Biol. Chem. 2017; 398(5-6): 637–651

Review Open Access

Ingrid R. Vetter* Interface analysis of small GTP binding complexes suggests preferred membrane orientations

DOI 10.1515/hsz-2016-0287 N-terminal (Arf family, ) or at the Received September 16, 2016; accepted December 12, 2016; C-terminal CAAX box (Ras, Rho, families, farnesylation ­previously published online December 21, 2016 or geranylgeranylation). Some G are additionally modified by palmitoyl groups at preceding the Abstract: Crystal structures of small GTP binding protein CAAX box (e.g. H-Ras, N-Ras, K-Ras4A, Rap2A-B), whereas complexes with their effectors and regulators reveal that in others the site is replaced by a polyba- one particularly flat side of the G domain that contains sic stretch (e.g. K-Ras4B, ) (Colicelli, 2004; Tsai et al., helix α4 and the C-terminal helix α5 is practically devoid 2015). As the prenyl groups are irreversibly attached, the of contacts. Although this observation seems trivial as membrane affinity conferred by those groups can be regu- the main binding targets are the switch I and II regions lated only by helper proteins that bind to and shield the opposite of this side, the fact that all interacting proteins, prenyl group from solvent, like the GDI-type proteins. In even the largest ones, seem to avoid occupying this area contrast, the palmitoyl groups are reversibly attached by (except for , that does not localize to membranes) is DHHC proteins at the and can be removed very striking. An orientation with this ‘flat’ side parallel by thioesterases. Candidate human thioesterases are APT1 to the membrane was proposed before and would allow and APT2 (Görmer et al., 2012). The closely related human simultaneous interaction of the lipidated C-terminus and protein LYPLAL1 has been shown to be unable to cleave positive charges in the α4 helix with the membrane while palmitoyl chains (Bürger et al., 2012). The involvement being bound to effector or regulator molecules. Further- of APTs in depalmitoylation is unclear, e.g. the ortholo- more, this ‘flat’ side might be involved in regulatory mech- gous protein to APT1/APT2 in toxoplasma can be knocked anisms: a Ras dimer that is found in different crystal forms out without major consequences (Kemp et al., 2013), and interacts exactly at this side. Additional interface analysis APT1/2 inhibition/knockdown did not show a significant of GTPase complexes nicely confirms the effect of different effect on N-Ras localization in NRAS mutant melanoma flexibilities of the GTP and GDP forms. Besides Ran pro- cells (Vujic et al., 2016), but inhibition of APTs (Rusch teins, guanine exchange factors (GEFs) bury et al., 2011; Zimmermann et al., 2013) indeed altered Ras the largest surface areas to provide the binding energy to localization (Dekker et al., 2010). Efforts to exploit the open up the switch regions for nucleotide exchange. palmitoylation machinery for therapeutic purposes are Keywords: buried interface; crystal structures; effector ongoing (Cox et al., 2015). Recently, other candidates complexes; G domain dimerization; GTPase; membrane for palmitoyl cleavage have been proposed, the ABDH17 interaction. family proteins (Lin and Conibear, 2015), and more might be found since the family has many uncharacterized members. Introduction From a structural viewpoint, the G domain (GTP binding domain) of the family of small GTP binding pro- Most small GTP binding proteins except Ran are post- teins is a versatile module that has evolved from an ances- translationally modified with prenyl groups at either an tral fold, the P-loop containing nucleoside triphosphate binding domain (Leipe et al., 2002; Ma et al., 2008). This fold is characterized by the presence of more or less flex- *Corresponding author: Ingrid R. Vetter, Max Planck Institute of Molecular Physiology, Department of Mechanistic Cell Biology, ible molecular ‘switch’ regions. According to the ‘loaded Otto-Hahn-Str. 11, D-44227 Dortmund, Germany, spring’ hypothesis (Vetter and Wittinghofer, 2001) the e-mail: [email protected] two switch regions are restrained in their dynamics by

©2017, Ingrid R. Vetter, published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. 638 I.R. Vetter: Interface analysis of GTP binding protein complexes the γ-phosphate of a GTP molecule, so that the entropic Post-translational modifications penalty for binding of effector molecules and GTPase acti- vating proteins (GAPs) especially to the switch I region is and other structural determinants decreased. Consequentially, GDP binding increases the of membrane orientation flexibility of the switches and attracts different interaction partners, the nucleotide exchange factors (GEFs) and the In the available crystal structures of small GTP binding guanine nucleotide dissociation inhibitors (GDIs), using proteins, the C-terminal hydrophobic modifications are different additional contact regions. rarely present as they render the proteins very poorly The functionality of the G domain has expanded soluble. Thus, prenylated usually crystallize during evolution by introduction of various insertions only in complex with GDI proteins that the (Wittinghofer and Vetter, 2011). The Rho/Rac/Cdc42 family hydrophobic group, like the RabGDI Ypt1 (1UKV; Rak has a small insertion of approx. 13 residues forming a et al., 2003), RhoGDIs [(1DOA; Hoffman et al., 2000), short α-helix between helix α4 and β-strand β4 that does 1HH4 (Grizot et al., 2001), 4F38 (Tnimov et al., 2012), not change the conformation upon nucleotide hydrolysis 5FR2; Kuhlmann et al., 2016)], PDE6δ (3T5G; Ismail et al., and is usually not directly involved in effector binding. 2011) or REP (Rab escort protein, 1VG0; Rak et al., 2004). Arf/Arl/Sar proteins undergo a large conformational The lipid modification site is separated from the G domain change of the G domain core where a register change of by a hypervariable region (HVR, 167-189, Figure 1B) that is two β-strands causes the N-terminus, usually an amphi- assumed to be flexible and is normally deleted for crys- philic, myristoylated helix, to detach from the protein core tallization, so that there are only a few crystal structures (therefore called ‘N-terminal switch’) so that it can inter- of full-length GTPases. For Ras proteins, one structure is act with membranes or effectors (Pasqualato et al., 2002; H-Ras-GDP 1-189 (4Q21; Milburn et al., 1990); the second Gillingham and Munro, 2007). Ran GTPases have an addi- one is K-Ras4B 1-189 (GDP, 4DSU, GSP, 4DSO; Maurer tional C-terminal helix that is destabilized if GTP is bound et al., 2012). In the H-Ras structure (4Q21), the C-terminal (called ‘C-terminal switch’; Vetter et al., 1999b). helix (α5) is visible up to Leu168, so this structure does Summaries of the structural features of G domain con- not contribute much additional information compared taining proteins can be found in the following references: to the truncated structures (usually comprising residues Vetter and Wittinghofer (1999), Paduch et al. (2001), Cher- 1-166). In the K-Ras structures (4DSU, 4DSO), the C-ter- fils and Zeghouf (2011), Wittinghofer and Vetter (2011) and minal helix continues up to residue Asp173, which is the Vetter (2014). In the following, structural characteristics of last residue with unambiguous electron density (Maurer complexes of small GTP binding proteins are highlighted, et al., 2012). The remaining residues 174–180 that contain with a focus on the implications for orientation at the the polybasic region, mostly , have only weak membrane. electron density, indicating their intrinsic flexibility, and

Membrane Membrane 47 161 161 C 128 135 172 180 165 135 128 165 169

N N 175

KKKKKK motif of K-Ras

AB

Figure 1: Proposed interaction of the three Ras proteins with the plasma membrane (orange). H-Ras in gray (5P21, GppNHp-bound), K-Ras in blue (4DSU, GDP-bound, 4DSO, GSP-bound, both look very similar), N-Ras in violet ­(GDP-bound, 3CON). (A) Positions carrying positive charges (128, 135, 161) are labeled and side chains shown as sticks, and residue 47 of the β-hairpin loop is indicated. (B) Orientation 110° rotated compared to (A). The hypervariable region (residues 167–189) forms an α-helix for residues 167–174 in the K-Ras structure (blue) and is probably flexible after residue 174, thus, the side chains of the polybasic region were modeled according to geometrical constraints. I.R. Vetter: Interface analysis of GTP binding protein complexes 639 are assumed to facilitate the interaction with the nega- position corresponding to Ras R161, but positions 128 and tively charged lipid head groups of membranes (Figure 135 are not conserved (at 128, e.g. R,K,E,Q,D, at position 1B) (Maurer et al., 2012). The farnesylated of the 135, e.g. R,K,D,Y). In the Rho family, positions correspond- CAAX box follows shortly after the positively charged ing to R161 and R135 in Ras are conserved as R/K, but posi- region at residue 185 (CVIM). Many Rho family GTPases tion 128 has no positive charge, but is often E,Q or D. In have a similar HVR compared to Ras (Vidal, 2010) in Arf proteins, position 135 is mostly , but the other terms of length and (positive) charges. Rab proteins two positions often have negative charges. A summary of tend to have a C-terminal­ helix that is longer than in the charge distributions in helices α4 and α5 can be found Ras protein structures [e.g. up to 13 residues in Rab11A in a recent review (Prakash and Gorfe, 2016). In addition, (3BFK, unpublished), Rab11B (2F9M; Scapin et al., 2006) Arf proteins, the major traffic regulators in the cell besides and Rab7 (1VG8; Rak et al., 2004)], and Rab proteins Ran and Rab proteins, have their lipid modification at the also have a longer linker between the helix end and the N-terminus instead of the C-terminus, and therefore will lipid modification site. For example, Rab11B (2F9M) has be described in some more detail in the following. 25 residues between the last in the structure Similar to Ran, the Arf-family GTPases feature an and the CC motif di-geranylation site, Rab7 (1VG8) has 18 additional switch mechanism that drastically remodels residues separating the helix end from the analogous CXC a part of the G domain. Where Ran has the C-terminal motif. This region is most likely flexible and thus is often helix attached to the G domain core in the GDP form deleted or not visible in the crystal structures. and detached in the GTP form, Arf proteins exhibit a In conclusion, the crystal structures indicate that the shift of one β-strand in the G domain core upon replace- lipid modification site at the very C-terminus of small GTP ment of GDP with GTP that pushes out the myristoylated binding proteins is attached via a linker region to the core ­N-terminus, the ‘interswitch toggle’ (Pasqualato et al., domain that can be either relatively short, stiff and highly 2002). The N-terminus often forms a short helix that, in charged (as in K-Ras) or relatively long, poorly conserved addition to myristoylation, is usually amphipathic (Huang and probably flexible (as in the Rab proteins), so that the et al., 2001; Vetter and Wittinghofer, 2001; Gillingham constraints on the specific membrane orientation of the and Munro, 2007; Donaldson and Jackson, 2011; Lin and G domain core will necessarily be different. Conibear, 2015). The linker following this helix, as well as In H-Ras, some basic residues in the core of the the C-terminus, often carries positive charges to facilitate G domain have been proposed to contribute to mem- membrane attachment (Liu et al., 2010). In some cases, brane association besides the lipid modification, namely the N-terminal helix can provide sufficient membrane R128, R135, and R161 (Abankwa et al., 2008). The posi- affinity even without myristoylation, e.g. in Sar1 and Arl6, tive charges at these positions are conserved in K-Ras where the lack of the lipid group is compensated by addi- and N-Ras: Position 161 is always , K-Ras has a tional hydrophobic residues at the N-terminus (Huang lysine at 128, and N-Ras has lysines both at positions 128 et al., 2001; Gillingham and Munro, 2007). The two attach- and 135. Indeed, if the C-terminal helix (α5) is oriented ment sites, i.e. the hydrophobic/amphiphilic N-terminus parallel to the membrane, the positive charges at those and the charged C-terminus, can restrain the orienta- three sites are optimally positioned for interaction with tion of Arf proteins at the membrane. Interestingly, NMR the membrane (Figure 1). This orientation has been pro- studies have led to a model where the C-terminal helix of posed as the favored one for the GTP form of H-Ras and Arf (that contributes to the plane of the ‘flat’ side) again N-Ras (Gorfe et al., 2007; Prakash and Gorfe, 2016), and lies almost parallel to the membrane (Liu et al., 2010). It this hypothesis was supported with more evidence later is thus tempting to assume that the N-terminus of Arf pro- (Kapoor et al., 2012; Šolman et al., 2015). Recently, it has teins takes over the role of the positively charged positions also been described as one of the dominant orientations 128 and 135 in Ras proteins as membrane anchor point. in K-Ras (Prakash et al., 2016), and possibly in other small In summary, the available structural information for GTPases, e.g. RheB, as highlighted in a recent review small GTP binding proteins contributes to explain the (Prakash and Gorfe, 2016). It is also notable that this side experimental evidence for the different modes of mem- of the G domain appears relatively flat, with the C-terminal brane binding. Besides the properties of the G domain helix defining one axis of this ‘plane’, and a line between itself, analysis of the overall shape of structures of effector the β-hairpin at residue 47 and residue 135 defining the complexes that often form in direct vicinity of the mem- other axis (Figure 1). brane might give some additional information about the In Ras-related GTPases, the three positive charges membrane orientation of those complexes, as described are not always conserved: Rab proteins have R/K at the in the following. 640 I.R. Vetter: Interface analysis of GTP binding protein complexes

Effector complexes of small GTP This orientation would allow simultaneous interactions of all and the C-terminal lipid modification with binding proteins – implications the membrane while still bound to the effector molecule. for membrane orientation and It also corresponds to one of the major two orientations observed for the oncogenic mutant G12D of K-Ras, called ­comparison of interaction surfaces ‘OS1’ in Prakash et al. (2016). There, it was already noted that the RafRBD effector domain would clash with the Effector proteins of small GTP binding proteins bind – membrane in a second predicted orientation (denoted per definition – to the GTP-bound form of the G domain. as ‘OS2’), in contrast to ‘OS1’. A third position features Superimposition of the available complex structures of helices α3 and α4 parallel to the membrane, as predicted membrane-interacting GTPases with their effectors reveal e.g. for K-Ras and Arf (Prakash and Gorfe, 2016). In this the interesting picture that one side of the G domain is orientation (Figure 2C), the main clashes with the mem- left completely free (Figure 2A). This could be attributed brane would be with the formin- and the LidA-complexes to the fact that the switch regions that indicate the pres- (the latter not shown in Figure 2C for clarity). Figure 2D ence or absence of the γ-phosphate are at the opposite depicts the same orientation, but with the formin com- side of the G domain, and the effector molecules natu- plexes omitted, now revealing potential membrane rally do cluster around the switch regions of the G domain clashes of the Arf6-effector JIP4 (2W83; Isabet et al., (Figure 2B). On the other hand, is still striking that even 2009), and the Rab6-effector R6IP1 (3CWZ; Recacha et al., the largest effector molecules would not clash with the 2009), that is recruited to Golgi membranes by active Rab6 putative membrane position, and that – probably not (Recacha et al., 2009). The coiled coil helix of JIP4 was coincidentally – this ‘flat’ side corresponds exactly to the proposed to be oriented parallel to the membrane (Isabet orientation predicted for the H-Ras GTP form (Figure 1). et al., 2009), which would also argue against membrane

ABMembrane

CD

Figure 2: Superimposition of effector complexes of small GTP binding proteins, except Ran complexes. The G domain is shown in green, the nucleotide in orange. (A) The G domain is oriented similar to Figure 1. In this position, effector molecules would not overlap significantly with the membrane. The position of the membrane next to the putative membrane-interacting side formed by helices α4, α5 and the β-hairpin of the G domain is indicated by the orange rectangle. (B) Enlargement of the G domain with switch I on the bottom (cyan ellipse) and switch II on the bottom right (orange ellipse) depicting the extensive contacts with the effector proteins. (C) Same set of complexes as in (A), but rotated so that helices α3 and α4 are next to the membrane. This would lead to clashes of the formin FH3 domains with the membrane (4YDH, 4YC7, 1Z2C, 3EG5, 4DVG). (D) Same orientation as in C, but with the formins omitted. Remaining clashes would be from Arf6-JIP4 (2W83, magenta helix) and Rab6-R6IP1 (3CWZ, orange domain) complexes. I.R. Vetter: Interface analysis of GTP binding protein complexes 641 interaction of the α3/α4 side in those complexes. In addi- function of the insert helix (Figure 3B) is still unclear. It tion, it is evident that the α4/α5/β-hairpin interface (the usually does not interact directly with effectors, with the ‘flat’ side) would bury a slightly larger interface with the exception of some minor contacts at the tip of the helix in membrane compared to the α3/α4 side, namely 2460Å some complexes (e.g. in Cdc42-FMNL2, 4YC7/4YDH; Kühn compared to 1847Å, i.e. an about 25% larger surface area et al., 2015), but the insert has been found to be essential, of the G domain, indicating a potentially more stable e.g. for activation of the Rho kinase (Zong et al., 2001). membrane association of the former. Interestingly, the insert helix lies almost parallel and Looking at the distribution of positive charges in very close to the membrane in the orientations shown in those complexes, the arginine/lysine clusters of the Figures 2 and 3, and also features some positive charges three positions corresponding to H-Ras R128, R135 and (Figure 3B). Indeed, it was speculated that the insert could R161 are clearly visible as an acute triangle (Figure 3B). also be important for fine-tuning the orientation of Rho As discussed by Prakash and Gorfe (2016), orientational proteins at the membrane (Vidal, 2010). Fittingly, effector restraints might also be relevant for G-domains of other complexes of Ran – that does not interact with ­membranes small GTP-binding protein families, and this idea is cor- during cargo transport through the nuclear pore – show roborated by several crystal structures as highlighted in the ‘flat’ side of the G domain completely blocked and the following. A distinctive basic cluster is notable in a buried by the huge helical solenoids of the transport pro- short helix close to the putative membrane that belongs teins (karyopherins) that cover almost the entire surface to the F2 lobe of the FERM domain of KRIT-1 in complex of the Ran protein. with Rap1B [(4DXA; Li et al., 2012), 4HDO/4HDQ (Gingras Analysis of the contact areas between the G domain et al., 2013)] with the motif ‘KKHK’ (Figure 3A). This motif and the effectors also reveals some characteristic differ- was postulated to be a NLS (Zawistowski et al., 2005), but ences between membrane-bound GTPases and Ran pro- in C2 domains, similar motifs are thought to confer affinity teins: Effector complexes of the former typically show to membranes (Thomas et al., 1999). Mutation of a lysine relatively small buried surface areas, corresponding to (K570) in the KKHK motif reduced the affinity to Rap1B the transient nature and moderate affinities of those as it is involved in a salt bridge to E45 of Rab1B (Gingras interactions (on average 785 Å2 for 74 structures contain- et al., 2013), but that would not exclude a possible addi- ing 139 sub-complexes (i.e. heterodimeric complexes), tional contribution to membrane interaction. The FERM analyzed with PISA, (Collaborative Computational domain has other interaction sites for the membrane as Project, Number 4, 1994). Values range from 422 Å2 (Rab6-­ well that would not be compatible with the orientation in golgin-GCC185 complex; Burguete et al., 2008) to 1860 Å2 Figure 3, so it would be interesting to obtain experimental (Rab27-SLAC2-A/melanophilin complex; Kukimoto-Niino support for this speculation, and, furthermore, to check if et al., 2008). In contrast, the karyopherins are snail-like similar motifs in other effector molecules might also facili- α-helical solenoids that wrap around the Ran G domain tate membrane attachment. In case of Rho proteins, the and bury up to 2503 Å2 in the interface (Crm1-RanQ69L

ABMembrane KKHK R128/R135 R161 H

C R135

R128

H

R161

Insert helix of Rho proteins

Figure 3: Superimposition of effector complexes of small GTP binding proteins, except Ran complexes, with positive residues highlighted as thin lines/blue color. The positions of the arginines R128/R135 and R161 and the C-terminus in Ras are indicated. ‘H’ is the β-hairpin loop at residue 47 (compare to Figure 1). (A) Side view with the putative position of the membrane in orange and the KKHK motif of KRIT-1 (4DXA, 4HDO, 4HDQ) high- lighted. (B) View as seen from the membrane side. 642 I.R. Vetter: Interface analysis of GTP binding protein complexes complex 3NC1; Güttler et al., 2010), leading to picomolar domains (RanBD), are designed to capture the (flexible) affinities. On average, 1997 Å2 are occluded in the inter- C-terminus of Ran-GTP and wrap it around themselves faces of Ran-effector-complexes (45 structures containing (Vetter et al., 1999b). Although the Ran binding domains 65 sub-complexes). In these complexes, nucleotide hydrol- are quite small with a molecular weight of approx. 16 kDa, ysis is almost completely abolished, which is essential for the ‘molecular embrace’ of Ran with a RanBD buries Ran function, as otherwise the karyopherin complexes almost as much surface as the large karyopherin com- would dissociate in the wrong cellular compartment or plexes (2350 Å2), along with having a very high affinity in during transit through the nuclear pore. Other effectors the low nanomolar range. Thus, the type of effector com- can have different effects on the GTP hydrolysis rate, they plexes formed by Ran is fundamentally different from the can either leave the rate unchanged or even accelerate it membrane-attached GTPases as Ran effectors cover much slightly (Vetter, 2014). In karyopherins, hydrolysis sup- larger surface areas of the G domain. pression is achieved by remodeling switch II so that the catalytic is in a hydrolysis incompetent posi- tion (Vetter et al., 1999a). Later it has been shown that the GAP (GTPase activating protein) and conserved Tyr39 in switch I of Ran proteins also contrib- utes to the slow hydrolysis in Ran effector complexes by GEF (guanine nucleotide exchange interfering with the optimal position of the catalytic water factor) complexes molecule (Rudack et al., 2015). In all Ran GTP forms, the (additional) C-terminal helix (α6) is detached from the Similar to the effectors (Figures 2 and 3), the super­ core G domain, and in case of the karyopherin complexes, imposition of the GAP complexes leaves the ‘flat’ side of it is usually disordered and not visible in the structures. In the G domain free (Figure 4A). Since many GAPs are mem- contrast, another type of Ran effectors, the Ran binding brane-bound or recruited to the membrane to stimulate

ABMembrane

CDMembrane

GEF /9 RhoGAP

Figure 4: Superimposition of GTPase-GAP and GTPase-GEF complexes. (A) GTPase-GAP complexes with the G domain in green, and RanGAP/Ran/RanBD1 in white/pink. (B) GTPase-GEF complexes with the G domain in green in in the same orientation as in (A). The protruding pink and cyan coiled coils belong to the Sec2p GEF of a Rab protein (2EQB, 2OCY). RCC1-Ran is yellow, overlaps with the ‘membrane’ (orange rectangle) are seen in the complexes with and DOCK8 that are depicted in (D). (C) RhoGAP complexes 1GRN, 1OW3, 1TX4, 2NGR, 3MSX,5C2J, 5HPY, 5IRC, 5JCP. (D) Cdc42-DOCK9 (2WMO, 2WMN, 2WM9), Rac1-DOCK9 (3B13, 2YIN), the minor overlaps with the ‘membrane’ could be resolved by a small rotation. I.R. Vetter: Interface analysis of GTP binding protein complexes 643

GTP hydrolysis and thus create the GDP-bound form of the the interface between Ran and RanGAP buries 1300 Å2, G domain, it would again seem logical to allow for simul- which is average for a GAP. The largest GAP-G domain taneous interaction of the G domain with the GAP and the interfaces are found in RasGAPs (1420 Å2, 1WQ1; Scheffzek membrane. The only exception is again the Ran-RapGAP- et al., 1997) and RapGAPs (Rap1·RapGAP 1094 Å2 3BRW; RanBD complex (Figure 4, gray and pink) where the linker Daumke et al., 2004; Scrima et al., 2008), with the record of the C-terminal helix of Ran and the N-terminus of RanBD holder Rap1·Plexin C1 with 1610–1734 Å2 (4M8N; Wang covers the ‘flat’ side of the G domain, but this complex is et al., 2013). Probably due to the fact that they do not have most likely not attached to membranes anyway. to unfold parts of the G domain, the GAP proteins tend to Analysis of the interfaces in GAP complexes reveals be slightly less ‘­engulfing’ than the GEFs described in the that ArfGAPs tend to have the smallest solvent excluded following. (GAPs bury on average 1205 Å2 in 36 structures areas of all GAPs, e.g. the ArfGAP/ankyrin domain of ASAP3 with 77 sub-complexes, GEFs 1407 Å2 in 69 structures with (3LVR/3LVQ; Ismail et al., 2010) buries only 562/715 Å2, 115 sub-complexes). corresponding to a relatively low affinity, and thus had to As expected from proteins that have to open the nucle- be crystallized as a fusion protein (Ismail et al., 2010). As otide for nucleotide exchange by binding to in most other GAP complexes, additional domains (like the often completely unfolded switch regions, GEF com- e.g. a PH domain that would facilitate membrane attach- plexes tend to bury the largest interface areas observed ment) are missing in the ArfGAP construct that could com- in small GTPase complexes, up to 2259 Å2 in the Cdc42- pensate for the low affinity by e.g. increasing the local DOCK9 complex (2WM9; Yang et al., 2009). In contrast to concentration of the GAP at the membrane. RhoGAPs all complexes described so far, superimposition of the GEF bury larger interfaces in their complexes, ranging from complexes (Figure 4B) reveals that some GEF domains 715 to 1184 Å2 (15 sub-complexes in 11 PDB structures), would clash with the membrane in the depicted orienta- similar to RabGAPs, which have related sequences and tion of the G domain, but not by a large amount. Inter- structures amongst each other (TBC domains), and bury estingly, they all belong to GEFs of the DOCK family, in 1036–1233 Å2 (6 sub-complexes in the two structures 4HLQ contrast to the DH-PH-domain containing GEF family. The and 2G77). Several RabGAPs have regions with positive largest clash is seen for the helical domain of the Cdc42- charges following their catalytic domains that are thought GEF DOCK9 (2WM9/2WMN/2WMO; Yang et al., 2009), to be involved in membrane localization (Kuhlmann et al., followed by the Rac1-DOCK9 complexes [(2YIN; Kulkarni 2016). et al., 2011), 3B13 (Hanawa-Suetsugu et al., 2012)], where Bacterial GAP mimics like the Legionella protein LepB DOCK9 is slightly rotated compared to the Cdc42 complex, are in the same range of interface areas [1098-1305Å2, and lastly the Cdc42 complex with the DHR-2 domain of 8 sub-complexes of 3 structures: 4JVS (Yu et al., 2013), 4I1O DOCK8 (3VHL; Harada et al., 2012). In all structures, (Gazdag et al., 2013), 4IRU (Mishra et al., 2013)] although the DHR-1 domain is missing that directly interacts with they are mechanistically very different: they stabilize the the membrane via its C2 domain fold, but the complete catalytic glutamine of the switch II region in analogy to DOCK1-Rac1 has been modeled (Premkumar et al., 2010). RanGAP, in contrast to the TBC GAPs that provide this In the model, the C-terminal­ helix of Rac is again almost glutamine in trans. Another bacterial GAP is the MglB parallel to the membrane, and the β-hairpin (Figure 1A, protein that regulates cell polarity in the bacterium Myxo- position 47) is close to the membrane. Thus, only a slight coccus xanthus (Miertzschke et al., 2011) together with the rotation is needed from the orientation in Figure 4 to avoid GTPase MglA. MglA forms a complex with a MglB homodi- the clash with the membrane. In summary, also the GAP mer (3T12; Miertzschke et al., 2011) and buries 1188 Å2, and GEF complexes would argue for the possibility of a which is in the lower range. As mentioned above, the preferred membrane interacting side of the G domain. RanGAP-Ran complex does not form at the membrane, but probably at the filaments of the mammalian nuclear pore where the RanBP2 protein is located. RanGAP has been crystallized in form of a ternary complex with Interactions with the GDP state Ran and a Ran binding domain of RanBP2 (1K5G/1K5D; Seewald et al., 2002). The Ran binding domains help to As expected from the more dynamic state of the switch sequester the Ran proteins that exit the pore in form of I and switch II regions in the GDP form, where they are karyopherin-complexes (Sarić et al., 2007) and subse- not restrained by the γ-phosphate of GTP, there are rela- quently facilitate GTP hydrolysis (Seewald et al., 2003). tively few proteins that bind to the GDP form of GTPases: In this complex, the Ran-RanBD1 buries 2730Å2 whereas in nuclear transport, NTF2 facilitates trafficking of the 644 I.R. Vetter: Interface analysis of GTP binding protein complexes

‘empty’ RanGDP through the FG-repeat meshwork of C-terminal region close to their main binding target, the the nuclear pore (Zahn et al., 2016), and the zinc finger prenyl moiety at the C-terminus. domains of Nup153 at the inner ‘basket’ of the nuclear RhoGDIs have a loop with positive charges (Figure 5B) pore might form a storage site for RanGDP waiting for its for membrane attachment, in addition to a negatively GEF RCC1 to exchange the nucleotide (3CH5; Schrader charged stretch at the N-terminus that is thought to bind et al., 2008). The buried areas are rather small, on to the C-terminal basic region of Rho/Cdc42/Rac proteins average 765 Å2 for NTF2 (two structures with six sub- close to the lipid modification site that provides addi- complexes), and 513 Å2 for the zinc fingers, respectively, tional membrane affinity for the complex (Tnimov et al., (seven structures with 11 sub-complexes) indicating a 2012). This would allow orienting the complex again with weak interaction consistent with the transient nature of the ‘flat’ side of the G-domain close to the membrane the sequestration at the nuclear basket for the zinc finger (Figure 5B). In contrast, the RabGDIs can not be posi- domains (Schrader et al., 2008). The interacting proteins tioned in a way that the mobile effector loop (residues are very different for membrane-localized G proteins: GDI 225–228; Ignatev et al., 2008) close to the prenyl binding proteins (GTP dissociation inhibitors) and the related site (Figure 5D, highlighted in red) and the ‘flat’ side of REP protein bind to the Rho and Rab families, and the Arf Rab can reach the membrane at the same time. But it effector Arfaptin binds to Rac1 in a GDI-like way both in would be feasible for the RabGDIs to bind to Rab if it were the GTP and GDP form (Tarricone et al., 2001). GDIs come in the position depicted in Figure 5D since there would be in three structural classes: RhoGDI, RabGDI and PDE6δ/ no clashes with the membrane, and subsequently extract UNC119 (Cherfils and Zeghouf, 2013). GDIs usually bind Rab by binding to the lipid anchor and the membrane as similarly to GDP and GTP forms of the G domain and postulated (Ignatev et al., 2008). therefore have to avoid contact to the switch I region Interestingly, some toxins and pathogens use a (Figure 5A and B). Instead, they concentrate their inter- similar mechanism to GDIs for binding to the GDP forms of actions to the vicinity of the switch II region and to the G proteins: SopB (4DID; Burkinshaw et al., 2012) and the

ABMembrane Membrane – + + N C

Switch I Switch II

CDMembrane Membrane

FIC domains Prenyl binding site (at the back side)

Figure 5: Superimposition of GTPase-complexes with GDIs and related proteins. (A) GDIs and GDI-like proteins including toxins and pathogens. (B) RhoGDIs (1CC0, 1DOA, 1DS6, 1HH4, 4F38) in yellow, cyan and magenta, with the G domain in green, switch I in blue and switch II in orange. The blue plus/minus signs indicate the charged regions close to the C- and N-termini of the GDI, the green plus sign represents the charges of the C-terminus of the G domain. (C) Fic domains of adenylylating proteins (3N3V, 4ITR). (D) RabGDIs (1UKV, 2BCG, 3CPH, 3CPJ). The prenyl binding site in the back is indicated in red. I.R. Vetter: Interface analysis of GTP binding protein complexes 645

C3-exoenzymes [2A9K/2A78; (Pautsch et al., 2005), 2BOV Interaction of effectors with the (Holbourn et al., 2005)] slow down nucleotide exchange by occupying similar surface regions as GDIs, which seems GDP state forced by high affinity natural since they have the same task of binding to the If the affinity to the effectors is sufficiently high, they can GDP form while coping with the relatively high flexibility bind the G domain even in the GDP-bound form. Exam- of the switch regions. SopB buries a slightly larger inter- ples are the Ran-karyopherin complexes (Villa Braslavsky face area than the GDIs, 1765 Å2 (4DID) compared to 1594 et al., 2000), e.g. RanA181C-Kap95P (3EA5; Forwood Å2 for RabGDI (1UKV; Rak et al., 2003). The Fic domains of et al., 2008) with a huge buried surface of up to 2182 adenylylating proteins found in pathogenic bacteria (e.g. Å2 that provides sufficient binding energy to force the IbpA, 3N3V; Xiao et al., 2010) are also attached to the more switch regions into the GTP conformation. Another inter- rigid parts of the G domain (Roy and Cherfils, 2015), but esting case are complexes of Ras with the Ras binding their α-helical domain extends to the switch I region to domain (RBD) of e.g. Raf kinase, where the RBD has access the that is adenylylated (Figure 5C). been mutated (A85K) for higher affinity (Ras-RBDA85K/ Examples for the third class of solubilizing proteins Rap1A E30DK31E, 3KUD/3KUC; Filchtinski et al., 2010). that bind to the C-terminal prenyl groups are PDE6δ/ The increased affinity enabled crystallization of the UNC119. An interaction solely via the prenyl group, i.e. H-Ras·GDP-RBD(A85K) complex that showed the switch I with only very few direct contacts between the G domain region forced into the GTP conformation by the RBD, but and PDE6δ, is structurally characterized in the RheB·GDP- coinciding with high temperature factors of the switch I PDE6δ complex (3T5G; Ismail et al., 2011). Accordingly, this region and the attached RBD, consistent with the higher interaction most likely imposes no steric restraints on the flexibility expected from switch I when the γ-phosphate relative orientations of the two domains. Arl proteins, that is missing (Filchtinski et al., 2010). This illustrates the are mostly not myristoylated themselves (Donaldson and delicate balance between effector affinity and flexibility/ Jackson, 2011), are involved in transport of hydrophobic dynamics of switch I. The dynamics of switch I can also prenylated cargo bound to e.g. the UNC119 domain (Ismail be influenced by small molecules like cyclen (Rosnizeck et al., 2012). In contrast to Arf proteins that attach to a et al., 2010), and some G domains have intrinsically membrane via their N-terminus that often forms an amphi- increased dynamics, e.g. Rac1b, caused by a destabiliz- phatic helix, Arl proteins do not detach the N-terminus in ing insert (Fiegen et al., 2004), so that this protein has a the GTP state, at least not completely. It was speculated drastically reduced affinity to the nucleotide and does not that this renders the Arl3-UNC119-cargo complex soluble even require a GEF protein. The last two cases of GTPase- until recruited to ciliar membranes by the specific mem- GDP complexes occur in Arl2, where the protein can be brane-bound GAP RP2 (Ismail et al., 2012), where cargo is purified in the GTP-bound form if co-expressed with its released by triggering GTP hydrolysis. A similar mechanism effector PDE6δ, but where the GTP is partially hydrolyzed of rendering prenylated cargo soluble is used by the PDE6δ in one of the crystal forms so that it contains 3 : 1 GDP/GTP domains that interact with high-affinity cargo like INPP5E as confirmed by HPLC (Hanzal-Bayer et al., 2002), and in which is released by Arl3 in cilia, perhaps aided by the scaf- the complex of Rab1 with the coiled-coil domain of LidA fold protein RPGR ( GTPase regulator) (3SFV; Cheng et al., 2012), an effector mimic, that is able to that can directly interact with PDE6δ (Wätzlich et al., 2013). bind both the GTP and GDP and renders Rab persistently In contrast, cargo that binds PDE6δ with low affinity can active (Cheng et al., 2012). be released by Arl2 everywhere in the cell (Fansa et al., 2016). Other domains, like the C2 domain of RPGRIP1, prob- ably compete with PDE6δ (Remans et al., 2014) for RPGR binding. Arfaptins behave like a GDI towards Rac1, i.e. they bind Rac in both nucleotide states and sequester Rac until it Analyzing the self-interaction of is displaced by the GTP-bound forms of Arf1 and Arf6 (Tarri- G domains cone et al., 2001), and thus might have a somewhat similar function to PDE6δ (Hanzal-Bayer et al., 2002). Dimerization of Ras proteins at the membrane has been pre- The only other types of GDP-containing complex struc- dicted by several groups now (e.g. Inouye et al., 2000; Gül- tures are either GEFs, or effectors (or mutants thereof) that denhaupt et al., 2012; Muratcioglu et al., 2015; Jang et al., have affinities that are sufficiently high to enable binding 2016). Besides e.g. molecular dynamics simulations, crystal to the GDP forms of some GTPases as described in the structures were also consulted to find putative dimeriza- following. tion interfaces, and a Ras dimer with an interface formed 646 I.R. Vetter: Interface analysis of GTP binding protein complexes by helices α4, α5 and the β-hairpin (i.e. the ‘flat’side) was slight differences in loops and side chain orientations in described in (Güldenhaupt et al., 2012) that occurs in four the wild type and mutant crystals in the different space different space groups and in the majority of the available groups. The largest area is 918Å2 in 2RGC (H-Ras Q61V- crystal structures (50 of 71 structures). It is important to note GppNHp; Buhrman et al., 2007), the smallest 772/766 Å2 that these dimers are mostly formed by crystallographic in ­1QRA(H-RasGTP)/2RGG(H-RasQ61I-GppNHp). N-Ras neighbors in the crystals, i.e. there are G domain monomers has very few amino acid substitutions in this dimer inter- in the asymmetric unit. An analysis of the buried interface face compared to H-Ras and K-Ras, only His131, Lys135 areas in the available crystal structures of solitary GTPases and Tyr166 are different to both H- and K-Ras. Although with PISA (Collaborative Computational Project, Number it should therefore be theoretically possible for N-Ras to 4, 1994) indeed reveals that the largest interface areas are form the same dimer, a different packing with a very small buried in this α4/α5/β-hairpin dimer, and that the same interaction area (335Å2) is found in the only available dimer actually occurs not only in seven different crystal crystal structure of N-Ras (3CON, unpublished). The very forms of H-Ras (2RGC; Buhrman et al., 2007), 2QUZ/4L9S different packing might have been caused by the in situ (Denayer et al., 2008; Ostrem et al., 2013), 3LO5 (Nassar partial with chymotrypsin during crystalliza- et al., 2010), 1QRA/2RGG (­Scheidig et al., 1999), 1AGP tion that left the switch II region either completely disor- (Franken et al., 1993), 1IAQ (Spoerner et al., 2001), 3KKM dered or even digested. (Shima et al., 2010), but also in one crystal form of K-Ras As described above, it would be interesting to know (4EPR; Sun et al., 2012) and one form of RhoE (1GWN; Gara- if full-length Ras proteins would be able to form the same vini et al., 2002). K-Ras 4EPR has the same crystal param- dimers as the truncated forms that are used for the over- eters as H-Ras 3KKM, with the one of the momomers very whelming majority of crystal structure determinations. slightly rotated compared to H-Ras (Figure 6). RhoE (1GWN) The closest to full length structures in the PDB are K-Ras shows a similar dimer, although rotated some more, in yet 4DSU and 4DSO (Maurer et al., 2012). Both GDP and GSP another crystal form. In Ran, the C-terminal helix and the forms crystallize as monomers and in the same crystal linker blocks the ‘flat’ side in the GDP form, but side chains form, with the C-terminal basic lysine stretch (residues 175– in the interface are so different that this dimer could not 180, Figure 1) mostly disordered and loosely packing into form anyway, not even in the GTP form. a neighboring, symmetry-related molecule, burying only Although the superimposed dimers are very similar 427 Å2. No interface in the crystals resembles the α4/α5/β- (Figure 6), the buried surface areas vary quite a bit due to hairpin dimer interface described above. One reason could

N

R161 C

C

R135

R128 N

Figure 6: Crystallographic H-Ras dimers of different crystal forms (1QRA yellow, 1IAQ cyan, 1AGP green, 4L9S violet, 2RGC orange) ­superimposed with the crystallographic K-Ras dimer 4EPR (magenta). Arginines R128, R135 and R161 of H-Ras are shown as sticks. I.R. Vetter: Interface analysis of GTP binding protein complexes 647 be a potential clash of the (extended) C-terminal helix: enthalpic binding energies. Large interaction surfaces are superimposing 4DSU on top of the 1QRA dimer would needed to provide sufficient affinity especially for the GEF cause Arg135 to clash with Glu168 of the second monomer. proteins that have to unfold the nucleotide-binding site to The C-terminal helix seems to be relatively rigid, the tem- facilitate nucleotide exchange, and for the Ran proteins to perature factors are only slightly increased, and only the achieve high affinity binding to karyopherins for secure polylysine tail is probably very flexible, so this could be transport through the nuclear pore. a possible explanation why the α­ 4/α5/β-hairpin dimer is A superimposition of all available complexes of not seen in the crystals. On the other hand, the clash is not GTPases revealed the quite surprising picture that the ‘flat’ severe, and might be circumvented by minor side chain side of the G domain formed by the C-terminal helix (α5) as adjustments or domain rotations. Another reason for the long axis and a second axis from R128/R135 in helix α4 of absence of the α4/α5/β-hairpin dimer in full-length K-Ras H-Ras to the β-hairpin at residue 47 does not participate in could be that the charged lysine tail causes this specific any interactions with effectors, GDIs, GAPs or GEFs, except packing as it might accidentally fit better into the neigh- in case of Ran. Ran proteins use their C-terminus not for boring molecule this way. membrane attachment, but for shielding the binding site In summary, the crystallographic α4/α5/β-hairpin for its effectors in the GDP-bound form. Helix α4 is the dimer that shows a direct face-to-face interaction of the least conserved part of small GTPases, it was found to be H-Ras/K-Ras/RhoE G domains might be physiologically the major membrane interacting element (together with relevant, although it probably has a very low affinity and the C-terminal part of helix α5) in K-Ras (Prakash et al., would need to be enhanced by other components, e.g. the 2015; 2016; Prakash and Gorfe, 2016), and it was specu- lipid modification. Indeed, an intact CAAX box was shown lated that helix α4 is an important determinant of specific- to be necessary for dimerization of K-Ras (Nan et al., 2015). ity for cellular localization (Abankwa et al., 2010). As this One could envision both a negative regulation of the inter- helix is practically never involved in direct contacts with action with membranes by the dimer blocking the ‘flat’ other proteins, it could achieve this function by influenc- side of the G domain, or, on the other hand, a dimerization ing the interaction with the membrane. could tilt the G domains away from the membrane while Analysis of potential dimerization interfaces of small staying anchored via the lipid modified C-terminus as pro- GTPases confirmed a possible interaction site exactly at posed, e.g. for the Ras GDP form (Gorfe et al., 2007) or for the ‘flat’ surface of the G domain that is never (except in K-Ras G12D in the GTP form (Prakash et al., 2016), and two Ran proteins) covered by interacting proteins. effector molecules could then bind at the same time. A Most publications, original work as well as reviews, dimerization of effectors is thought to be required for acti- contain only comparisons to a limited set of related struc- vation of, e.g. Raf-1 kinase (Freeman et al., 2013), and Ras tures, so it might be fruitful in the future to look more dimers were shown to form to activate MAPK (Nan et al., often at the larger picture of the structures of this extended 2015), so a direct dimerization via the ‘flat’ face could be a family of small GTP binding proteins. In this way, crystal possible mechanism. Other dimers have been postulated structures can help to understand even the membrane (Muratcioglu et al., 2015; Jang et al., 2016; Sayyed-Ahmad interactions of these most versatile and ancient signaling et al., 2016), but from the crystallographic point of view proteins, in spite of the impossibility to crystallize them at the interaction that buries the largest surface area is the a membrane. most likely one. A dimer via the effector interacting β2- strand has just been regarded as most likely not physi- Acknowledgments: The review summarizes the work that ologically relevant (Sayyed-Ahmad et al., 2016). was achieved within the SFB 642. The German Science Foundation (DFG) is kindly acknowledged for continued financial funding. I would also like to thank all colleagues Conclusions and future directions who helped with data collection, as well as the beamline staff of the synchrotrons ESRF, Grenoble, France, SLS, Vil- It is evident that the crystal structures of small GTP ligen, Switzerland and PETRA, Hamburg, Germany. binding proteins and their complexes have contributed tremendously to elucidate the detailed interactions with their effectors and regulators. 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