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.     

The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

Structural fingerprints of the Ras-GTPase activating proteins neurofibromin and p120GAP

Ras specific GTPase activating proteins (GAPs), neurofibromin and p120GAP, bind GTP bound Ras and efficiently complement its active site. Here we present comparative data from mutations and fluorescence-based assays of the catalytic domains of both RasGAPs and interpret them using the crystal structures. Three prominent regions in RasGAPs, the arginine-finger loop, the phenylalanine-leucine-arginine (FLR) region and alpha7/variable loop contain structural fingerprints governing the GAP function. The finger loop is crucial for the stabilization of the transition state of the GTPase reaction. This function is controlled by residues proximal to the catalytic arginine, which are strikingly different between the two RasGAPs. These residues specifically determine the orientation and therefore the positioning of the arginine finger in the Ras active site. The invariant FLR region, a hallmark for RasGAPs, indirectly contributes to GTPase stimulation by forming a scaffold, which stabilizes Ras switch regions. We show that a long hydrophobic side-chain in the FLR region is crucial for this function. The alpha7/variable loop uses several conserved residues including two lysine residues, which are involved in numerous interactions with the switch I region of Ras. This region determines the specificity of the Ras-RasGAP interaction.     

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.     

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.     

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.     

How the Pseudomonas aeruginosa ExoS toxin downregulates Rac

Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.     

Millisecond molecular dynamics simulations of KRas-dimer formation and interfaces

Ras dimers have been proposed as building blocks for initiating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cellular signaling pathway. To better examine the structure of possible dimer interfaces, the dynamics of Ras dimerization, and its potential signaling consequences, we performed molecular dynamics simulations totaling 1 ms of sampling, using an all-atom model of two full-length, farnesylated, guanosine triphosphate (GTP)-bound, wild-type KRas4b proteins diffusing on 29%POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)-mixed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes. Our simulations unveil an ensemble of thermodynamically weak KRas dimers spanning multiple conformations. The most stable conformations, having the largest interface areas, involve helix α₂ and a hypervariable region (HVR). Among the dimer conformations, we found that the HVR of each KRas has frequent interactions with various parts of the dimer, thus potentially mediating the dimerization. Some dimer configurations have one KRas G-domain elevated above the lipid bilayer surface by residing on top of the other G-domain, thus likely contributing to the recruitment of cytosolic Raf kinases in the context of a stably formed multi-protein complex. We identified a variant of the α₄-α₅ KRas-dimer interface that is similar to the interfaces obtained with fluorescence resonance energy transfer (FRET) data of HRas on lipid bilayers. Interestingly, we found two arginine fingers, R68 and R149, that directly interact with the beta-phosphate of the GTP bound in KRas, in a manner similar to what is observed in a crystal structure of GAP-HRas complex, which can facilitate the GTP hydrolysis via the arginine finger of GTPase-activating protein (GAP).     

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 crystal structure of rna1p: a new fold for a GTPase-activating protein.

rna1p is the Schizosaccharomyces pombe ortholog of the mammalian GTPase-activating protein (GAP) of Ran. Both proteins are essential for nuclear transport. Here, we report the crystal structure of rna1p at 2.66 A resolution. It contains 11 leucine-rich repeats that adopt the nonglobular shape of a crescent, bearing no resemblance to RhoGAP or RasGAP. The invariant residues of RanGAP form a contiguous surface, strongly indicating the Ran-binding interface. Alanine mutations identify Arg-74 as a critical residue for GTP hydrolysis. In contrast to RasGAP and RhoGAP, Arg-74 could be substituted by lysine and contributed significantly to the binding of Ran. Therefore, we suggest a GAP mechanism for rna1p, which constitutes a variation of the arginine finger mechanism found for Ras GAP and RhoGAP.     

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.     

Understanding the catalytic mechanism of GTPase-activating proteins: demonstration of the importance of switch domain stabilization in the stimulation of GTP hydrolysis

Cdc42, a member of the Rho family of GTP-binding proteins, has been implicated in a variety of biological activities, including the organization of the actin cytoskeleton, changes in cell morphology and motility, intracellular trafficking, cell cycle progression, and cellular transformation. The cycling of Cdc42 between its on (GTP-bound) and off (GDP-bound) states is essential for its stimulation of cell growth and transformation, with an important aspect of this cycle being the regulation of the GTP hydrolytic activity of Cdc42 by its GTPase-activating protein (Cdc42GAP). On the basis of the structural determinations of the Cdc42-Cdc42GAP complex, as well as the Ras-RasGAP complex, it has been proposed that an arginine residue provided by the GAP (called the "arginine finger") stabilizes charges developing on the guanine nucleotide during the transition state for GTP hydrolysis and is an important contributor to GAP-stimulated catalysis. However, the 85 kDa regulatory subunit (p85) of the phosphoinositide 3-kinase (PI-3K) is homologous with the Cdc42GAP and contains the essential arginine residue, but is ineffective as a GAP. This argues that the introduction of the arginine finger is insufficient for GAP activity and that the GAP must fulfill an additional function, one possibility being the engagement and stabilization of the conformationally sensitive switch regions of Cdc42. In the study presented here, we have tested this idea by examining three residues within the Cdc42GAP, which are missing in the GAP homology domain of the 85 kDa regulatory subunit (p85) of the PI 3-kinase and are involved in specific interactions with switch domain residues of Cdc42. We show that the mutation of all three residues, as well as individual mutations of each of these residues, yields GAPs that are defective in stimulating GTP hydrolysis. We further demonstrate that the switch I residue tyrosine 32 plays an important role in GAP interactions and in the regulation of both intrinsic and GAP-stimulated GTP hydrolysis. Taken together, these findings indicate that stabilizing the switch domains of GTP-binding proteins is an important part of GAP-stimulated catalysis, and that the inability of p85 to participate in these interactions may at least in part explain its ineffectiveness as a GAP.     

Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf

Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.     

Why does mutation of Gln61 in Ras by the nitro analog NGln maintain activity of Ras‐GAP in hydrolysis of guanosine triphosphate?

Interpretation of the experiments showing that the Ras‐GAP protein complex maintains activity in guanosine triphosphate (GTP) hydrolysis upon replacement of Glu61 in Ras with its unnatural nitro analog, NGln, is an important issue for understanding details of chemical transformations at the enzyme active site. By using molecular modeling we demonstrate that both glutamine and its nitro analog in the aci‐nitro form participate in the reaction of GTP hydrolysis at the stages of proton transfer and formation of inorganic phosphate. The computed structures and the energy profiles for the complete pathway from the enzyme‐substrate to enzyme‐product complexes for the wild‐type and mutated Ras suggest that the reaction mechanism is not affected by this mutation. Proteins 2015; 83:2091–2099. © 2015 Wiley Periodicals, Inc.     

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.     

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.     

QM/MM modeling the Ras-GAP catalyzed hydrolysis of guanosine triphosphate

The mechanism of the hydrolysis reaction of guanosine triphosphate (GTP) by the protein complex Ras-GAP (p21(ras) - p120(GAP)) has been modeled by the quantum mechanical-molecular mechanical (QM/MM) and ab initio quantum calculations. Initial geometry configurations have been prompted by atomic coordinates of a structural analog (PDBID:1WQ1). It is shown that the minimum energy reaction path is consistent with an assumption of two-step chemical transformations. At the first stage, a unified motion of Arg789 of GAP, Gln61, Thr35 of Ras, and the lytic water molecule results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low-barrier transition state TS1. At the second stage, Gln61 abstracts and releases protons within the subsystem including Gln61, the lytic water molecule and the gamma-phosphate group of GTP through the corresponding transition state TS2. Direct quantum calculations show that, in this particular environment, the reaction GTP + H(2)O --> GDP + H(2)PO(4) (-) can proceed with reasonable activation barriers of less than 15 kcal/mol at every stage. This conclusion leads to a better understanding of the anticatalytic effect of cancer-causing mutations of Ras, which has been debated in recent years.     

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins

We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.     

The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP.

Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).     

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.