Unravelling the mechanism of dual‐specificity GAPs

The molecular mechanism by which dual‐specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G‐proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady‐state kinetic assays and time‐resolved Fourier‐transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1^IP4BP and RASAL is mediated by the extra GTPase‐activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch‐II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re‐orientation requires specific interactions between switch‐II of Rap and helix‐α6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.     

The arginine finger loop of yeast and human GAP is a determinant for the specificity toward Ras GTPase.

In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21. It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase. Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase. The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop. In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase. Analysis of these results and modeling of Ira2p.Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms.     

The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues

The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb.     

The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.     

Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction

Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 10(5)-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we have investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general we find a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.     

The Legionella pneumophila GTPase Activating Protein LepB Accelerates Rab1 Deactivation by a Non-canonical Hydrolytic Mechanism

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.     

Structural changes induced in p21Ras upon GAP-334 complexation as probed by ESEEM spectroscopy and molecular-dynamics simulation

BACKGROUND: The means by which the protein GAP accelerates GTP hydrolysis, and thereby downregulates growth signaling by p21Ras, is of considerable interest, particularly inasmuch as p21 mutants are implicated in a number of human cancers. A GAP "arginine finger," identified by X-ray crystallography, has been suggested as playing the principal role in the GTP hydrolysis. Mutagenesis studies, however, have shown that the arginine can only partially account for the 10(5)-fold increase in the GAP-accelerated GTPase rate of p21. RESULTS: We report electron spin-echo envelope modulation (ESEEM) studies of GAP-334 complexed with GMPPNP bound p21 in frozen solution, together with molecular-dynamics simulations. Our results indicate that, in solution, the association of GAP-334 with GTP bound p21 induces a conformational change near the metal ion active site of p21. This change significantly reduces the distances from the amide groups of p21 glycine residues 60 and 13 to the divalent metal ion. CONCLUSIONS: The movement of glycine residues 60 and 13 upon the binding of GAP-334 in solution provides a physical basis to interpret prior mutagenesis studies, which indicated that Gly-60 and Gly-13 of p21 play important roles in the GAP-dependent GTPase reaction. Gly-60 and Gly-13 may play direct catalytic roles and stabilize the attacking water molecule and beta,gamma-bridging oxygen, respectively, in p21. The amide proton of Gly-60 could also play an indirect role in catalysis by supplying a crucial hydrogen bonding interaction that stabilizes loop L4 and therefore the position of other important catalytic residues.     

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.     

RasGAPs: a crucial regulator of extracellular stimuli for homeostasis of cellular functions

Ras and its GTPase activating proteins (GAPs) are among the crucial regulators of extracelluar ligands. Information about these regulators has been elucidated during the course of studies in signal transduction over the last two decades. RasGAPs such as p120GAP and neurofibromin have been studied extensively for their roles as either "negative" regulators or effectors of Ras. Accumulating evidence suggests that these molecules are crucial regulators of extracellular stimuli that serve to maintain the homeostasis of cellular functions. This compendium highlights cellular functions of RasGAPs and their signaling characteristics from the viewpoint of homeostasis, including our recent finding of the phenotype of R-RasGAP mutant mice whose GAP activity is down-regulated.     

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.     

Rap-specific GTPase activating protein follows an alternative mechanism.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.     

The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.     

Structural analysis of the GAP-related domain from neurofibromin and its implications.

Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most frequent genetic disorders in man. It acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which despite very limited sequence homology is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed which is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions.     

DRoP: A Water Analysis Program Identifies Ras-GTP-Specific Pathway of Communication between Membrane-Interacting Regions and the Active Site

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.     

Computational characterization of the chemical step in the GTP hydrolysis by Ras‐GAP for the wild‐type and G13V mutated Ras

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H₂O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053. © 2015 Wiley Periodicals, Inc.     

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.     

Mechanisms of isoform-specific residue influence on GTP-bound HRas,KRas, and NRas

HRas, KRas, and NRas are GTPases with a common set of effectors that control many cell-signaling pathways, including proliferation through Raf kinase. Their G-domains are nearly identical in sequence, with a few isoform-specific residues that have an effect on dynamics and biochemical properties. Here, we use accelerated molecular dynamics (aMD) simulations consistent with solution x-ray scattering experiments to elucidate mechanisms through which isoform-specific residues associated with each Ras isoform affects functionally important regions connected to the active site. HRas-specific residues cluster in loop 8 to stabilize the nucleotide-binding pocket, while NRas-specific residues on helix 3 directly affect the conformations of switch I and switch II. KRas, the most globally flexible of the isoforms, shows greatest fluctuations in the switch regions enhanced by a KRas-specific residue in loop 7 and a highly dynamic loop 8 region. The analysis of isoform-specific residue effects on Ras proteins is supported by NMR experiments and is consistent with previously published biochemical data.     

GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins

GAP1(IP4BP) is a member of the GAP1 family of Ras GTPase-activating proteins (Ras GAPs) that includes GAP1(m), CAPRI, and RASAL. Composed of a central Ras GAP domain, surrounded by amino-terminal C(2) domains and a carboxyl-terminal pleckstrin homology/Bruton's tyrosine kinase domain, GAP1(IP4BP) has previously been shown to possess an unexpected GAP activity on the Ras-related protein Rap, besides the predicted Ras GAP activity (Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527-530). Here we have shown that GAP1(IP4BP) is indeed an efficient Ras/Rap GAP, having K(m)s of 213 and 42 microm and estimated k(cat)s of 48 and 16 s(-1) for Ras and Rap, respectively. For this dual activity, regions outside the Ras GAP domain are required, as the isolated domain (residues 291-569) retains a pronounced Ras GAP activity yet has very low activity toward Rap. Interestingly, mutagenesis of the Ras GAP arginine finger, and surrounding residues important in Ras binding, inhibit both Ras and Rap GAP activity of GAP1(IP4BP). Although the precise details by which GAP1(IP4BP) can function as a Rap GAP remain to be determined, these data are consistent with Rap associating with GAP1(IP4BP) through the Ras-binding site within the Ras GAP domain. Finally, we have established that such dual Ras/Rap GAP activity is not restricted to GAP1(IP4BP). Although GAP1(m) appears to constitute a specific Ras GAP, CAPRI and RASAL display dual activity. For CAPRI, its Rap GAP activity is modulated upon its Ca(2+)-induced association with the plasma membrane.     

Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange

Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.     

Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis

The crystal structure of the complex of ARF1 GTPase bound to GDP and the catalytic domain of ARF GTPase-activating protein (ARFGAP) has been determined at 1.95 A resolution. The ARFGAP molecule binds to switch 2 and helix alpha3 to orient ARF1 residues for catalysis, but it supplies neither arginine nor other amino acid side chains to the GTPase active site. In the complex, the effector-binding region appears to be unobstructed, suggesting that ARFGAP could stimulate GTP hydrolysis while ARF1 maintains an interaction with its effector, the coatomer complex of COPI-coated vesicles. Biochemical experiments show that coatomer directly participates in the GTPase reaction, accelerating GTP hydrolysis a further 1000-fold in an ARFGAP-dependent manner. Thus, a tripartite complex controls the GTP hydrolysis reaction triggering disassembly of COPI vesicle coats.