proteins, turning off signal transduction. Eventually, GDP is released from Kras, and GTP, which is more abundant in the cell, will bind, resetting the cycle (3). The Kras protein is farnesylated at the C terminus and localized to the cytosolic face of the plasma membrane, where it regulates many signal transduction pathways (4).

The nucleotide-mediated signal transduction switch is further mediated by GTPase Activating Proteins (GAPs) and Guanine Nucleotide Exchange Factors (GEFs). GAPs catalyze the hydrolysis of GTP, increasing the reaction rate by six orders of magnitude. The binding of GAP rapidly switches Kras signaling off by speeding up GTP hydrolysis (5-7). Conversely, the GEFs, such as (SOS), catalyze dissociation of GDP from Kras, which causes signaling to be switched on when a new molecule of GTP binds (8). Ras activation by GEF occurs through a transduction pathway associated with the epidermal receptor (EGFR) (3). Extracellular signals bind to receptors on the EGFR, which transmits the signal across the membrane and activates GEF proteins, that then activate Kras. As mentioned above, active Kras triggers the activation of serine/threonine kinase cascades, which result in cell growth. This signal continues until the GTP is hydrolyzed, either intrinsically or when catalyzed by GAP (Figure 1-2).

2

Figure 1-1: Simplified diagram of Kras signaling including the Epidermal Growth Factor Receptor (EGFR), Son of Sevenless (SOS), which is a Guanine Nucleotide Exchange Factor, and Raf, an effector for the mitogen-activated (MAPK) pathway. Raf is a MAPKKK, MEK is a MAPKK, and ERK is a MAPK.

3

Figure 1-2: Cycle of activation and deactivation of Kras. GTP= ; GDP=guanosine diphosphate; GAP= GTPase activating protein; GEF= guanine nucleotide exchange factor.

4 Many aspects of the Kras signaling process have been well studied. Several laboratories have published crystal structures of Kras and various G12 mutants, in addition to Kras in complex with GAP (9,10). The Ras family proteins contain a conserved G domain of 166 amino acids and a highly variable C-terminal domain, which anchors the protein to various membranes, depending on functionalization (1). Kras is farnesylated, while other members of the family, such as Nras or Hras, are palmitoylated (4). Kras has five important binding motifs, which bind to GTP, conferring specificity for GTP over ATP and creating the binding surface for cofactors and effector proteins. Two of these binding motifs, called Switch I and Switch II, are especially important for regulation and downstream signaling because dramatic conformational changes occur when GTP is hydrolyzed to GDP (11) (Figure 1-3). When Kras is bound to GTP, Switch I adopts a conformation that has a high affinity for GAPs and effectors such as Raf. When GTP is hydrolyzed, both switches shift, causing Switch I to lose its binding capabilities and Switch II to gain affinity for GEFs (12). The structure of rasGAP has also been well studied. This 120 kDa protein contains a 334-amino acid catalytic subunit, which is purely alpha-helical. Studies have demonstrated the mechanism of GTPase catalysis by GAP through co-crystallization of Kras and GAP, with non-hydroyzable analogs of GTP (6). Additionally, crystal structures of the complex - bound to GDP with AlF4 taking the place of the γ-phosphate have aided mechanistic studies (11).

1.2 Mechanism of GTP Hydrolysis

GTP bound to Kras is stabilized by a Mg2+ ion that resides inside the nucleotide binding pocket (13,14). The magnesium ion, aligned by the residues threonine 35 and serine 17, interacts with the phosphate groups of GTP. Interactions from the magnesium ion cause the β- phosphate to be twisted into an unfavorable “eclipsed” state, in which the oxygen atoms of the two phosphate groups are aligned with each other. This twist stretches the bonds between the phosphates and makes the γ-phosphate slightly more vulnerable to nucleophilic attack by water, which is aligned by the glutamine 61 residue in the active site (15). The contributions of Kras and magnesium are enough to catalyze hydrolysis of GTP, but the process is greatly accelerated by GAP (16). When GAP binds to GTP-bound Kras, an arginine (residue 789) is inserted into the nucleotide-binding pocket (Figure 1-4). Hydrogen bonds form between the guanidinium

5 protons of arginine and the oxygens of the α- and γ-phosphates. The position of the arginine causes the γ-phosphate to be twisted into the same aligned conformation as the α- and β- phosphates. This motion further lengthens the bond between the β and γ phosphate, making it more susceptible to nucleophilic attack by water. The positively-charged arginine side chain also stabilizes the partial negative charge that occurs in the transition state of the phosphoryl transfer reaction (16). By weakening the phosphate bond and stabilizing the transition state, GAP lowers the activation energy for the hydrolysis reaction, increasing the rate by 6 orders of magnitude (17).

Figure 1-3: Crystal Structures of Kras bound to GTPγN (shown as red sticks). On the left, a ribbon diagram, and on the right, a rotated view of the structure as a surface model. Mg2+ shown in magenta, the P-loop (phosphate binding) shown in green, Switch I shown in orange, Switch II shown in blue, and the G12 residue shown as sticks in yellow. C-terminal membrane-associating domain not shown. Pymol rendering of structure deposited in the PDB, code 3GFT (Tong, et al. 2009).

6

Figure 1-4: Pymol rendering of GDP-bound Ras in complex with rasGAP. Ras shown in light green, and GAP shown in light red, with residues and atoms of interest colored differently: Orange: Ras Q61. Blue: GDP. Pink: Mg2+. Red: GAP R789. Yellow: Ras G12. R789 of GAP is inserted into the nucleotide binding pocket of Ras, where it interacts with the phosphates of GTP/GDP, lowering the activation energy of hydrolysis and speeding up the reaction. The glycine 12 of Ras is within van der Waals distance of R789. Amino acid substitutions for this glycine would occupy more space in the pocket and cause steric clashes with R789. PDB code 1WQ1 (9).

7 1.3 Oncogenic Mutations in Kras

Many point mutations in Kras have been detected in human cancers. These mutations result in over-activated Kras, causing increased time spent in the “on” signaling state (18). The most common oncogenic mutations occur in the GTP-binding pocket and interfere with hydrolysis. A point mutation of the 12th amino acid, glycine, to nearly any other amino acid, is one of the most common oncogenic mutations across all human cancers. This mutation requires a single nucleotide substitution and results in impaired ability to hydrolyze GTP due to steric interference (19). The added steric bulk of a sidechain other than a single hydrogen (glycine) obstructs the space normally occupied by the arginine from GAP. While hydrolysis unaided by GAP can continue normally, the G12 mutation blocks the contributions from GAP needed to speed up GTP hydrolysis. This interference causes Kras to be locked into the GTP bound state significantly longer than normal, resulting in overactive signaling and ultimately uncontrolled cell growth and proliferation (3).

Point mutations in Kras are highly implicated in many types of tumor-forming cancers. A study by the Cancer Genome Project found point mutations in Kras in 80% of cases studied. Of this 80%, 90% of the mutations were at the G12 position. Additionally, Kras mutations were detected in 60% of colon cancer patients and 30% of patients (20). The study also found an association between Kras mutations and a history of smoking and tobacco use. Cancers involving Kras mutants are associated with poor prognosis due to added difficulty in treatment and lack of a way to specifically target the mutationally-activated Kras. While a mutation of G12 to any amino acid but proline can cause activation leading to cancer, the specific amino acid may have an effect on the degree of activation (2). The COSMIC database (Catalogue Of Somatic Mutations In Cancer) lists the distribution of specific G12 mutations in found in cancer patients. The database lists 25,000 cancer cases, with aspartate, valine, alanine, and cysteine being the most prevalent substitutions for G12 (21).

1.4 Project Goals

Cancers exhibiting Kras mutations are notoriously difficult to target and treat. Many different approaches have been taken, from using monoclonal antibodies to target Kras to interfering with Kras-GEF binding interactions. The problem with current methods of treatment

8 is that they do not specifically target mutationally-activated Kras (18,22). For example, drugs made to stop nucleotide exchange from GDP to GTP would stop growth signals in both healthy and mutant Kras. This non-specific effect could result in the death of healthy cells, which causes problems when the drug gets to clinical trials (8, 23). The goal of this project is to study a novel method of inactivating the Kras signaling pathway. The project will attempt to redesign the GAP protein in a way that restores catalysis of GTP hydrolysis. Redesigning GAP will be accomplished by making point mutants to the active site of GAP. The catalytic arginine will be moved to different positions with the hope that, by inserting the arginine into the active site from a different angle, the steric interference caused by the G12 mutation can be avoided. If a GAP mutant can be made that is able to catalyze hydrolysis of Kras-bound GTP, that protein would have potential as an anti-tumor biologic (24). The advantage of this novel approach to down- regulating Kras signaling is that the mutant GAP would not abnormally affect wild-type Kras. The theoretical redesigned GAP protein would be able to switch “off” the signaling of mutant Kras, allowing the signaling cycle to return to that of a healthy cell.

The oncogenic activation of Kras is due to the steric bulk of the substituted amino acid at Kras G12 interfering with the ability of Arg 789 in GAP to stabilize the transition state. GAP mutations were chosen using rational design methods in an attempt to reposition the arginine. Two sets of engineered mutants were created. The first set incorporated an additional arginine into the binding pocket-associating loop of GAP. Residues in the loop upstream of R789 were mutated to arginine one at a time. For the second set of engineered mutants, R789 was removed and replaced in positions 785, 786, 787, or 788. The goal of the redesign was to have the arginine residue insert into the nucleotide binding pocket from an angle opposite that of the Kras G12 mutation. The work was based on the hypothesis that avoiding interference from the G12 mutation would allow GAP to catalyze hydrolysis of GTP. Support for this hypothesis can be found by examining the structure of the Rap/rapGAP active site. Rap is a GTPase in the Ras family with high homology to Kras. The catalytic residue of rapGAP is inserted into the nucleotide pocket from the side, at an opposing angle to the glycine 12 position. G12 mutations in Rap do not interfere with GTP hydrolysis ability, which supports the design strategy to be used for Kras (25).

9 1.5 References

1. Fernández-Medarde, A., & Santos, E. (2011). Ras in cancer and developmental diseases. Genes & Cancer, 2(3), 344–58. doi:10.1177/1947601911411084 2. Jancík, S., Drábek, J., Radzioch, D., & Hajdúch, M. (2010). Clinical relevance of KRAS in human cancers. Journal of Biomedicine & Biotechnology, 2010, 150960. doi:10.1155/2010/150960 3. Malumbres, M., & Barbacid, M. (2003). RAS : the first 30 years. Nature Reviews. Cancer, 3(6), 459–65. doi:10.1038/nrc1097 4. Jackson, J. H., Cochrane, C. G., Bourne, J. R., Solski, P. A., Buss, J. E., & Der, C. J. (1990). Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proceedings of the National Academy of Sciences of the United States of America, 87(8), 3042–6. 5. Cherfils, J., & Zeghouf, M. (2013). Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiological Reviews, 93(1), 269–309. doi:10.1152/physrev.00003.2012 6. Hettich, L., & Marshall, M. (1994). Structural analysis of the Ras GTPase activating protein catalytic domain by semirandom mutagenesis: implications for a mechanism of interaction with Ras-GTP. Cancer Research, 54(20), 5438–44. 7. Scheffzek, K., Lautwein, A., Kabsch, W., Ahmadian, M. R., & Wittinghofer, A. (1996). Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras. Nature, 384(6609), 591–6. doi:10.1038/384591a0 8. Sun, Q., Burke, J. P., Phan, J., Burns, M. C., Olejniczak, E. T., Waterson, A. G., … Fesik, S. W. (2012). Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angewandte Chemie (International Ed. in English), 51(25), 6140–3. doi:10.1002/anie.201201358 9. Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmüller, L., Lautwein, A., Schmitz, F., & Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science (New York, N.Y.), 277(5324), 333–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9219684

10 10. Scheffzek, K., Lautwein, A., Scherer, A., Franken, S., & Wittinghofer, A. (1997). Crystallization and Preliminary X-Ray Crystallographic Study of the Ras-GTPase- Activating Domain of Human p120GAP, 318(September 1996), 315–318. 11. Maurer, T., Garrenton, L. S., Oh, A., Pitts, K., Anderson, D. J., Skelton, N. J., … Fang, G. (2012). Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS- mediated nucleotide exchange activity. Proceedings of the National Academy of Sciences of the United States of America, 109(14), 5299–304. doi:10.1073/ pnas.1116510109 12. Hall, B. E., Bar-Sagi, D., & Nassar, N. (2002). The structural basis for the transition from Ras-GTP to Ras-GDP. Proceedings of the National Academy of Sciences of the United States of America, 99(19), 12138–42. doi:10.1073/pnas.192453199 13. Du, X., Frei, H., & Kim, S. (2000). The mechanism of GTP hydrolysis by Ras probed by fourier transform infrared spectroscopy * GTP or GDP bound to Ras mutants . IR spectra revealed, 275(12), 8492–8500. 14. Mazhab-Jafari, M. T., Marshall, C. B., Smith, M., Gasmi-Seabrook, G. M. C., Stambolic, V., Rottapel, R., … Ikura, M. (2009). Real-time NMR study of three small GTPases reveals that fluorescent 2’(3')-O-(N-Methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics. Journal of Biological Chemistry, 285(8), 5132–5136. doi:10.1074/jbc.C109.064766 15. Rudack, T., Xia, F., Schlitter, J., Kötting, C., & Gerwert, K. (2012). Ras and GTPase- activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations. Proceedings of the National Academy of Sciences of the United States of America, 109(38), 15295–300. doi:10.1073/pnas.1204333109 16. Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmüller, L., Lautwein, A., Schmitz, F., & Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science (New York, N.Y.), 277(5324), 333–8. 17. Res, A. H. C., Biomol, A. J. J., Natl, D. M. P., Sci, A., John, J., Sohmen, R., … Goody, R. S. (1990). Kinetics of interaction of nucleotides with nucleotide-free H-ras. Cancer Research, (1979), 6058–6065. 18. Adjei, A. A. (2001). Blocking oncogenic Ras signaling for cancer therapy. Journal of the National Cancer Institute, 93(14), 1062–74.

11 19. Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., & Shokat, K. M. (2013). K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature, 503(7477), 548–51. doi:10.1038/nature12796 20. Moore, P. S., Sipos, B., Orlandini, S., Sorio, C., Real, F. X., Lemoine, N. R., … Smad, D. P. C. (2001). Genetic profile of 22 pancreatic carcinoma cell lines. Virchows Arch, 798– 802. doi:10.1007/s004280100474 21. Forbes, S. a, Bindal, N., Bamford, S., Cole, C., Kok, C. Y., Beare, D., … Futreal, P. A. (2011). COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Research, 39(Database issue), D945–50. doi:10.1093/nar/gkq929 22. Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., … Waldmann, H. (2013). inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling. Nature, 497(7451), 638–42. doi:10.1038/nature 12205 23. Fleming, J. B., Shen, G.-L., Holloway, S. E., Davis, M., & Brekken, R. a. (2005). Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy. Molecular Cancer Research : MCR, 3(7), 413–23. doi:10.1158/1541-7786.MCR-04-0206 24. Carter, P. J. (2011). Introduction to current and future protein therapeutics: a protein engineering perspective. Experimental Cell Research, 317(9), 1261–9. doi:10.1016 /j.yexcr.2011.02.013 25. Scrima, A., Thomas, C., Deaconescu, D., & Wittinghofer, A. (2008). The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues. The EMBO Journal, 27(7), 1145–53. doi:10.1038/emboj.2008.30

12

Chapter 2:

Gene Cloning and Site-Directed Mutagenesis of Kras and rasGAP

13 2.1 Introduction

This chapter outlines the experiments performed to successfully clone the Kras and rasGAP genes into a vector for over-expression. The genes were amplified and cloned from human cDNA, and once the clones were confirmed by sequencing, the recombinant proteins were overexpressed. This chapter also describes methods used to make site-directed mutations in both the Kras and rasGAP genes.

The Kras protein has two domains, a G domain of 166 amino acids, and a C terminal extension for localization to the plasma membrane (1). The full length Kras gene (564 nucleotides, coding for 188 amino acids) was used initially for this study, but the resulting over- expressed protein was insoluble. To make the protein more stable in solution for subsequent study, the cloning primers were redesigned to amplify only the first 507 nucleotides (169 amino acids) of the gene. Studies have shown that removal of the C-terminal domain has no effect on GTPase activity or effector-protein binding (2), and also makes the protein more amenable to structural studies. Additionally, during the course of the project, it was reported in the literature that mutating the cysteine at position 118 to serine dramatically increased solubility (3). This mutation was incorporated into all subsequent clones. Similarly, the full-length rasGAP protein (>1000 amino acids) was not used. Studies have demonstrated that the GAP domain, a 334- amino acid section of the protein, is the minimal unit necessary for catalysis of ras-mediated hydrolysis (4).

The genes of interest were amplified with PCR from human cDNA, using primers designed to target the genes. The primers were designed to incorporate restriction sites that can be acted on by endonucleases to create “sticky ends” in the DNA (5). When designing the primers, restriction enzymes were chosen such that there were no internal restriction sites in the genes of interest. Once the gene was amplified and digested using the restriction enzymes, the DNA was purified and ligated into a cloning vector using a DNA ligase that joins the sticky ends of the gene with corresponding sticky ends in the vector DNA. The pET28 vector was used as the over-expression plasmid in this study. This vector contains a multiple cloning site for digestion using various restriction enzymes, genes for kanamycin resistance for selection, and the lac operon, which allows induction of (6). When the lac operon binds to lactose, or an analog such as isopropyl β-D-1 thiogalactopyranoside (IPTG), T7 polymerase is

14 expressed. T7 polymerase then transcribes the gene of interest, which has been cloned downstream of a T7 promoter (7). An additional feature of the pET28 vector is the option of a 6X-histidine tag, at either the C- or N-terminus for affinity purification. The 6X-histidine tag can be separated from the over-expressed protein by thrombin cleavage for tag removal after protein purification (6).

The final goal of the project was to create GAP mutants that restored GTPase activity to oncogenic Kras mutants. This project required cloning mutant genes of both Kras and rasGAP. Two methods of mutagenesis were employed. For production of Kras G12 mutants, the gene amplification primers were extended to incorporate mutations (8). Because the mutation occurs only 36 nucleotides into the gene, one or two nucleotide substitutions could be introduced by modifying the primer sequences. A library of Kras G12 mutants was created, consisting of the eight most frequently occurring G12 mutations in cancer. For mutations deeper within the gene such as Kras C118S, a second strategy was used, called overlap-extension mutagenesis. This method involves construction of two additional primers, which incorporate the desired mutation. These primers include approximately 15 nucleotides of the wild-type gene sequence on both sides of the desired mutation site. An initial round of the polymerase chain reaction was used to amplify individual halves of the gene: from the forward primer to the first mutation primer and from the second mutation primer to the reverse primer. The resulting DNA was purified, and the corresponding halves were combined and subjected to a second polymerase chain reaction. This round of PCR did not use primers: it allowed the two halves, which overlap, to anneal to each other and act as self primers for strand elongation. Finally, for a third polymerase chain reaction, forward and reverse primers were added to amplify the now-mutated gene (9). Some mutants required multiple site-directed mutations. For this study, mutations were made one at a time, with previously-mutated genes used as templates for further mutation.

2.2 Methods

2.2.1 Primer Design

Primers were designed to amplify the Kras and rasGAP genes from human cDNA clones purchased from the Thermo Scientific Mammalian Gene Collection (MGC). Primer design was accomplished using the web-based software Primer Prim’er (Rutgers), by supplying the

15 nucleotide sequence of the gene for cloning and choosing restriction enzymes and a vector. To construct primers for Kras G12 mutants, the forward primer from the Primer Prim’er program was lengthened according to the complement of the gene sequence to include about 40 nucleotides from the sequence. Nucleotide substitutions were selected by consulting a codon usage table for E. coli. Substitutions were selected that required the fewest changes to the sequence and that resulted in a frequently used codon (10). Restriction sites for the endonucleases NdeI and XhoI were incorporated into the forward and reverse primers, respectively. A stop codon was incorporated into the reverse primer, so that the 6X-histidine tag would be attached at the N-terminus and not the C-terminus.

For mutations requiring overlap extension mutagenesis, primers were designed to incorporate 15 nucleotides on each side of the desired mutation, with high frequency codons chosen for the mutation. The forward primer for the internal mutation (termed F’) was created from the complement of the gene of interest. The reverse primer for the internal mutation (R’) was created by taking the complement of the F’ primer. Some of the GAP mutations required two nearly adjacent site-directed mutations. For these mutants, the first mutation was created, cloned, and sequenced, and was used as a template for the second mutation. Due to the proximity of the two mutation sites, the nucleotide substitution for the first mutation was built into the primer for the second mutation. For example, to make the GAP R789A T786R mutant, the GAP R789A mutation was first made, and cloned into a vector and sequenced. This mutant was then used as a template for the GAP T786R mutation, with the codon for A789 built into the mutagenesis primers. All primers were purchased from Integrated DNA Technologies (Coralville, IA) (Figure 2-1).

2.2.2 Gene Amplification

PCR reactions were carried out on a GeneAmp PCR System 9700 (Applied Biosystems) in 50 µL reaction volumes. Initial screening of PCR conditions for specificity of amplification was carried out using DreamTaq Master Mix (Fisher), 1 µL of each 10 mM primer, and 2 µL of DNA template at 10 ng/µL. Completed PCR reactions were analyzed using a 30 mL 1.2% agarose gel, made in TAE buffer with 1 µL ethidium bromide (Fisher) as an intercalating agent for visualization. Agarose gels were run in TAE buffer at 80 volts for 40 minutes and imaged using an Alpha Imager (Innotech) to check for DNA bands of the correct length compared to a

16 Figure 2-1: A) The forward and reverse primers used to amplify the Kras (1-169) and GAP (714-1047) genes. The forward and reverse primers use the NdeI and XhoI restriction sites, respectively. B) The forward primers used to amplify the Kras (1-169) gene incorporating various Kras G12 mutations. C) The forward and reverse primers used to create the Kras C118S mutation in Kras (1-169) by overlap extension. D) The forward and reverse primers used to various GAP (714-1047) mutants by overlap extension. Note: For site-directed mutants, all nucleotide substitutions are highlighted in yellow. For (D), green highlighting represents GAP R789A mutation already incorporated into the clone.

A) Kras (1-169) F: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAG R: GCCCGCTCCGAGCTACTTTTCTTTATGTTTTCGAATTTCTC GAP (714-1057) F: CCCGCCCGCATATGGAAAAAATCATGCCAGAAGAAG R: GCCCGCTCGAGCTACCTGACATCATTGGTTTTTGTATAC

B) Kras G12A: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTGCTGGCGTA Kras G12C: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTTGTGGCGTA Kras G12D: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTGACGGCGTA Kras G12E: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTGAAGGCGTA Kras G12N: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTAACGGCGTA Kras G12R: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTCGTGGCGTA Kras G12S: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTTCTGGCGTA Kras G12V: CCCGCCCGCATATGACTGAATATAAACTTGTGGTAGTTGGAGCTGTTGGCGTA

C) Kras (1-169) C118S F’: GTCCTAGTAGGAAATAAATCTGATTTGCCTTCTAGAACA R’: TGTTCTAGAAGGCAAATCAGATTTATTTCCTACTAGGAC

17 D) GAP T785R F’: ATGGAAGATGAAGCCCGTACCCTATTTCGAGCCACA R’: TGTGGCTCGAAATAGGGTACGGGCTTCATCTTCCAT GAP T786R F’: GAAGATGAAGCCACTCGCCTATTTCGAGCCACAACA R’: TGTTGTGGCTCGAAATAGGCGAGTGGCTTCATCTTC GAP L787R F’: GATGAAGCCACTACCCGCTTTCGAGCCACAACACTT R’: AAGTGTTGTGGCTCGAAAGCGGGTAGTGGCTTCATC GAP F788R F’: GAAGCCACTACCCTACGTCGAGCCACAACACTTGCA R’: TGCAAGTGTTGTGGCTCGACGTAGGGTAGTGGCTTC GAP R789A: F’: GAAGCCACTACCCTATTTGCAGCCACAACACTTGCA R’: TGCAAGTGTTGTGGCTGCAAATAGGGTAGTGGCTTC GAP T785R with R789A F’: ATGGAAGATGAAGCCCGTACCCTATTTGCAGCCACA R’: TGTGGCTGCAAATAGGGTACGGGCTTCATCTTCCAT GAP T786R with R789A F’: GAAGATGAAGCCACTCGCCTATTTGCAGCCACAACA R’: TGTTGTGGCTGCAAATAGGCGAGTGGCTTCATCTTC GAP L787R with R789A F’: GATGAAGCCACTACCCGCTTTGCAGCCACAACACTT R’: AAGTGTTGTGGCTGCAAAGCGGGTAGTGGCTTCATC GAP F788R with R789A F’: GAAGCCACTACCCTACGTGCAGCCACAACACTTGCA R’: TGCAAGTGTTGTGGCTGCACGTAGGGTAGTGGCTTC

18 GeneRuler 1kb Plus DNA ladder (Fisher). Once optimal PCR conditions were established, gene amplification was repeated using the Phusion Hi-Fidelity Taq Polymerase (Thermo Fisher). The Hi-Fidelity Taq had a significantly decreased rate of random nucleotide mismatches. PCR reactions were carried out using 10 mM dNTP mix from Thermo Scientific, 1 µL of 25 mM

MgCl2 (New England Biolabs), Hi-Fidelity 5X buffer (Thermo Fisher), and concentrations of template and primer mentioned above.

For overlap extension mutagenesis, PCR was carried out using concentrations from above, with primer pairs F,R’ and F’, R. The products of these PCR reactions were run for 1 hour at 80 volts on a 50 mL 1% agarose gel, made with TAE buffer and with SYBR Green I (Lonza) for visualization. The gel was visualized using a black light, and bands corresponding to the correct length were excised from the gel. The gel slices were solubilized by heating in home- made QG buffer (5.5 M guanidine thiocyanate (Promega), 20 mM Tris, pH 6.6). DNA was precipitated by adding isopropanol, and the solution was loaded onto EconoSpin DNA binding columns (Epoch Life Science). DNA binding columns were washed with QG buffer and ethanol and allowed to dry before eluting the bound DNA with 30 µL nanopure water. DNA concentration was determined using a NanoDrop 2000 Spectrophotometer (Thermo Scientific). The two overlapping halves of the gene were combined in a 1:1 molar ratio and used as template DNA for the overlap extension round of PCR. This PCR reaction followed the same recipe as stated above with the exception of the primers. After overlap extension, F and R primers were added to the same PCR tubes for the third and final round of PCR. PCR products were analyzed on an agarose gel as described previously.

All PCR products were cleaned up using a similar method as the gel extraction procedure described: QG buffer was added to the reaction mix, DNA was loaded onto a DNA binding column, washed with ethanol, and eluted with water.

2.2.3 Endonuclease Digestion

After quantification of DNA concentration with the NanoDrop spectrophotometer, genes (subsequently referred to as inserts) were subjected to digestion by restriction endonucleases. The reaction included 20 µL of DNA from the PCR cleanup, 1 µL each of Fast Digest NdeI and XhoI (Fisher), 3 µL 10X Fast Digest Buffer (Fisher), and 5 µL water. Reactions were incubated at 37 °C overnight. The vector plasmid pET28b (Novagen) (used for both Kras and GAP

19 cloning) purified from transformed DH10β E. coli, was similarly digested. After overnight incubation, the reaction mixtures were run on an agarose gel and extracted, as previously described in section 2.2.2.

2.2.4 Ligation

Digested insert was ligated into digested pET28 plasmid by reaction with T4 DNA ligase (Promega). The molar ratio of insert to plasmid for the ligation reaction was 6:1. For the reaction mixture, 1 µL T4 DNA Ligase, at 3 units/µL, was combined with 2 µL 10X Ligase buffer (Promega) and DNA amounts determined by concentration, with water added to make the final volume 20 µL. Ligation mixtures were incubated at room temperature overnight.

2.2.5 Transformation and Plasmid Isolation

After incubation overnight, ligation mixtures were transformed into competent E. coli DH10β cells (Stratagene). Competent cells were prepared by repeated washing with cold 0.1 M

CaCl2, and stocks were stored at -80 °C (11). For transformation, 200 µL of gently thawed competent cells were mixed with 10 µL of each ligation mixture in culture tubes and placed on ice for 20 minutes. The cells were then heat shocked for 50 seconds at 42 °C, and then placed on ice for two minutes. Then, 200 µL of home-made SOC medium was added to the culture tubes, and the tubes were placed in an incubating shaker for 1 hour at 250 rpm and 37 °C. After incubation, 200 µL of the culture was plated on an agar plate with 30 µg/mL kanamycin (Gold Biotechnology) and spread with sterilized glass beads. Plates were incubated at 37 °C overnight. Colonies that grew were picked up using a pipette tip and placed in a culture tube with 3 mL of LB media with 30 µg/mL kanamycin. After these cultures grew in a shaking incubator overnight at 37 °C, 400 µL was removed from the culture and stored at -80 °C after adding 100 µL 50% glycerol and stored at -80 °C.

To isolate the plasmid from the E. coli cloning host, the PureYield Plasmid MiniPrep System was used (Promega). To harvest the cells, 2 mL of the cell cultures were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed. The Resuspension Buffer A was added to the cell pellet according to kit instructions, along with RNase A (Fisher). Tubes were vortexed to resuspend cells, and then the Lysis Buffer B was added. After mixing and incubating at room temperature, the Neutralization Buffer C was added. The resulting solution was centrifuged for 20 minutes at 12,000 rpm. The supernatant was loaded onto EconoSpin DNA

20 binding columns (Epoch), washed with ethanol, and eluted with de-ionized water. Plasmid concentration was determined using a NanoDrop spectrophotometer (Thermo).

2.2.6 Colony Screening and Sequencing

To ensure that insert ligation was successful and that the insert was the correct length, the purified plasmids were subjected to restriction digest using NdeI and XhoI (Fisher). For these reactions, 5 µL of plasmid DNA was mixed with 1 µL of each enzyme, 1 µL of 10X Fast Digest Buffer (Fisher), and 2 µL of de-ionized water. After overnight incubation at 37 °C, 5 µL of each sample was loaded onto a 1.2% agarose gel and run for 40 minutes. If ligation was successful there would be a 500 bp band for Kras or a 1000 bp band for GAP. Comparison of insert size with a 0.5kb DNA ladder (Fisher) confirmed correct cloning.

To confirm that the plasmids contained the correct genes and the correct desired mutations, plasmids were sequenced by di-deoxy sequencing. For the cycle sequencing reaction mixture, 5 µL of plasmid DNA was combined with 2 µL 5X Dilution Buffer, 1 µL of primer, 1 µL Big Dye Ready Reaction Mix (CBFG Miami University), and water. For Kras sequencing, the T7 primer was used as the PCR primer. Di-deoxy sequencing can accurately read 600-1000 base pairs, and the rasGAP sequence is just over 1000 base pairs. For this reason, two reactions were set up for each GAP plasmid, one with T7 terminator and one with T7 promoter as the primer. After the cycle sequencing, the reaction mix was purified: 1 µL of a solution of 1.5 M

NaOAc/0.25 M Na2EDTA and 30 µL 100% EtOH were added, and the solutions were mixed. After incubation at room temperature for 15 minutes, the tubes were centrifuged at 12,000 rpm for 15 minutes. The supernatant was removed, and the pellet was washed with 50 µL of 70% EtOH, after which the tubes were centrifuged again. After drying, the samples were taken to the Center for Bioinformatics and Functional Genomics (CBFG) at Miami University for sequencing. Sequencing data were analyzed using the 4Peaks software (Nucleobytes) for MacOS, translated using Expasy Translator, and aligned using ClustalW.

21 2.3 Results and Discussion

2.3.1 Gene Amplification and Mutagenesis

For both Kras and GAP, agarose gels showed that the desired gene was amplified. The Kras insert, 507 bp, ran just above the 500 marker on the DNA ladder, and the GAP insert, 1,002 bp, ran right next to the 1000 base pair marker. The intensities of the bands on the gel showed that a good yield was achieved, and the absence of any extraneous bands demonstrated high specificity and purity (Figure 2-2). Similar results were observed for the Kras G12 mutants (not shown).

After the first round of PCR for the overlap extension GAP mutants, agarose gel analysis of the PCR products showed bands corresponding to both halves of the GAP gene (Figure 2-3 A). There are several bands in each lane on this gel, demonstrating nonspecific binding of the primers. However, these non-specific bands were not a problem because the pieces of the insert were cut out of the gel and purified before moving on to the next PCR step. Similar results were seen for the Kras C118S mutation, with bands matching the expected sizes of the two halves of the gene. After the final round of PCR, the products were analyzed on a gel to determine if the overlapped insert was the correct size. Figure 2-3 B shows the final PCR product of the Kras C118S mutant insert, at the correct size of 500 base pairs, with good yield and purity.

2.3.2 Endonuclease Digestion of Insert and Vector

All inserts and the vector were digested with NdeI and XhoI, run on an agarose gel, and extracted. To confirm double digestion of the pET28 vector, which is crucial for ligation, the vector was digested with one enzyme at a time, and the products were compared to the double- digested vector and the uncut vector. The uncut vector showed two bands with different apparent lengths due to different mobilities. The band that ran further represents uncut vector, which is supercoiled and thus migrates faster through the gel. The slower band represents nicked (non-supercoiled) circular DNA. Both the single digests and the double digest showed a linearized vector (represented by a band that moves slower than the supercoiled vector), suggesting that both restriction enzymes are active and have correctly cut the vector (Figure 2-4).

2.3.3 Colony Screening and Sequencing

22 Plasmid DNA isolated from cultures of individual colonies was subjected to NdeI/XhoI double digestions. If ligation was successful, the insert should be cut out of the plasmid and appear at a predicted location on an agarose gel. Transformed colonies were screened in this way before sequencing; plasmids with the correct length insert were then sequenced. Some of the screened GAP mutants are shown in Figure 2-5, with the appropriate 1000-base pair insert cut out of the much larger plasmid. Genes and mutants that have been successfully cloned and confirmed by sequencing are listed in Table 2-1. Translated sequences for Kras and GAP are shown in Figure 2-6, with annotations describing relevant amino acids.

2.4 Conclusions

PCR amplified genes were successfully digested and ligated into pET28 vectors. Site- directed mutagenesis was carried out by overlap extension PCR. The vectors were transformed into the E. coli DH10β cloning host. Positive transformants were cultured and the plasmid was isolated. Upon restriction digestion, clones showed bands for the ligated insert and were then sequenced. Genes and mutations successfully cloned and confirmed by sequencing are listed in Table 2-1.

23

A) B)

1kb

0.5kb

Figure 2-2: A) 1.2% agarose gel showing amplification of the GAP gene (1,002 base pairs). Lane 1: 1 kb Plus ladder. Lane 2: GAP insert. B) 1.2% agarose gel showing amplification of the Kras gene (507 base pairs). Lane 1: Kras insert. Lane 2: 1 kb Plus ladder.

24

A) B)

1kb 1kb

700bp 700bp

500bp 500bp 400bp 400bp 300bp 300bp

200bp 200bp

Figure 2-3: 1.2% agarose gels demonstrating amplification of genes for overlapping PCR mutagenesis: A) Use of internal primers with mutations incorporated to carry out mutagenesis by overlap extension PCR. Lane 1: 1 kb Plus DNA ladder. Lanes 2-5: GAP insert piece from F to R’ (225 bases) for GAP R789A, GAP L787R, GAP T786R, and GAP T785R, respectively. Lanes 6-9: GAP insert piece from F’ to R (800 bases) for GAP R789A, GAP L787R, GAP T786R, and GAP T785R, respectively. B) Agarose gel showing full-length mutated product of the Kras gene. Lanes 1-2: Kras C118S insert (507 bases). Lane 3: 1 kb Plus DNA ladder.

25

Figure 2-4: Agarose gel showing successful digestion of pET28 plasmid DNA as evidenced by change in gel mobility. Lane 1: Undigested plasmid, showing bands for supercoiled and nicked DNA. Lane 2: NdeI digest. Lane 3: XhoI digest. Lane 4: NdeI/XhoI double digest

.

Figure 2-5: Successful restriction enzyme screening of GAP mutants. GAP clones were subject to a double digest to confirm the size of the insert. An agarose gel showing linearized plasmid DNA at roughly 5 k base pairs and the GAP insert at 1k base pairs. Lanes 1-3: L787R GAP mutant clones. Lanes 4-6: T786R GAP mutant clones. Lanes 7-9: T785R GAP mutant clones.

26 A)

MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTA GQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNK CDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEK

B)

MEKIMPEEEYSEFKELILQKELHVVYALSHVCGQDRTLLASILLRIFLHEKLESLLLCTLN DREISMEDEATTLFRATTLASTLMEQYMKATATQFVHHALKDSILKIMESKQSCELSPSK LEKNEDVNTNLTHLLNILSELVEKIFMASEILPPTLRYIYGCLQKSVQHKWPTNTTMRTR VVSGFVFLRLICPAILNPRMFNIISDSPSPIAARTLILVAKSVQNLANLVEFGAKEPYMEG VNPFIKSNKHRMIMFLDELGNVPELPDTTEHSRTDLSRDLAALHEICVAHSDELRTLSNE RGAQQHVLKKLLAITELLQQKQNQYTKTNDVR

C)

MGSSHHHHHHSSGLVPRGSH

Figure 2-6: Amino acid sequences of gene products: A) Sequence of Kras 169. Glycine 12 is highlighted in blue, and cysteine 118, later mutated to serine, is highlighted in green. B) Sequence of GAP 334. Catalytic arginine (789) is highlighted in yellow. Residues to be mutated to arginine are highlighted in gray. C) The N-terminal cloning extension consisting of the start codon (M), 6X-histidine tag (HHHHHH), and TEV protease site for 6X-histidine tag cleavage (LVPRGS).

27

Table 2-1:

Gene: Successfully cloned inserts:

Kras 169 WT C118S G12A G12C G12D

G12E G12N G12R G12S G12V

GAP 334 WT R789A T785R T786R L787R

R789A, T785R R789A, T786R R789A, L787R R789A, F788R

28 2.5 References

1. Jancík, S., Drábek, J., Radzioch, D., & Hajdúch, M. (2010). Clinical relevance of KRAS in human cancers. Journal of Biomedicine & Biotechnology, 2010, 150960. doi:10.1155/2010/150960 2. Res, A. H. C., Biomol, A. J. J., Natl, D. M. P., Sci, A., John, J., Sohmen, R., … Goody, R. S. (1990). Kinetics of interaction of nucleotides with nucleotide-free H-ras. Cancer Research, (1979), 6058–6065. 3. Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., & Shokat, K. M. (2013). K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature, 503(7477), 548–51. doi:10.1038/nature12796 4. Wiesmuller, L. (1996). Structural differences in the minimal catalytic domains of the GTPase-activating proteins p120GAP and neurofibromin. Journal of Biological Chemistry, 271(27), 16409–16415. doi:10.1074/jbc.271.27.16409 5. Nathans, D., & Smith, H. O. (1975). Restriction endonucleases in the analysis and restructuring of dna molecules. Annual Review of Biochemistry, 44, 273–93. doi:10.1146/annurev.bi.44.070175.001421 6. Rosenberg, A. H., Lade, B. N., Dao-shan, C., Lin, S.-W., Dunn, J. J., & Studier, F. W. (1987). Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56(1), 125–135. doi:10.1016/0378-1119(87)90165-X 7. Beckwith, J. R. (1967). Regulation of the lac operon. Science, 156(3775), 597–604. doi:10.1126/science.156.3775.597 8. Higuchi, R., Krummel, B., & Saiki, R. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Research, 16(15), 7351–7367. doi:10.1093/nar/16.15.7351 9. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., & Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77(1), 51– 9. 10. Sharp, P. M., Cowe, E., Higgins, D. G., Shields, D. C., Wolfe, K. H., & Wright, F. (1988). Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a

29 review of the considerable within-species diversity. Nucleic Acids Research, 16(17), 8207–11. 11. Morrison, D. A. (1977). Transformation in Escherichia coli: cryogenic preservation of competent cells. Journal of Bacteriology, 132(1), 349–351.

30

Chapter 3:

Expression, Purification, and Characterization of Kras and rasGAP

31 3.1 Introduction

This chapter focuses on experiments carried out to express and purify the proteins of interest. The methods presented below represent a generalized method of expression and purification and do not reflect the great deal of optimization needed for each different protein or mutant. When over-expressing proteins in E. coli, there are many variables that can be slightly altered to improve over-expression and protein characteristics. These variables include over- expression temperature, time allowed for over-expression, density of the culture at time of induction, method of induction, buffer composition and buffer pH, to name a few (1).

The pET28 plasmids containing Kras and GAP inserts were transformed into E. coli BL21 (DE3) Codon Plus competent cells. The BL21 strain is a widely used expression host, which is deficient in several proteases that can degrade heterologous proteins. The BL21 Codon Plus strain is often used for over-expression of eukaryotic proteins because its codon usage better matches that of eukaryotic cells. The Codon Plus strain contains tRNAs for codons that are not generally found in bacteria, which allow better potential for gene expression. The E. coli BL21 strain is also resistant to chloramphenicol, so all media can be prepared with both chloramphenicol and kanamycin, to select for both the bacteria strain and the presence of the pET28 plasmid (2). Induction of the cell culture by the addition of IPTG turns on the lac operon, which is an element of the pET28 plasmid. The lac operon is linked to T7 Polymerase, a polymerase enzyme derived from the T7 phage that transcribes genes with a T7 promoter (3). The insert gene has been ligated next to a T7 promoter, so it is expressed by T7 polymerase.

All protein constructs in this study were fused to a 6X-histidine tag to aid in purification. This chain of six histidine residues has a high affinity for nickel ions, and this property is exploited for protein purification. Crude cell lysates are passed through a resin containing immobilized nickel ions (4). Proteins with a 6X-histidine tag tightly bind to the resin, and everything else flows through the resin. Proteins that bind weakly are washed off using a dilute solution of imidazole, which also has a high affinity for nickel and competes with proteins for binding (5). Once extraneous proteins are washed off the column, the protein is eluted with a concentrated solution of imidazole. Elution results in a protein solution with a high concentration of imidazole. For some applications, after purification, the 6X-histidine tag must be removed. The 6X-histidine tag is not part of the natural protein and can interfere with

32 packing during protein crystallization, for example (6). The 6X-histidine tag is linked to the protein by a thrombin cleavage site. The cleavage site is a specific series of amino acids that is recognized by the serine protease thrombin. Thrombin is added to the protein sample, and once the tag is removed, the mixture is passed through a nickel affinity resin again. This time, the protein of interest flows through the resin, and the 6X-histidine tag fragment is trapped, resulting in pure untagged protein.

The Kras protein changes conformation when it hydrolyzes bound GTP to GDP (7), as described in Chapter 1. For the purposes of this study, it is important to know the conformational state of Kras. One method that can be used to determine the conformational state is two-dimensional NMR spectroscopy, 1H-15N HSQC specifically. This technique allows detection of insensitive spin I = 1/2 15N nuclei by transferring magnetization from the more sensitive 1H nucleus to the nitrogen (8). Protein samples must be isotopically-enriched with 15N, which is easier to study than the more abundant spin I = 1 14N nucleus. The HSQC experiment gives the correlation between nitrogen and hydrogen atoms connected by a chemical bond. Thus, a cross peak is seen on the spectrum for each unique proton attached to a nitrogen. The chemical shift of each cross peak is characteristic of the local environment of the amide. Consequently, when the local environment of the amide changes due to conformational change, e.g., upon Kras binding GTP, the chemical shift of the representative peaks change (9). This phenomenon was used to visualize the conformational state of Kras. Other laboratories have assigned the 1H-15N HSQC spectrum of Kras and have identified which peaks shift upon hydrolysis of GTP (10). 1H-15N HSQC was also used as a confirmation of proper Kras folding, and as a confirmation of rasGAP activity. The GAP protein should dramatically speed up hydrolysis of GTP to GDP, so when GAP is added to the NMR sample, the conformation (as determined by the HSQC peaks) should switch rapidly. The addition of GAP was not expected to interfere with data quality, because the GAP protein was not isotopically-labeled and it was only added at catalytic amounts.

The current gold standard of protein structure determination is X-ray crystallography. By measuring the diffraction of X-rays that pass through a protein crystal, an electron density map can be generated that provides a scaffold for the three-dimensional structure of a protein. This method requires the growth of protein crystals, which can be elusive. Proteins crystallize when their concentration is gradually increased, allowing them to regularly stack. Many conditions

33 must be optimized for the growth of a high quality protein crystal, including buffer pH and salt concentration, temperature, and protein concentration and purity. Because crystallization is a rare event, trays of dozens of separate crystallization conditions are set up. This study used the hanging drop crystallization method, which involves placing a reservoir of buffer in the bottom of a well, and mixing a drop of concentrated protein with a small drop of buffer onto a glass slide which is inverted over the well. Through vapor diffusion between the drop and the reservoir, the protein concentration gradually increases until the solution is supersaturated. At this point, the protein may precipitate or crystallize.

3.2 Methods

3.2.1 Transformation, Expression, and Cell Lysis

The following general procedure was used for all proteins: Competent cell stocks of the expression host, E. coli BL21 (DE3) Codon Plus (Life Technologies) were prepared as previously described (2). For transformation mixtures, 100 µL portions of competent cells were mixed with 1 µL purified plasmid DNA. After 30 minutes of incubation on ice, the mixtures were heat shocked at 42 °C for 50 seconds and returned to ice for 2 minutes. Then, 100 µL of SOC medium was added, and the tubes were moved to a shaking incubator at 250 rpm and 37 °C for 1 hour. After incubation in the shaker, cultures were plated onto LB agar plates with 30 µg/mL kanamycin (Gold Biotechnology), spread with sterilized glass beads, and incubated at 37 °C overnight.

The next day, individual colonies were inoculated into 3 mL LB medium with 30 µg/mL kanamycin and 33 µg/mL chloramphenicol (Fisher), and incubated at 37 °C overnight in an incubator shaking at 250 rpm. The following day, the 3 mL pre-cultures were inoculated into 1

L of LB, with the same antibiotics added, and grown in a shaking incubator. The OD600 was checked regularly, and when the absorbance reached 0.8 units, cultures were induced with 0.5 mL of 1 M IPTG (Gold Biotechnology) and moved to a room temperature shaker (22 °C). Cultures were incubated at room temperature with 250 rpm shaking overnight. In the morning, the cultures were centrifuged for 20 minutes at 5,000 xg and the supernatant was poured off.

34 Cell pellets were resuspended in 20 mL “Kras Buffer” (20 mM Tris, pH 7.8, 100 mM NaCl, 10% glycerol) and PMSF protease inhibitor (Thermo) was added to inhibit serine proteases from E. coli. The cell suspension was passed through a French pressure cell press at 1,000 psi three times, and the lysate was clarified by centrifugation at 20,000 rpm for 30 minutes.

3.2.2 Nickel Affinity Purification

Protein lysate was passed through a Nickel-NTA Superflow column (Qiagen) pre- equilibrated with 50 mL of Kras buffer (described above). The column was washed with 50 mL of Kras buffer, followed by 50 mL of wash buffer number 1 (Kras buffer with 30 mM imidazole (Fisher)), and then 25 mL of wash buffer number 2 (Kras buffer with 60 mM imidazole). The protein was eluted with 50 mL of 300 mM imidazole in Kras buffer and collected in 7 mL fractions. Protein concentrations of the wash buffers and elution fractions were assessed using a home-made Bradford reagent (Brilliant Blue (Acros Organics), phosphoric acid, ethanol). The absorbance readings of the samples at 595nm were compared to that of a standard curve created using bovine serum albumin (Fisher) with a Beckman DU-640 spectrophotometer. Protein purity was visualized by running the samples on 15% SDS-polyacrylamide gels with 20 mM dithiothreitol (Gold Biotechnology) as a reducing agent. Gels were stained with Coomassie blue overnight. If necessary, protein samples were concentrated in dialysis bags with a MWCO of 12,000-14,000 Daltons (Fisher) by covering the dialysis bags in Peg 20,000 (Alfa Aesar) for several hours. For some applications, it was necessary to remove the 6X-histidine tag from the protein, using thrombin (Fisher). For the thrombin digestion, thrombin was added to a protein sample at 3 units per mg of protein, with 25 mM CaCl2 and 0.01% sodium azide (RICCA) and incubated at room temperature overnight. To purify, the mixture was passed through an equilibrated Nickel-NTA column as described above, but the flow-through was collected.

3.2.3 Kras Expression in Minimal Media

For analysis by nuclear magnetic resonance spectroscopy, the Kras protein needed to be labeled with 15N in M9 medium. Expression in M9 medium was first tested with non- isotopically labeled M9 to check expression levels. Cultures from frozen stocks were inoculated into a 3 mL pre-culture of LB with the appropriate antibiotics and incubated overnight. The pre- culture was then inoculated into a further 25 mL pre-culture. This culture was incubated

35 overnight and was inoculated into 1 liter of M9 medium (MgSO4, metals mix, NPS, vitamin mix, 15 glucose) (11) with the appropriate antibiotics and 0.5 g NH4Cl. Once the cells grew to OD600= 0.8, the culture was induced with 0.5 mL of 1M IPTG, and the culture was moved to a room temperature shaker to express overnight. From this point, the culture was treated as described above for over-expression in LB. Once over-expression was verified, the protein was over- expressed in minimal medium with isotopically-labeled 15N (Cambridge Isotope Laboratories). 15 PS solution was substituted for NPS, and 0.5 g N NH4Cl was added to the medium. All other methods were the same as described above. Once the protein was purified, it was dialyzed into NMR buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol). Protein samples were then concentrated by dialysis against PEG-20,000. Samples for NMR were concentrated to 10 mg/mL and placed in Shigemi tubes with 10% D2O and 0.01% sodium azide (RICCA).

3.2.4 Crystal Screening

Proteins were concentrated by dialysis against PEG 20,000. Crystal trial plates were set up at concentrations from 10 mg/mL to 35 mg/mL for Kras and from 5 mg/mL to 20 mg/mL for GAP. Crystal trials were set up using the hanging drop method in 48-well plates with Crystal Screen buffer kits (Hampton Research). Trials were set up for individual proteins, mutants, and Kras/GAP complexes at a 1:1 molar ratio. For crystallization, all of the Kras proteins in a sample must have the same nucleotide binding state because of the resulting conformation change. Kras was loaded with excess nucleotide: either GDP or the non-hydrolyzable analog, GDPNP (Sigma). At the time of writing, no protein crystals had formed.

3.2.5 Kras Nucleotide Binding State Determination

Kras was over-expressed in M9 medium as described above, and NMR samples were prepared. 1H-15N HSQC spectra were recorded on an 850 MHz Bruker Avance III Spectrometer. High resolution 1H-15N HSQC spectra were recorded over the course of an hour. Twenty minute spectra were recorded when speed of acquisition was more important than spectral quality. Kras 1H-15N HSQC peaks have been assigned (10), and several amide cross peaks shift depending on nucleotide binding state. The cross peak for Y157 was chosen due to its clear shift between states and its isolation from other peaks in the spectrum. Spectra were recorded directly after sample preparation, and then again after several days, and the Y157 peak was compared.

36 Additionally, GAP was added to a second sample of newly-prepared 15N-labeled Kras at a 1:40 molar ratio, and the spectrum was recorded. Data were processed using TopSpin 3.0.

3.3 Results and Discussion

3.3.1 Protein Expression and Purification

6X-histidine-tagged fusion proteins were purified using a nickel affinity column. Eluates were collected in five fractions of 7 mL each, labeled E1-E5. Protein concentration was determined by using the Bradford assay, and protein purity was assessed using SDS-PAGE. Protein was typically found at the highest concentration and purity in the E1 and E2 fractions. Protein of lower purity was found in the 60 mM imidazole washes, and protein was also found at very low concentrations in some E3 fractions. For Kras, GAP, and all mutants, several conditions for over-expression were screened. Over-expression temperature, time, and IPTG concentrations were varied to achieve maximum protein yield. Interestingly, some BL21 transformants with identical sequence-confirmed clones expressed proteins at very different amounts. For each protein, multiple clones were screened for expression if yield was low. The Kras protein and various mutants typically yielded 20-30 mg of protein per liter of culture (about 1 mg/mL in E1 and E2) Figures 3-1, 3-2). After incorporation of the C118S mutation into the Kras sequence, over-expression improved to 40-50 mg per liter (Figure 3-4). Kras ran as a dimer on a denaturing gel in the absence of a reducing agent. When the reducing agent DTT was added to the sample, the band corresponding to the dimer disappeared (Figure 3-1). The GAP protein and its mutants also over-expressed well. GAP clones typically yielded 70-100 mg of protein per liter (between 3 and 5 mg/mL in E1 and E2) (Figures 3-3, 3-5). E1 and E2 fractions for both proteins showed very high purity. These fractions could be concentrated using dialysis against PEG 20,000 to prepare samples for crystal screening. Crystal trial plates were set up at concentrations from 10 mg/mL to 35 mg/mL for Kras and from 5 mg/mL to 20 mg/mL for GAP.

3.3.2 Thrombin Cleavage

High salt concentrations can inhibit the activity of serine proteases, so protein preparations were dialyzed against Kras buffer (described above) to remove imidazole. To determine the amount of thrombin needed to remove the 6X-histidine tag, various ratios of

37

A) B) C)

55kDa

43kDa

34kDa 26kDa

26kDa

17kDa

17kDa

Figure 3-1: SDS-PAGE gel analysis of wild-type over-expressed Kras purified from E. coli. Gels are 15% acrylamide (Fisher) and run with Pre-Stained Rec Protein Ladder (Fisher). A) Purified Kras (21 kDa) running at the expected size. The gel shows a higher molecular weight band corresponding to dimer formation. Lane 1: Kras E1 elution. Lane 2: Rec protein ladder. B) The high molecular weight band disappears on addition of the reducing agent DTT. Lane 1: Kras in denaturing buffer (no DTT). Lane 2: Kras with 20 mM DTT. C) Thrombin cleavage of fused 6X-histidine tag. Cleavage carried out with 3 units of thrombin per mg protein, incubated at room temperature overnight. Lane 1: Rec protein ladder. Lane 2: Unprocessed Kras. Lane 3: Kras after thrombin cleavage, running at a slightly lower molecular weight.

38

34kDa

26kDa

17kDa

Figure 3-2: SDS-PAGE gel showing elution profile of wild-type Kras over-expressed in E. coli. Lane 1: 30 mM imidazole wash. Lanes 2-5: 300 mM imidazole eluates collected in sequential 7 mL fractions, corresponding to fractions 1 through 4. Lane 6: Rec protein ladder.

39

55kDa

43kDa

34kDa

26kDa

Figure 3-3: PAGE gel showing elution profile of wild-type GAP (38 kDa) over-expressed in E. coli. Lane 1: Rec protein ladder. Lane 2: Total crude lysate. Lane 3: 30 mM imidazole wash. Lanes 4-7: 300 mM imidazole eluates collected in sequential 7 mL fractions, corresponding to fractions 1 through 4.

40

55kDa

43kDa

34kDa

26kDa

17kDa

Figure 3-4: SDS-PAGE gel demonstrating expression of various proteins and thrombin cleavage. Lanes 1-3: Kras mutant G12R 300 mM imidazole elution fractions 1 through 3. Lane 4: Rec protein ladder. Lane 5: 60 mM wash of Kras mutant C118S. Lanes 6-8: Kras mutant C118S 300 mM imidazole elution fractions 1 through 3. Lane 9: Wild-type GAP. Lane 10: Thrombin cleavage product of wild-type GAP.

41

43kDa

34kDa

26kDa

Figure 3-5: SDS-PAGE gel demonstrating expression of GAP mutations. Lane 1: Rec protein ladder. Lane 2: 60 mM imidazole wash of GAP T786R. Lanes 3-5: GAP mutant T786R 300 mM imidazole elution fractions 1 through 3. Lane 6: 60 mM imidazole wash of GAP T785R. Lanes 7-9: GAP mutant T786R 300 mM imidazole elution fractions 1 through 3. Lane 10: Wild-type GAP to use as molecular weight comparison.

42 thrombin to protein were screened for both Kras and GAP. Thrombin/protein mixtures were incubated for varying amounts of time and analyzed by SDS-PAGE. It was found that a ratio of 3 units thrombin : 1 mg protein was sufficient for total 6X-histidine tag cleavage. Thrombin and the his tag were removed from the protein sample by passing the sample through a nickel-NTA resin. The 6X-histidine tags bind to the resin and the now untagged protein passes through. (Figure 3-1)(Figure 3-4)

3.3.3 Binding State Determination by 1H-15N HSQC

M9 minimal medium expression of Kras yielded about 20 mg of protein per liter. The HSQC spectra recorded for Kras matched the published spectra very well (10). Clear differences could be seen in the chemical shift of the Y157 cross peak a day after sample preparation. Directly after protein purification and sample preparation, Y157 peaks representative of both the GTP and GDP bound conformational states were visible, suggesting that there was a mixture of binding states (Figure 3-6). A spectrum recorded a day later showed only the Y157 peak representative of GDP bound Kras, suggesting that all of the GTP had been hydrolyzed (Figure 3-7). This result is rational: GTP concentration is higher in the of cells, so Kras will be predominantly GTP bound when purified. Over the course of purification, some GTP will become hydrolyzed, as evidenced by the presence of both peaks. Finally, after several days, enough time has elapsed for the slow intrinsic catalytic ability of Kras to hydrolyze all of the GTP.

A second study involved two parallel sample preparations of 15N-labeled Kras. A spectrum of each sample was recorded. To one sample, GAP was added to the NMR tube at a 1:40 molar ratio, and an equal volume of buffer was added to the other sample. The 1H-15N HSQC of both samples was recorded directly after addition of GAP or buffer. The Kras+GAP sample only showed the peak representative of GDP bound Kras, while the sample without GAP still showed both peaks (Figure 3-8). This result suggests that the GAP-catalyzed hydrolysis, causing all GTP to be cleaved into GDP within the time frame of the data collection. This finding demonstrates that the GAP protein is behaving as expected and has the proper catalytic activity.

43 3.4 Conclusions

After cloning into the pET28 vector, constructs were transformed into an E. coli expression host, and the protein was over-expressed. Cells were harvested and lysed, and protein was purified using affinity chromatography. For many clones, several over-expression conditions had to be screened to attain high over-expression levels. Also, the 60 mM imidazole wash step was added to increase protein purity in the elutions. A set of Kras proteins and mutants was over-expressed reasonably well, with good purity. A set of GAP proteins and mutants was over-expressed very well, with outstanding purity. Kras, GAP, and mutants that were successfully over-expressed are shown in Table 3-1. Thrombin cleavage of the 6X- histidine tag was also successful. Kras was also over-expressed in M9 minimal medium, with yields comparable to those of over-expression in LB.

1H-15N HSQC studies verified the structure of Kras based on similarity to spectra previously reported in the literature (10). The nucleotide-binding state of freshly-prepared protein was determined to be the GTP-bound state, as measured by cross peak shifts. The GTPase activity of Kras was confirmed by monitoring the chemical shift of the Y157 cross peak at different times. Additionally, catalytic activity of GAP was confirmed. Catalysis occurred at enzymatic amounts of GAP (1:40 molar ratio of GAP to Kras), and addition of GAP caused shift of the Y157 cross peak to the GDP-bound position immediately. This data suggests that GAP sped up the hydrolysis reaction by several orders of magnitude, which is consistent with the literature (7).

Table 3-1:

Gene: Successfully Over-Expressed Proteins:

Kras 169 WT C118S G12A G12D

G12N G12R G12S

GAP 334 WT R789A T785R T786R

R789A, T785R R789A, T786R R789A, L787R

44

Y157

Figure 3-6: 1H-15N HSQC spectrum of wild-type Kras just after protein purification and sample preparation. Spectrum recorded on an 850MHz Bruker Avance III Spectrometer. Protein concentration was approximately 10 mg/mL, or 0.5 mM. The Y157 amide is represented by a pair of peaks (circled) due to the presence of both GTP-bound and GDP-bound protein in the sample.

45

Y157

Figure 3-7: 1H-15N HSQC spectrum of wild-type Kras recorded 24 hours after sample preparation. All GTP has been hydrolyzed by Kras. The spectrum was much clearer due to the presence of a single conformational state. The cross peak assigned to Y157 is circled. Y157 is the cross peak that was used to determine nucleotide-binding state.

46 Figure 3-8: Overlay of 1H-15N HSQC of Kras. In red, a spectrum was recorded immediately after sample preparation. The spectrum shows the presence of both GTP-bound and GDP-bound conformations, demonstrated by the presence of both Y157 peaks (shown larger in inset). The peak intensities suggest that the GTP-bond form has a higher population than the GDP- bound form. Directly following measurement of the first spectrum, purified GAP protein was added to the NMR tube. The spectrum was recorded again, shown in blue. This spectrum only shows the GDP-bound peak, suggesting that hydrolysis of GTP was complete.

47 3.5 References

1. Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology, 10(5), 411–421. doi:10.1016/S0958-1669(99)00003-8 2. Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72(2), 211–22.doi:10.1007/s00253-006-0465-8 3. Tabor, S., & Richardson, C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proceedings of the National Academy of Sciences, 82(4), 1074–1078. doi:10.1073/pnas.82.4.1074 4. Porath, J. (1992). Immobilized metal ion affinity chromatography. Protein Expression and Purification, 3(4), 263–281. doi:10.1016/1046-5928(92)90001-D 5. Schmitt, J., Hess, H., & Stunnenberg, H. G. (1993). Affinity purification of histidine- tagged proteins. Molecular Biology Reports, 18(3), 223–230. doi:10.1007/BF01674434 6. Jenny, R. J., Mann, K. G., & Lundblad, R. L. (2003). A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expression and Purification, 31(1), 1–11. doi:10.1016/S1046-5928(03)00168-2 7. Hall, B. E., Bar-Sagi, D., & Nassar, N. (2002). The structural basis for the transition from Ras-GTP to Ras-GDP. Proceedings of the National Academy of Sciences of the United States of America, 99(19), 12138–42. doi:10.1073/pnas.192453199 8. Markwick, P. R. L., Malliavin, T., & Nilges, M. (2008). Structural biology by NMR: structure, dynamics, and interactions. PLoS Computational Biology, 4(9), e1000168. doi:10.1371/journal.pcbi.1000168 9. Widmer, H., & Jahnke, W. (2004). Protein NMR in biomedical research. Cellular and Molecular Life Sciences : CMLS, 61(5), 580–99. doi:10.1007/s00018-003-3382-3 10. Vo, U., Embrey, K. J., Breeze, A. L., & Golovanov, A. P. (2013). 1H, 13C and 15N resonance assignment for the human K-Ras at physiological pH. Biomolecular NMR Assignments, 7(2), 215–9. doi:10.1007/s12104-012-9413-y 11. Marley, J., Lu, M., & Bracken, C. (2001). A method for efficient isotopic labeling of recombinant proteins. Journal of Biomolecular NMR, 20(1), 71–75. doi:10.1023/A:1011254402785

48

Chapter 4:

Measuring GAP-Mediated Catalysis of GTP Hydrolysis by Kras

49 4.1 Introduction

In an effort to redesign the GAP catalytic site to restore rapid GTP hydrolysis to oncogenic Kras mutants, a set of GAP mutants were created using recombinant technology. These mutants needed to be screened to determine if they could cause accelerated GTP hydrolysis with mutant Kras (G12X). This screening could be accomplished using 1H-15N HSQC NMR by monitoring the cross peak shifts as previously described, but screening using NMR would require a large amount of 15N-labeled Kras and a significant amount of instrument time, which are both prohibitive especially when screening a large number of mutants (1). Therefore, we attempted to identify a relatively simple assay that would allow for high throughput screening of engineered GAP mutants. Standard assays for determining GTPase activity involve radio labeled phosphate, fluorescently-labeled GTP, or a phosphate-binding assay (2). Radio isotopes are expensive to work with, and the lab was not equipped to handle radioactivity (3). Alternatively, GTP can be labeled with a fluorescence probe at the γ- phosphate, and when the γ-phosphate is removed by hydrolysis, there is a drop in fluorescence intensity. However, the fluorescent probe adds steric bulk to the nucleotide and may interfere with normal chemistry of the active site (4). Additionally, this project focuses on the structure and mechanism within the active site, and the incorporation of a fluorescent probe would add a confounding variable to the experiments. Thus, the best option for determining GTPase activity was the use of a phosphate binder. Phosphate assays are an indirect measurement of GTP hydrolysis: when GTP is hydrolyzed, a phosphate is released and can be picked up by a secondary compound, inducing a color change or other observable effect (5). The release of phosphate as measured by this assay can be correlated to the GTPase activity of Kras. Kras binds to GTP in a 1:1 ratio, and nucleotide loading ensures that all Kras is GTP-bound. Thus, the amount of GTP in the sample can be quantified. This molar concentration of GTP can be compared to the phosphate concentration returned from the GTPase assay (calculated from the regression equation derived from the standard curve). In this way, data can be expressed as a percentage of GTP hydrolyzed after varying amounts of time. A disadvantage to this technique is the high background levels of phosphate that are possible with impure protein samples. Background phosphate signal can come from phosphate in the protein solution or contaminating GTPase proteins. All inorganic phosphate must be removed from the sample to minimize the background, and several controls must be used to standardize the data.

50 This study utilized the PiColorLock Gold Phosphate Assay from Innova Biosciences (Cambridge, England). This is a sensitive phosphate assay that results in a color change from yellow (λmax=446 nm) to green (λmax=635 nm). When phosphate is released from the hydrolysis of GTP, it binds to an ammonium molybdate complex in an acidic environment. This binding results in the formation of a large phospho-molybdate complex, which can bind malachite green dye. Binding of the dye to the molybdate complex induces the color change (6,7) (Figure 4-1). The strong acidity of the assay reagent stops GTPase activity, so precise time points can be taken. The PiColorLock Gold assay kit from Innova includes a stabilizing reagent to prevent acid hydrolysis of GTP. This assay allows high throughput time-course measurements of GTP hydrolysis, so that GTPase activity with and without GAP mutants can be compared.

The GTPase assay required several positive and negative controls. First, due to potential of high background, readings were taken of all reagents and proteins separately. A positive control, wild-type Kras with wild-type GAP, was used to demonstrate the activity of GAP and the validity of the assay. A negative control, wild-type GAP with G12A mutant Kras, was expected to show no increase in activity upon addition of GAP due to the mutation. Additionally, mutant GAPs were tested with wild-type Kras to determine if the mutation had any effect on the wild-type activity. Due to variability in phosphate backgrounds, assays were run side by side with and without GAP, to determine if there was a difference in activity when GAP was added. Also, the timing of the addition of reagents and absorbance readings were important and were tightly-controlled. A great deal of optimization went into tailoring this assay for the project, from determining amounts of protein needed to what minimum assay volume could be used. The original assay kit instructions suggest using 50 µL of the color-forming reagent for each assay. However, the data for this project would require multiple time points taken from a single sample. Time course data were generated for a number of protein/mutant combinations, which would have resulted in a huge amount of assay reagent being used. Consequently, this assay was optimized so that only 2 µL of the color reagent was needed for each time point.

Initial assay data were inconclusive, with high amounts of contamination present in the samples. GTP had to be added to the reaction mixtures as a substrate, and inorganic phosphate contamination from slow degradation of GTP was unavoidable. One source of problems was that while GTP hydrolysis was catalyzed by GAP, the exchange of GDP for GTP happened very slowly. The solution to this problem was to load Kras with GTP before beginning the assay, and

51 only measuring the first GTPase cycle. Thus, the assay was conducted without relying on turnover of Kras/GTP. By not adding additional GTP to the system, the background signal was lowered, and the effect of GAP was seen immediately, because every Kras protein was already loaded with GTP. Nucleotide loading was accomplished by stripping magnesium from the active site with EDTA, which prevented spontaneous nucleotide hydrolysis, and then adding a large excess of GTP (8). The GTPase hydrolysis reaction then began upon addition of magnesium. However, this loading procedure required removal of the magnesium-chelating agent as well as all unbound GTP. To accomplish this, the protein sample was desalted. Desalting involved a small-scale size exclusion column, which traps small molecules in a porous gel while allowing large molecules, such as proteins, so flow past the small pores. Large molecules are eluted from the column first, and small molecules such as salts are eluted much later (9). Desalting served a dual purpose for this assay: it removed the chelating agent and unbound GTP, and it also removed any inorganic phosphate that would contaminate the assay. Finally, after nucleotide loading and desalting, protein samples were ready for the GTPase assay.

As reported in the literature, the presence of GAP accelerates hydrolysis by several orders of magnitude (8). The experiments in this chapter verified the activity of both the GAP and Kras proteins and allowed us to screen the engineered GAP mutants. The goal of these experiments was to find an engineered GAP mutant that increased the hydrolysis rate of GTP bound to G12 mutant Kras.

4.2 Methods

4.2.1 Kras GTP Loading and Desalting

To accurately determine GTPase activity without relying on turnover, Kras had to be loaded with GTP. To accomplish nucleotide loading, GTP at a final concentration of 5 mM and EDTA (Fisher) at a final concentration of 10 mM were added to 3 mL of purified Kras protein at a concentration above 1 mg/mL (wild-type or mutant) and the sample was mixed gently for 3 minutes.

52

Figure 4-1: General mechanism of GTPase colorimetric assay. Phosphate released by the enzymatic reaction binds to an ammonium molybdate complex in acidic conditions. This complex binds to a malachite green dye, causing a color change (7).

53 Beforehand, PD-10 Columns (GE) containing 8.3 mL of Sephadex G-25 Medium were equilibrated with 15 mL of Kras buffer (described previously). After mixing, the loaded Kras protein was passed through the PD-10 column. After the protein sample passed through, the column continued to be washed with filtered Kras buffer. Elutions fractions were collected every 1 mL. The protein generally eluted after 4 or 5 mL. Immediately after desalting, protein fractions were moved to ice. To ensure that the PD-10 columns effectively removed imidazole and phosphate, the imidazole content of each fraction was measured by US/Vis spectrophotometry (230 nm), and the phosphate content was measured using the assay described below. Protein concentration was assessed using the Bradford reagent. To confirm the effectiveness of the GTP loading procedure, a 1H-15N HSQC spectrum of the eluent of 15N- labeled Kras was recorded. Knowing the percentage of Kras bound to GTP allowed for the interpretation of GTP hydrolysis data described below. Additionally, a set of 1H-15N HSQC spectra were collected every twenty minutes to monitor GTP hydrolysis.

4.2.2 Optimization of a Colorimetric GTPase Assay

A colorimetric GTPase assay, PiColorLock Gold (Innova Biosciences) was used to indirectly measure GTPase activity by phosphate release. The assay was optimized and scaled down to improve throughput and reduce costs. First, the PiColorLock Gold reagent from the assay kit was mixed with the Accelerator reagent in a 100:1 volume ratio and mixed. This reagent (subsequently called Gold Mix) was allowed to warm to room temperature before use. The Kras protein sample was normalized to 1 mg/mL by concentration or dilution with Kras buffer. To set up the reaction mixture, 100 µL of GTP-loaded Kras was mixed with 5 µL of either GAP protein or buffer control, at room temperature. The GAP was added at a 1:40 molar ratio. To start the reaction, 4 µL of 0.5 M MgCl2 (Tris buffered pH 7.8) was added to the mixture. After addition of magnesium and mixing, an 8 µL portion of the mixture was removed and added to 2 µL of the Gold Mix in a separate tube. This solution was mixed and allowed to sit for 5 minutes. Following the initial time point, additional 8 µL portions of the reaction mixture were removed and tested every minute for 5 minutes. In some cases, the spacing of time points was spread out over a long period of time. After the Gold Mix + protein sample mixtures sat for 5 minutes, 1 µL of the Stabilizer reagent from the kit was added. Then, after 10 minutes of incubation, the color change of the solutions was quantified using a NanoDrop 2000 spectrophotometer blanked with Kras buffer (λ= 635 nm) (Thermo Scientific). This procedure

54 was repeated for every protein combination to be tested for GTPase activity. For all sets of assay samples, OD635 was measured for buffer, protein, and MgCl2 solutions as controls.

A standard curve was created using 0.1 mM phosphate standard solution (Innova Biosciences) to determine the accuracy, reliability, and dynamic range of the assay. This assay was used to measure the catalyzed (by GAP) and uncatalyzed GTP hydrolysis of Kras for wild- type Kras and wild-type GAP, initially. For comparison, GAP was tested with G12A mutant Kras. To screen engineered GAP mutants, each mutant was tested with both wild-type and G12A mutant Kras. All data points were collected in triplicate. To account for the fact that different protein samples may have different amounts of background phosphate, the initial absorption reading was subtracted from all subsequent readings, so that the time course would begin at zero. By monitoring the change in absorbance, and not the absolute absorbance, variability in background contamination is less of a problem because the background will be the same for every time point for a sample. Data were also normalized to reflect a percentage of total GTP hydrolysis. Normalization was accomplished by using Kras protein samples at 0.5 mg/mL for the assay, which corresponds to 0.025 mM. The loading procedure described above was able to fully load Kras with GTP (as shown by NMR), so due to the 1:1 binding ratio, the GTP concentration was also 0.025 mM. The absorbance readings obtained from the phosphate assay were converted to phosphate concentrations using the linear regression equation obtained from the standard curve. These concentrations were used to determine what percentage of GTP was hydrolyzed.

4.3 Results

4.3.1 Kras GTP Loading and Desalting

Kras samples were loaded with GTP and desalted to remove unbound GTP, free phosphate, EDTA, and imidazole. Protein yield from the PD-10 columns was 80-90%, at 90% of the original sample concentration. Good separation of protein from small molecules was achieved. The elution traces for protein, phosphate, and imidazole are shown below (Figure 4- 2). To confirm GTP binding, a 1H-15N HSQC spectrum was recorded for an 15N-labeled sample immediately after desalting. The spectrum had only one peak for Y157, at the shift assigned to

55

3.5 ColorLock Gold (OD 635)

3 Bradford (OD 595nm) OD 230nm 2.5

2

1.5

1

0.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fraction number (1mL)

Figure 4-2: Elution traces from PD-10 Desalting Columns (GE). Protein elution determined by the Bradford assay, OD measured at 595 nm and shown in red. Phosphate elution determined by PiColorLock Gold (Innova Biosciences), OD measured at 635 nm and shown in blue. Imidazole elution determined desalting 300 mM imidazole Kras buffer, without protein, and measuring absorption at 230 nm, shown in green. The Y axis is relative absorbance. Protein was well separated from all small molecules.

56

Y157

Figure 4-3: 1H-15N HSQC of 15N-labeled Kras recorded after GTP. The spectrum recorded immediately after loading and desalting is shown in red, overlaid with the spectrum of Kras with GTP completely hydrolyzed (blue). The absence of a Y157 peak representative of GDP-bound Kras indicated that GTP loading was complete (peaks circled).

57

Y157 Y157 (GDP) (GTP)

Initial

40 min

1 hr 20 min

2 hr

2 hr 40 min

3 hr 20 min

4 hr

4 hr 40 min

Figure 4-4: Y157 peaks shown for sequential 1H-15N HSQC spectra. Spectra were recorded every twenty minutes; rows of this figure represent every other spectrum. Comparison of the two peaks indicated that GTP hydrolysis took place gradually over about 5 hours. The contour line threshold was normalized to the first spectrum.

58 GTP-bound Kras. The absence of a second peak for Y157 indicated that all Kras was bound to GTP. This spectrum is shown below, overlaid with the spectrum collected after all GTP hydrolysis was complete (Figure 4-3). Additionally, spectra were recorded every 20 minutes after the initial spectrum was collected. These data provided a time course of the GTPase reaction by comparing peak intensities and demonstrated that complete hydrolysis of GTP bound to Kras took about 5 hours.

4.3.2 Establishment of a Standard Curve

After creating various dilutions of 0.1 mM standard phosphate solution, phosphate concentrations were measured in triplicate according to the procedure described above. Figure 4- 5 shows the averaged triplicates with error bars. The R2 value of the linear regression is 0.9949. This linear trend of the data suggests that the assay is still accurate and reproducible when scaled down. Most of the data collected was well within the dynamic range of this technique, between 0 and 50 µM. The linear regression equation from this curve, y=0.0052x+0.0268, was used to determine phosphate concentration from absorbance data.

4.3.3 GTPase Activity of WT and G12A Kras with WT GAP

First, an activity curve was generated for C118S Kras (used as wild-type), with a comparison of phosphate release for Kras alone and Kras with GAP at a 40:1 molar ratio (Fig 4-

6). A mixture of MgCl2, GAP, and Kras buffer was monitored for the same time period to confirm that there was no phosphate generation without protein mediation. The data show that there was no unexplained phosphate production without the presence of Kras from GTP degradation over the course of the experiment. Kras alone slowly hydrolyzed of GTP, as expected. The addition of GAP dramatically increased the rate of phosphate production. In this experiment after 2 minutes, the sample with GAP produced 20 times more phosphate than the sample without GAP. At 2 minutes, amount of phosphate released by the sample with GAP reached a phosphate maximum, while the Kras sample continued to show a slow increase in phosphate concentration. Eventually, the amount of phosphate in the Kras sample reached the sample plateau that the sample of Kras+GAP did (data not shown). These data are in agreement with the information obtained by NMR: GAP causes almost instantaneous hydrolysis of GTP, in comparison to Kras alone (Figure 4-6).

59

0.7

0.6

0.5

0.4

0.3

Absorbance 635nm 0.2

y = 0.0052x + 0.0268 R² = 0.99492 0.1

0 0 20 40 60 80 100 120 Pi Concentration (μM)

Figure 4-5: Standard curve for the PiColorLock Gold phosphate assay, showing absorbance at 635 nm on a NanoDrop spectrophotometer. Error bars reflect one standard deviation in each direction.

60

Figure 4-6: GTPase activity of wild-type (C118S) Kras with and without GAP. For the “without GAP”, buffer was used as a replacement. Data are expressed as the percentage of GTP in the sample that has been hydrolyzed. This percentage was calculated by comparing the starting GTP concentration to the concentration of phosphate produced by the reaction. Time points occur every minute. The hydrolysis ability of Kras increases 20-fold when GAP is added at a 1:40 GAP:Kras molar ratio. Phosphate signal of the Kras+GAP sample plateaued after 2 minutes, suggesting that GTP was fully hydrolyzed to GDP. Error bars reflect one standard deviation in each direction.

61

Figure 4-7: GTPase activity of Kras G12A with and without GAP. Data are expressed as the percentage of GTP in the sample that has been hydrolyzed. Time points reflect data collected every minute. The Kras G12A protein alone showed similar activity to Kras G12A + GAP. Error bars reflect one standard deviation in each direction.

62 An identical procedure was carried out to study the hydrolysis rates of the Kras G12A mutant. Without GAP, the Kras G12A sample showed slightly more activity than C118S Kras, but the overall trends of phosphate production looked similar (Figure 4-7). Sources of error that could account for the difference include the error of the Bradford assay in determining protein concentration and the possibility of less than 100% GTP loading. When the Kras G12A was mixed with GAP, no increase in GTPase activity was observed. The GAP to Kras ratio was increased from 1:40 to 1:10, and there was still no increase in GTP hydrolysis (data not shown). This finding agrees with the model explaining the oncogenic activation of Kras: G12 mutant Kras is can intrinsically hydrolyze GTP slowly, but the reaction is not catalyzed by GAP. These data are summarized in Figure 4-8, which shows GTPase activity of each combination of proteins after 2 minutes. GAP was able to catalyze GTP hydrolysis by wild-type (C118S) Kras, but not by Kras G12A. In all experiments, the background level of phosphate remained constant, suggesting that any change in phosphate concentration could be attributed to GTP hydrolysis. Absorbance data were processed to reflect percentage of GTP hydrolysis. The starting GTP concentration was determined from the protein concentration in the assay, which is fully-loaded with GTP in a 1:1 ratio as demonstrated by NMR. Then, the phosphate concentrations calculated from the absorbance data were compared to the starting GTP concentration to determine percentage of hydrolysis because the hydrolysis of one mole of GTP corresponds to the release of one mole of phosphate.

4.3.4 GAP Mutant Screening for Restoration of Rapid GTP Hydrolysis

Each designed and over-expressed GAP mutant (listed in Table 3-1) was screened using the GTPase assay described above to determine whether the mutation affected on the ability of the GAP to facilitate GTP hydrolysis with wild-type and Kras G12A. First, the set of six mutants was screened against wild-type Kras to establish a baseline of how the mutations made affected the GTP hydrolysis by non-oncogenic Kras in comparison to wild-type GAP. Reactions with Kras alone and wild-type GAP + Kras were run side by side using the same protein preparation as controls. The controls behaved as expected, with the addition of GAP causing a dramatic increase in GTP hydrolysis. Figure 4-9 shows the amount of phosphate detected by the colorimetric assay after 3 minutes of reaction time. The bars are shown relative to the Kras+WT GAP sample, to provide a comparison to the wild-type system. The GAP R789A mutant protein had little activity, releasing a similar amount of phosphate to the sample of Kras without GAP.

63

100

90

80

70

60

50

Minutes 40

30

20 Percentage GTP Hydrolysis After 2 10

0 buffer+GAP C118S Kras C118S Kras+GAP G12A Kras G12A Kras+GAP

Figure 4-8: Hydrolysis of GTP bound to Kras after 2 minutes. Data are expressed as the percentage of GTP in the sample that has been hydrolyzed. WT Kras shows a 20-fold increase in activity upon addition of GAP, but Kras G12A shows no change. Buffer and GAP were used as a negative control.

64 The GAP R789A mutation removes the arginine residue that is necessary for catalysis of GTP hydrolysis (10), so it is not surprising that the data show that removing this residue causes GAP to have no catalytic activity. The data suggest that the changes made to the catalytic site of GAP affected the ability of GAP to catalyze GTP hydrolysis. The T786R mutant, which has two arginines in the catalytic site, retained most of the catalytic activity of the wild-type GAP (96%). However, all other mutants had significantly lower catalytic activity than wild-type GAP. The T785R mutant, also with two arginines in the catalytic site, retained 28% of the activity of the wild-type. Three GAP mutants were over-expressed in which the R789 was removed and replaced elsewhere. These three mutants all showed significantly decreased catalytic activity: 18%, 17%, and 6% relative to wild-type Kras and GAP, for GAP L787R R789A, GAP T786R R789A, and GAP T785R, R789A, respectively. Kras alone was shown to have 5 to 7% of the activity of the Kras + GAP mixture (Figure 4-9). A possible explanation for the decreases in catalytic activity is the unavailability of the arginine residue. In the mutants with little to no activity, the arginine residue may be pointing away from the nucleotide-binding pocket. For the double arginine mutants, the positively-charged side chains may be repelling each other. This repulsion could help or hinder the availability of an arginine for catalysis or have some unknown effect. Without structural information, we can only guess how the changes made to the catalytic site affect protein function.

Next the engineered GAP mutants were tested for catalytic ability on the Kras G12A mutant. These results are shown in Figure 4-10, shown as relative activity after 2 minutes with respect to the wild-type Kras/GAP system. The Kras G12A alone had activity similar to that of wild-type Kras, which is expected because the G12A mutation does not affect intrinsic hydrolysis, only the GAP-catalyzed reaction (3). When wild-type GAP was added, there was no increase in GTP hydrolysis. Similarly, the R789A GAP mutant, in which the catalytic arginine is removed as a negative control, had no catalytic activity. Of the five engineered GAP mutants tested, four caused no significant change in GTPase activity. However, the GAP T786R mutant caused a slight increase in hydrolysis compared to the wild-type GAP. This mutant resulted in 20% of the activity of the wild-type system, which is an increase over the sample of Kras G12A and wild-type GAP. These data suggest that this T786R mutation is able to restore some catalytic activity in comparison to wild-type GAP.

65

1.2

1

0.8

0.6

0.4 Activity Relative to WT GAP

0.2

0 WT Kras WT GAP GAP R789A GAP T786R GAP T785R GAP L787R, GAP T786R, GAP T785R, R789A R789A R789A

Figure 4-9: Activity of GAP mutants on wild-type Kras. After 3 minutes, phosphate levels of mixtures of C118S Kras and various GAPs were measured. GTPase activity is shown relative to that of the WT Kras/WT GAP system. The first bar shows the activity of wild-type Kras without GAP. One GAP mutant, T786R, caused similar GTP hydrolysis as the wild-type GAP. Other mutants had significantly decreased activity. The bars represent an average of three measurements, with error bars showing one standard deviation in each direction.

66

3

20 2.5

2 15

1.5 10 Relative Avtivity 1

5 0.5 Percentage of WT Kras + WT GAP Activity (%)

0 0 G12A Kras G12A Kras + GAP R789A GAP T786R GAP T785R GAP L787R, GAP T786R, GAP T785R, WT GAP R789A R789A R789A Figure 4-10: Activity of GAP mutants on GTP bound to Kras G12A relative to wild-type GAP on Kras G12A. The first bar shows intrinsic activity of Kras G12A, and the second bar shows activity of wild-type GAP with Kras G12A. The following bars show activity of various GAP mutants against GTP bound to Kras G12A. The data has been normalized to the activity of the Kras G12A + WT GAP sample for visualization of improvements upon this activity. The secondary axis on the right shows comparisons between the activities of GAP mutants on Kras G12A with the activity of wild-type GAP on wild-type Kras. The GAP T786R mutant showed slight ability to hydrolyze Kras G12A-bound GTP compared to wild-type GAP. The bars represent an average of three measurements, with error bars showing one standard deviation in each direction.

67 4.4 Conclusions

The data presented in this chapter provide a proof of concept for this project. Kras, wild- type and G12A mutant, was successfully loaded with GTP, as demonstrated by NMR. GTP loading was necessary due to the slow intrinsic exchange rate of GDP release. Also, adding GTP to the reaction mixtures caused high background phosphate levels. By ensuring that all Kras was GTP-bound, this study was able to monitor the initial cycle of GTP hydrolysis, without relying on enzymatic turnover, which made it simpler to see the effects caused by GAP. Confirmation of loading by NMR also allowed the concentration of GTP in the sample to be calculated using the protein concentration. To remove small molecules such as unbound GTP, imidazole, EDTA, and phosphate that could interfere with the assay, the protein samples were desalted after GTP loading. Desalting with PD-10 columns effectively separated protein from small molecules.

The PiColorLock Gold assay kit reproducibly measured phosphate concentrations from 1 µM to 100 µM as a scaled down reaction. The procedure was optimized so that only 2 µL of the color-forming reagent was necessary for each sample. The original procedure called for 50 µL of the reagent and for reading the absorbance of the resulting solution in a plate reader. Optimization lead to significantly decreased reagent usage, decreasing costs, and improving throughput. Samples were read on a NanoDrop spectrophotometer, which only requires 2 µL of sample for a reading.

The activity of the wild-type Kras/GAP system was measured. Due to the unavailability and high price of other methods of activity determination, the phosphate method was used. This method gives an indirect measure of GTP hydrolysis: GTP is hydrolyzed, which releases an equivalent of inorganic phosphate. Extraneous sources of phosphate were ruled out by desalting the samples and subtracting the background phosphate concentration from all subsequent readings. This method provides data in the form of a change in phosphate concentration over time. This assay could not be carried out in real time due to the highly acidic conditions of the color-forming reagent. So, small portions of a reaction mixture were removed and added to the reagent at regular time intervals. The first set of experiments showed that addition of GAP dramatically increased phosphate production. This finding agrees with the literature: GAP is able to catalyze the hydrolysis of GTP by Kras (3). The second set of experiments showed that GAP had no catalytic activity on G12A mutant Kras, due to steric interference between the

68 G12A mutation and the catalytic arginine of GAP (11). but the intrinsic activity of Kras G12A was unchanged.

The engineered GAP mutants described in earlier chapters were then tested against wild- type or Kras G12A for ability to catalyze GTP hydrolysis. The GAP R789A mutant did not catalyze hydrolysis, because the arginine is the catalytic residue in GAP which is responsible for stabilizing the transition state for GTP hydrolysis (11). Of the five GAP mutants screened for catalytic activity, one retained its activity on wild-type Kras and had a small amount of activity (20% of the activity of WT GAP) on the Kras G12A mutant. The other mutants tested had little activity on wild-type Kras, and even less activity on Kras G12A mutant. These differences are likely due to interactions between the arginine and other amino acids of both proteins, determined by the new location of the arginine residue. These data provide a proof of concept for this project: mutants can be designed and produced that have an effect on the ability of GAP to catalyze hydrolysis of GTP bound by either wild-type or mutant Kras. For different mutants, different amounts of activity were seen, suggesting that the mutations created have altered the chemistry at the active site of the Kras/GAP complex. Without detailed structural information, we cannot determine what interactions are causing the differences, but the work in this thesis shows that this experimental design shows promise for future work. Future studies will attempt to acquire crystallographic structure data to analyze how the chemistry of the active sites has changed.

4.5 References

1. Vo, U., Embrey, K. J., Breeze, A. L., & Golovanov, A. P. (2013). 1H, 13C and 15N resonance assignment for the human K-Ras at physiological pH. Biomolecular NMR Assignments, 7(2), 215–9. doi:10.1007/s12104-012-9413-y 2. Rojas, R. J., Kimple, R. J., Rossman, K. L., Siderovski, D. P., & Sondek, J. (2003). Established and emerging fluorescence-based assays for G-protein function: Ras- superfamily GTPases. Combinatorial Chemistry & High Throughput Screening, 6(4), 409–18.

69 3. Res, A. H. C., Biomol, A. J. J., Natl, D. M. P., Sci, A., John, J., Sohmen, R., … Goody, R. S. (1990). Kinetics of interaction of nucleotides with nucleotide-free H-ras. Cancer Research, (1979), 6058–6065. 4. Mazhab-Jafari, M. T., Marshall, C. B., Smith, M., Gasmi-Seabrook, G. M. C., Stambolic, V., Rottapel, R., … Ikura, M. (2009). Real-time NMR study of three small GTPases reveals that fluorescent 2’(3')-O-(N-Methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics. Journal of Biological Chemistry, 285(8), 5132–5136. doi:10.1074/jbc.C109.064766 5. Shutes, A., & Der, C. J. (2005). Real-time in vitro measurement of GTP hydrolysis. Methods, 37(2), 183–189. doi:10.1016/j.ymeth.2005.05.019 6. Feng, J., Chen, Y., Pu, J., Yang, X., Zhang, C., Zhu, S., … Liao, F. (2011). An improved malachite green assay of phosphate: mechanism and application. Analytical Biochemistry, 409(1), 144–9. doi:10.1016/j.ab.2010.10.025 7. Geladopoulos, T. P., Sotiroudis, T. G., & Evangelopoulos, A. E. (1991). A malachite green colorimetric assay for activity. Analytical Biochemistry, 192(1), 112–6. 8. Li, S., & Nakamura, S. (1997). Activation of R-Ras GTPase by GTPase-activating Proteins for Ras , Gap1 m , and p120GAP *. Journal of Biological Chemistry, 272(31), 19328–19332. 9. Hagnauer, G. L. (1982). Size exclusion chromatography. Analytical Chemistry, 54(5), 265–276. doi:10.1021/ac00242a025 10. Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmüller, L., Lautwein, A., Schmitz, F., & Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science (New York, N.Y.), 277(5324), 333–8.

70

Chapter 5:

Conclusions and Future Directions

71 5.1 General Remarks

Mutations in Kras have been shown to have high occurrences in many types of tumor- forming cancers. Specifically, nucleotide substitutions in the 12th codon of the gene causes over- activation of cell growth pathways (1). Clinically, cancers related to Kras mutations have proven more resistant to treatment, and result in worse prognosis for the patient (2). The Kras protein has been widely studied and characterized, and its mechanism of action is well known. Several studies are currently attempting to design small molecules or siRNAs to inhibit specific functions of the Kras signaling system to decrease cancer growth or improve response to treatment (3-5). These studies seek to inactivate Kras directly, in a non-specific manner. Attempts to target mutant Kras and inactivate the signaling pathway without permanently stopping all signaling have been largely unsuccessful (6).

5.2 Conclusions

5.2.1 Cloning and Mutagenesis of Kras and GAP

Chapter two detailed the cloning of genes to be studied in this project. Mutations were incorporated into the genes by overlap extension PCR. This study began working with wild-type full length Kras, then truncated the gene to the soluble G domain, and then incorporated a cysteine to serine mutation for improved solubility. Clinically relevant G12 mutations were incorporated into the Kras gene. The GAP gene was also subjected to mutagenesis by overlap extension PCR. Mutations were chosen that shifted the arginine of GAP down the catalytic loop one amino acid at a time, with alanine taking the original position of the arginine. Genes were cloned into pET vectors for transformation into E. coli cloning and expression hosts.

5.2.2 Expression and Purification of Kras and GAP

Chapter three describes the expression and purification of wild-type Kras and GAP, as well as several mutants of each gene. The genes were expressed in E. coli and purified using nickel affinity chromatography. High yield and purity was attained, as determined by the Bradford assay and SDS-PAGE analysis. 1H-15N HSQC NMR experiments were performed to determine the nucleotide binding state of Kras after purification. These experiments found that Kras could be bound to either GTP or GDP directly after purification, but due to the intrinsic

72 GTP hydrolysis ability of Kras, after a day the entire sample was bound to GDP. Additionally, when GAP was added to the Kras sample at a 1:20 molar ratio, the conformation immediately fully changed to the GDP bound state, demonstrating that Kras and GAP interact as expected.

5.2.3 Optimizing a Phosphate Assay to Monitor GTP Hydrolysis

A colorimetric phosphate assay from Innova Biosciences was successfully adapted to fit the needs of this study. Kras protein samples were loaded with GTP and desalted in preparation for the assay, which was used to measure the release of phosphate from GTP hydrolysis over time. GTP loading of Kras was confirmed by 1H-15N HSQC NMR. Kras exhibited a slow rate of GTP hydrolysis, which was increased 20-fold when GAP was added. Additionally, G12A mutant Kras was tested. The G12A mutant was demonstrated to be unresponsive to catalysis by GAP.

5.2.4 Screening Engineered GAP Mutants for Restoration of GTPase Catalysis

The GTPase assay described above was used to determine if the GAP mutants created were able to restore rapid GTP hydrolysis to Kras. The GAP mutants were screened against both wild-type and Kras G12A. The GAP R789A mutant was established as a negative control, due to loss of the catalytic arginine needed for activity. The majority of the mutants had little to no activity compared to the wild-type system. One mutant, T786R, retained 96% of WT GAP’s activity on WT Kras, and demonstrated a small amount of activity on G12A mutant Kras. This mutant added to Kras G12A demonstrated four times more GTPase activity than wild-type GAP, but still 5 times less than wild-type GAP with wild-type Kras. This data, presented in chapter four, provides a proof of concept that altering the location of the catalytic arginine of GAP has effects on GAP’s ability to catalyze GTP hydrolysis. Also, it was shown that through engineering of the active site, GAP can be made to have an effect on G12A mutant Kras.

5.3 Future Directions

The results outlined in this thesis have provided the basis for a much larger, more comprehensive study. The scope of this thesis was testing a set of five GAP mutants, along with relevant controls, for restoration of GTPase activity. These mutations were chosen through a combination of rational design, analyzing the protein structures, and guesswork. This study has provided a proof of concept: the changes made to GAP affect its ability to catalyze hydrolysis of

73 GTP bound to Kras. The protocols for screening mutants have been established and optimized, so in the future, a much larger and more comprehensive array of mutations can be made. One of the most important aspects of future work will be designing the GAP mutants to be cloned and expressed. Computational modeling software can be used to help determine what mutations might be worthwhile: by making the amino acid substitutions and running energy minimization programs, better decisions can be made on what mutations to make (7, 8). Programs such as Visual Molecular Dynamics (VMD) and Rosetta Design have been used to successfully redesign enzymes to have different specificities or activities, by identifying likely conformations of the amino acids in question based on the surrounding environment and sequence (9-12). The mutations made in this study simply moved the catalytic arginine around within the loop of GAP and replaced the original arginine with an alanine. Further engineering will involve altering the sequence of amino acids around the catalytic arginine to influence the structure of the loop and optimize the torsion angles of the side chains for better availability to the nucleotide binding pocket of Kras. Also, there can be multiple strategies for redesigning GAP. The first, used in this study, involves moving the catalytic arginine in hopes of avoiding steric interference between the arginine and the G12 mutation. A second strategy for design would be trying to move the problematic G12 mutated side chain out of the way of the catalytic arginine. For example, G12D is a clinically relevant oncogenic Kras mutation. The negatively charged side chain of aspartic acid may be able to be moved out of the way through salt bridge interactions with a positive side chain introduced somewhere else. This strategy would involve creating GAP mutants complementary to each specific G12 mutation.

The GAP mutants created in this study affected Kras-mediated GTP hydrolysis, but we can only speculate as to why or how. To help in the design of future mutants to screen, and to explain how the changes are affecting activity, structural information is needed. Another significant future direction for the study will be acquisition of crystal structures of various combinations of Kras and GAP. Crystal structures of wild-type Kras in complex with wild-type GAP have been published, as well as structures for G12 mutant Kras, but structures of mutant Kras in complex with GAP have yet to be solved (13). Also, structures of mutant or wild-type Kras in complex with the various GAP mutants created in this study will help us learn what changes the amino acid substitutions are causing. This information will give us more insight toward how to effectively redesign GAP and may also provide new information on the

74 mechanism of GAP catalysis of Kras-mediated GTP hydrolysis. In addition to structures, binding assays will be useful in determining whether the redesigned GAP proteins can still bind to Kras.

If more GAP mutants can be designed that exhibit mutant Kras inactivation by restoring GTP hydrolysis, these mutants can be tested for clinical potential. Mutants able to catalyze GTP hydrolysis in vitro may be tested using an in vivo system. One possible strategy for delivering engineered GAP mutants to cancer cells is through the use of an oncolytic virus. Several studies have shown that specially designed viruses will selectively replicate in cancer cells (14). These viruses can be used to deliver the DNA coding for the GAP mutants to cancer cells and cause expression of the genes (15). For example, adenoviruses have been engineered that preferentially replicates in tumor cells and is able to cause expression of transgenes (16). This strategy of gene therapy is one possibility for implementing the results of this work in a clinical setting.

Closing Remarks:

The Kras protein is currently being widely studied as a target for cancer treatment due to its high frequency of mutation in many types of cancers. Current attempts to deactivate signaling focus on targeting the Kras binding pocket or downstream effector binding sites with small molecules or siRNA (6). However, these methods affect all Kras proteins, mutant or wild-type, and can stop healthy Kras-mediated signals. This study presents a novel strategy of Kras deactivation, by engineering the catalytic site of GAP to be effective on mutant Kras. The advantage to this approach is that, in theory, there will be no additional effect on wild-type Kras, and the signaling of mutant Kras will be returned to normal levels. This study has established a low cost method for assaying GTP hydrolysis, and has created a GAP mutant that is able to restore a fraction of catalytic activity to Kras G12 mutant.

5.4 References

1. Fernández-Medarde, A., & Santos, E. (2011). Ras in cancer and developmental diseases. Genes & Cancer, 2(3), 344–58. doi:10.1177/1947601911411084

75 2. Jancík, S., Drábek, J., Radzioch, D., & Hajdúch, M. (2010). Clinical relevance of KRAS in human cancers. Journal of Biomedicine & Biotechnology, 2010, 150960. doi:10.1155/2010/150960 3. Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., & Shokat, K. M. (2013). K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature, 503(7477), 548–51. doi:10.1038/nature12796 4. Maurer, T., Garrenton, L. S., Oh, A., Pitts, K., Anderson, D. J., Skelton, N. J., … Fang, G. (2012). Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS- mediated nucleotide exchange activity. Proceedings of the National Academy of Sciences of the United States of America, 109(14), 5299–304. doi:10.1073/pnas.1116510109 5. Fleming, J. B., Shen, G.-L., Holloway, S. E., Davis, M., & Brekken, R. a. (2005). Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy. Molecular Cancer Research : MCR, 3(7), 413–23. doi:10.1158/1541-7786.MCR-04-0206 6. Adjei, A. A. (2001). Blocking oncogenic Ras signaling for cancer therapy. Journal of the National Cancer Institute, 93(14), 1062–74. 7. Toscano, M. D., Woycechowsky, K. J., & Hilvert, D. (2007). Minimalist active-site redesign: teaching old enzymes new tricks. Angewandte Chemie (International Ed. in English), 46(18), 3212–36. doi:10.1002/anie.200604205 8. Carter, P. J. (2011). Introduction to current and future protein therapeutics: a protein engineering perspective. Experimental Cell Research, 317(9), 1261–9. doi:10.1016/j.yexcr.2011.02.013 9. Kaufmann, K. W., Lemmon, G. H., Deluca, S. L., Sheehan, J. H., & Meiler, J. (2010). Practically useful: what the Rosetta protein modeling suite can do for you. Biochemistry, 49(14), 2987–98. doi:10.1021/bi902153g 10. Liu, Y., & Kuhlman, B. (2006). RosettaDesign server for protein design. Nucleic Acids Research, 34(Web Server issue), W235–8. doi:10.1093/nar/gkl163 11. Street, A. G., & Mayo, S. L. (1999). Computational protein design. Structure, 7(5), R105–R109. doi:10.1016/S0969-2126(99)80062-8 12. Lutz, S. (2010). Beyond directed evolution--semi-rational protein engineering and design. Current Opinion in Biotechnology, 21(6), 734–43. doi:10.1016/j.copbio.2010.08.011

76 13. Scheffzek, K., Ahmadian, M. R., & Wittinghofer, a. (1998). GTPase-activating proteins: helping hands to complement an active site. Trends in Biochemical Sciences, 23(7), 257– 62. 14. Hermiston, T. (2000). Gene delivery from replication-selective viruses: arming guided missiles in the war against cancer. The Journal of Clinical Investigation, 105(9), 1169– 72. doi:10.1172/JCI9973 15. Kirn, D., Martuza, R.L., Zwiebel, J. (2001). Replication-selective virotherapy for cancer: Biological principles, risk management and future directions. Nature Medicine. Jul2001, 7(7), 781-787. 16. Breitbach, C. J., Burke, J., Jonker, D., Stephenson, J., Haas, A. R., Chow, L. Q. M., … Kirn, D. H. (2011). Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature, 477(7362), 99–102. doi:10.1038/nature10358

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