The Arginine Finger of Rasgap Helps Gln-61 Align the Nucleophilic Water in GAP-Stimulated Hydrolysis of GTP

The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP

Haluk Resat†, T. P. Straatsma, David A. Dixon†, and John H. Miller

Paciﬁc Northwest National Laboratory, Richland, WA 99352

Edited by William N. Lipscomb, Harvard University, Cambridge, MA, and approved February 16, 2001 (received for review October 24, 2000) The Ras family of GTPases is a collection of molecular switches that sufficiently conclusive to decide whether Gln-61 is the base. link receptors on the plasma membrane to signaling pathways that However, glutamine is a very weak base and there is no polar regulate cell proliferation and differentiation. The accessory group in the vicinity that can significantly alter its pKa. There- GTPase-activating proteins (GAPs) negatively regulate the cell fore, this mechanism was argued to be highly unlikely (15, 16, signaling by increasing the slow intrinsic GTP to GDP hydrolysis 21). It has been suggested that Gln-61 is not the base but plays rate of Ras. Mutants of Ras are found in 25–30% of human tumors. a crucial structure-stabilizing role by being able to act as a The most dramatic property of these mutants is their insensitivity hydrogen bond donor and acceptor (18, 21). Because there is no to the negative regulatory action of GAPs. All known oncogenic other likely candidate, it was advocated that the GTP itself is the mutants of Ras map to a small subset of amino acids. Gln-61 is general base (18, 22). This hypothesis was supported by linear particularly important because virtually all mutations of this resi- free-energy relationships for mutant Ras proteins and by pKa due eliminate sensitivity to GAPs. Despite its obvious importance determinations using a combination of experimental approaches for carcinogenesis, the role of Gln-61 in the GAP-stimulated GTPase (18, 22). Later, Sondek et al. (23) used the x-ray structure of a activity of Ras has remained a mystery. Our molecular dynamics related protein, transducin-␣, complexed with GTP analog Ϫ simulations of the p21ras–p120GAP–GTP complex suggest that the GDP-AlF4 , to propose a variant of this mechanism in which local structure around the catalytic region can be different from Gln-61 participates in a proton shuttle from a water molecule in that revealed by the x-ray crystal structure. We ﬁnd that the the active site to the ␥-phosphate. carbonyl oxygen on the backbone of the arginine ﬁnger supplied In agreement with experimental results (24), recent computer in trans by p120GAP (Arg-789) interacts with a water molecule in simulations using the empirical valence bond method (25) the active site that is forming a bridge between the NH2 group of showed that part of the catalytic effect of RasGAPs derives from the Gln-61 and the ␥-phosphate of GTP. Thus, Arg-789 may play a direct interaction between an inserted arginine finger (Arg-789 dual role in generating the nucleophile as well as stabilizing the in the case of p120GAP) and the charge distribution of the transition state for POO bond cleavage. transition state for hydrolysis of GTP to GDP. The same

function is expected for arginine residues of G␣ proteins based BIOCHEMISTRY ow molecular weight GTP-binding proteins, like p21Ras, act on the similarity of their active-site structure (23) to that of the as molecular switches in cellular signaling pathways that complex between p120GAP and RasGTP (26). However, the L ␥ control cell proliferation and differentiation (for extensive re- arginine finger inserted by p120GAP approaches the -phos- views see refs. 1–7). In the GTP-bound on (active) state, Ras phate of GTP from the opposite direction of that observed for Arg-178 in the crystal structure of G␣ complexed with GTP interacts with effector molecules and transmits signals to the next Ϫ i1 downstream component. The hydrolysis of GTP to GDP analog GDP-AlF4 (23). This different orientation allows the switches Ras to the off (inactive) state. Guanine nucleotide backbone carbonyl of Arg-789 to interact with the NH2 group of exchange factors catalyze the dissociation of GDP from Ras and the Gln-61 side chain (26). We observed this configuration in the thus promote the loading of GTP to regenerate the active state. early part of our 1-ns molecular dynamics (MD) simulation of a The accessory GTPase-activating proteins (GAPs) negatively Ras–GTP–RasGAP complex started from the resolved part of regulate the GTP-bound state by increasing the slow intrinsic the crystal structure of p21ras bound by GDP-AlF3 and com- hydrolysis rate of Ras by factors of up to 105 (8–12). Oncogenic plexed with p120GAP (26). After about 350 ps of simulation, our mutants of Ras are found in 25–30% of human tumors (3, 13, 14). model underwent a structural rearrangement to a conformation Mutations of Gln-61 increase the activation barrier for Ras in which both the backbone carbonyl of Arg-789 and the side hydrolysis of GTP by 1.5–2 kcal͞mol, thereby decreasing the rate chain NH2 of Gln-61 are interacting with a crystallographic water molecule believed to be the precursor of the nucleophile by at least one order of magnitude depending on the mutant (15, ␥ 16). The most dramatic property of these mutants is their that attacks the -phosphate of GTP. We refer to this water insensitivity to the negative regulatory action of GAPs. Muta- molecule as the ‘‘nucleophilic’’ water. tions of Gln-61 have been found that reduce the rate of Methods GAP-stimulated hydrolysis by as much as 106-fold (17, 18), which corresponds to an increase in the activation barrier by 8.5 The starting structure used in the MD simulations was taken kcal͞mol. There is little doubt that the oncogenic properties of from the x-ray crystal structure (Protein Data Bank code: Gln-61 mutations are caused by their impact on the intrinsic and 1WQ1) of the p21ras bound by the GTP analog GDP-AlF3 and GAP-stimulated rates of GTP hydrolysis; however, the biochem- ical origins of these effects are still not entirely clear. This paper was submitted directly (Track II) to the PNAS ofﬁce. The proton-transfer step before nucleophilic attack on the ␥-phosphate of GTP is a critical point in understanding the Abbreviations: GAP, GTPase-activating protein; MD, molecular dynamics. intrinsic and GAP-stimulated GTPase activity of Ras. It was †To whom reprint requests should be addressed at: Paciﬁc Northwest National Laboratory, P.O. Box 999, Mailstop K1-83, Richland, WA 99352. E-mail: [email protected] or suggested initially that Gln-61 facilitates the nucleophilic attack ␥ [email protected]. on the terminal -phosphate by acting as the general base in the The publication costs of this article were defrayed in part by page charge payment. This hydrolysis (19, 20). Experiments measuring the intrinsic hydro- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. lysis functionality for Ras mutated at position 61 are not §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.091506998 PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 ͉ 6033–6038 Downloaded by guest on September 29, 2021 in complex with the p120GAP (GAP-334; ref. 26). Only the equilibration,¶ data collection was performed for 1 ns using a 2-fs Ras-binding domain of p120GAP (residues 718-1037) was in- time step. cluded in our simulations. Because of the lack of resolution, the x-ray structure was reported with residues 981–990 of p120GAP Results modeled as alanines and tyrosine (Tyr)-952 modeled as a Structural Rearrangement of the Active Site. With the exception of ʈ phenylalanine. The missing parts of these residues as well as the the C terminus of the p120GAP, the complex maintained its polar hydrogens were added to our starting structure by using the general shape throughout the simulation. After about 350 ps, the internal coordinate definitions of the CHARMM program package catalytic region underwent a structural rearrangement that significantly changed the hydrogen-bonding pattern in the active (27). To obtain a model with GTP bound to Ras, AlF3 was ␥ site. No further major structural rearrangement was observed converted to be the -phosphate PO3 terminal group. Protonation states of residues were determined by calculating during the production run. Detailed investigation of the structure around the catalytic site revealed only one water molecule pKa shifts with the algorithm developed by Antosiewitz et al. ‡ in the vicinity of the ␥-phosphate group of GTP. This water, (28). These pKa shift calculations indicated that all of the aspartic acid, glutamic acid, arginine, and lysine residues were W230 in x-ray crystal structure (26), did not exchange with the bulk waters during the simulation. The crystal structure (26) of charged as in a standard assignment. Cysteine residues that are the Ras–GTP–RasGAP complex suggests that hydrogen bonds not disulfide bonded and tyrosine residues were found to be allow W230 to form a bridge between the side-chain carbonyl of neutral. All three p21ras histidines were charged (doubly pro- Gln-61 and the ␥-phosphate of GTP. By our ‘‘not-too-strict’’ tonated) but only 3 of the 12 histidines (His-812, -986, and -1005) criteria,** this configuration was present during roughly the first of p120GAP were charged. Ambiguity regarding the pKa of a 350 ps of our simulation. Fig. 1 shows that this bond formed only reference compound makes the protonation state of GTP more occasionally and for short durations after the structural rear- uncertain than that of the amino acids. An experimental pKa Ϯ ␥ rangement of the active site. value of 2.9 0.1 has been measured for the -phosphate of GTP The hydrogen-bonding diagram reported by Scheffzek et al. in p21ras (18). Because the environment of phosphate oxygens (26) shows the NH2 group of Gln-61 forming a hydrogen bond in the p21ras-p120GAP-GTP complex is probably more posi- with the GTP ␥-phosphate. Even though multiple equivalent Ϫ ␥ tively charged than in p21ras alone, a formal charge of 4 for sites (three -phosphate oxygens and two hydrogens in the NH2 GTP is a reasonable first approximation. Based on this assump- group of Gln-61) favor this type of interaction, the main ten- tion, MD simulations and energy minimization were carried out dency that we observed for the NH2 of Gln-61 was an indirect to relax the starting structure before calculating pKa shifts (from interaction with GTP via a hydrogen-bond chain through the ␥ the reference compound) for the two -phosphate oxygens not nucleophilic water. The side-chain NH2 group of Gln-61 also 2ϩ involved in Mg coordination. The resulting pKa shift values of contacts the carboxylate of the nearby Glu-63. The crystal Ϫ3.4 and Ϫ2.8 were consistent with the assumption that the structure suggests that a hydrogen bond exists between the ␥ -phosphate in the p21ras–p120GAP–GTP complex is not backbone carbonyl of Arg-789 and the NH2 group of Gln-61. protonated. This bond was observed at the beginning of our simulation, but Simulations were performed with the NWCHEM computa- around 220 ps, Gln-61 moved away from Arg-789 and the tional chemistry package (30) by using the AMBER all-atom force hydrogen bond between Gln-61 and Arg-789 was later replaced field (31). Parameters for GTP were obtained by using an with a hydrogen bond between the Arg-789 backbone and the approach consistent with the AMBER force-field development.§ nucleophilic water as shown in Fig. 2. Missing hydrogen atoms were placed at standard positions. Ten W230 plays a key role in the local dynamics around the sodium ions were added to the unit cell to achieve the charge catalytic region by forming an extensive network of hydrogen Ϸ neutrality. The x-ray crystal structure contains 35 detected bonds. At 350 ps into the production run of our MD simula- waters molecules that were kept as part of our starting structure. The system was solvated further by placing the biomolecular ¶The system was equilibrated in several stages. First, before the system was solvated, the complex into a box of solvent molecules and deleting the waters hydrogen atom positions were energy minimized while the heavy atoms were ﬁxed. After that overlap with the solute molecules. This setup resulted in a solvating the system, the solute atoms were kept ﬁxed and solvent-molecule positions system of 7,856 solute atoms and 12,780 solvent molecules for a were optimized by energy minimization followed by a short MD simulation. To reduce the bias, the velocities were assigned randomly at regular intervals by using a Boltzmann total of 46,196 atoms. The Particle Mesh Ewald (PME) method distribution. Solute-proton and solvent-molecule positions were equilibrated further by was used to deal with the long-range electrostatic interactions. A running a 5-ps MD simulation and gradually increasing the temperature to 298 K by cutoff radius of 0.9 nm was imposed on nonbonded short-range randomly assigning the velocities at regular intervals. This equilibration step was followed by energy minimization of the whole system and equilibration MD simulations to relax the interactions between atoms. The SHAKE algorithm was used to structure. Once the structure was reasonably relaxed, the Particle Mesh Ewald option was constrain bond lengths involving hydrogen atoms at most stages turned on to deal with long-range electrostatics. Constant temperature and pressure of the equilibration and during the data-collection run. After equilibration MD simulations were run for 22 ps during which velocities were reassigned at regular intervals. The equilibration run was continued for another 190 ps without reassigning the velocities.

‡Following the approach detailed in ref. 28, a dielectric constant of 20 was assigned to the ʈThe 32 C-terminal residues lost their helical secondary structure during the production region occupied by the proteins, GTP, and the magnesium ion. Calculations were carried phase of the simulation. Omitting the unresolved parts of p120GAP probably caused an out by using the UHBD program (29). The dielectric constant of the surrounding solvent artificial unwinding of the C-terminal helix, which is a small domain on the side of the media was assumed to be 80. The united atom CHARMM force-field charges were used. The larger Ras-binding domain and far from the catalytic site. To confirm that unwinding of the probe radius was 1.4 Å, and dielectric boundary smoothing and successive focusing were C terminus did not have any important effect on the properties of the remainder of the used. The histidines were modeled as having protonatable groups at their ␧ nitrogen sites. system, we ran a short simulation with the C-terminal helix truncated. This second The pH was set to 7.0. The ␥-phosphate oxygens of GTP were not protonated. As discussed simulation with a shortened GAP component produced results similar to the first simula- in Methods, the protonation properties of the oxygens were investigated separately. tion, which assured us that the properties of the catalytic region were unaffected by the unwinding of the C terminus during our production run. §Ab initio electronic-structure calculations using the 6–31G* basis set were carried out for GTP at a geometry derived from the x-ray crystal structure of p21Ras bound to the slowly **In our analysis of hydrogen bonds, the criterion used to determine whether a bond hydrolyzing GTP analogue guanosine 5Ј-[␤,␥-imido]triphosphate (GppNp) (19). The elec- existed was that the distance between the heavy atoms be less than 3.2 Å and that the trostatic potential at the molecular surface was generated from the Hartree–Fock wave donor H-acceptor angle be at least 90°. Use of stricter hydrogen-bond criteria, R Ͻ 3.2 Å function. A restrained electrostatic surface potential (RESP) fit then was used to obtain the and a minimum angle of 145°, showed that the hydrogen bond between the Gln-61 side atomic charges. The short-range force-field parameters for GTP were assigned using chain CAO group and the nucleophilic water formed only occasionally, even during the analogy to published results for nucleic acids and proteins (31). early part of our MD simulation.

6034 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.091506998 Resat et al. Downloaded by guest on September 29, 2021 Fig. 1. The solid line represents the number of hydrogen bonds between the Gln-61 side-chain carbonyl and the nucleophilic water as a function of simulation time. The dashed lines represent the distance in nm between the Gln-61 side-chain carbonyl oxygen and each of the catalytic-water hydrogen atoms.

tion, W230 changed its location relative to the Gln-61 side chain, lost its hydrogen bond with the side-chain carbonyl of Gln-61,** and formed a more persistent hydrogen bond with the NH2 group of Gln-61. Before this transition, W230 interacted mainly with the side-chain carbonyl of Gln-61 and the GTP ␥-phosphate oxygens. A weaker and irregular interaction with the backbone of Thr-35 was present also. This pattern of interactions, shown schematically in Fig. 3A, resembles that reported in the analysis of the x-ray crystal structure (26). Simultaneous with switching between the Gln-61 side-chain groups, the nucleophilic water

also forms a hydrogen bond with the backbone carbonyl oxygen BIOCHEMISTRY of Arg-789. The pattern shown in Fig. 3B of water interacting with GTP, Gln-61 NH2, and the Arg-789 backbone carbonyl, in addition to less frequent interactions between Gln-61 NH2 and GTP, dominated the final 500 ps of our simulation. The structural change that occurs in our simulation around 350 ps can be viewed as the Gln-61 side chain swinging away from the active site and allowing the nucleophilic water to approach the arginine residue inserted by RasGAP. This interpretation is

Fig. 3. Schematic diagrams of interaction patterns during the ﬁrst 250 ps (A) and last 500 ps (B) of the MD simulation. Thick solid arrows represent the interactions that always exist. Interactions shown by thick broken arrows are strong and almost always exist. Interactions shown by thick dotted arrows are important, and the thin dashed arrows show weaker interactions that are infrequent.

consistent with the x-ray crystal structures (19, 21, 26) where residues 61–64 exhibit large B factors, suggesting high mobility and possible multiple side chain positioning. MD simulations of p21Ras (32–35) also have indicated that Gln-61 is very mobile, frequently forming and breaking hydrogen bonds with the nearby groups.

Structural Characterization by Using Cluster Analysis. To find the dominant hydrogen bond configurations, we have extended the Fig. 2. The solid line represents the number of hydrogen bonds between the analysis of the MD trajectory by clustering the bonding patterns. Arg-789 backbone carbonyl and the nucleophilic water. The dashed lines Seven key hydrogen bonds were identified in the catalytic region represent the distance in nm between the Arg-789 backbone carbonyl oxygen and were used to characterize the state of the system. These and each of the nucleophilic-water hydrogen atoms. seven bonds were between: (i) W230 H and Gln-61 CAO (side

Resat et al. PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 ͉ 6035 Downloaded by guest on September 29, 2021 Table 1. Hydrogen bond (HB) properties during the MD simulation HB no. 1 HB no. 2 HB no. 3 HB no. 4 HB no. 5 HB no. 6 HB no. 7

W230 H–Gln-61 Gln-61 Gln-61 W230 W230 Gln-61 Thr-35

Conﬁguration %* CAO NH2–W230 O NH2–GTP OG H–GTP OG H–Arg-789 CAO NH2–Arg-789 CAO NH–W230 O

During the ﬁrst 250 ps of the simulation 132YNNYN Y N 228YNYYN Y Y 316YNNYN N N 412YNNYN Y Y 58YNYYNYN During the last 500 ps of the simulation 156NYNYY N N 210YYNYN N N 37NYNYNNN 47NYYYYNN 57NNYYYNN 66NNNYYNN

Y and N respectively denote presence or absence of the hydrogen bond. *Shows the percentage of snapshots in which the conﬁguration was observed.

chain); (ii) Gln-61 NH2 and W230 O; (iii) Gln-61 NH2 and GTP water-mediated interaction. Configurations 2 and 3 are missing OG (␥-phosphate oxygens); (iv) W230 H and GTP OG; (v) W230 the W230–Arg-789 interaction whereas configurations 5 and 6 A H and Arg-789 C O (backbone); (vi) Gln-61 NH2 and Arg-789 are missing the W230–Gln-61 interaction. The dominance of CAO (backbone); and (vii) Thr-35 NH (backbone) and W230 O. configurations with direct interaction between Arg-789 and the In all cases, the key hydrogen bonds involve either Gln-61 or the nucleophilic water (83% total probability) shows that, in addi- crystallographic water, W230, which remained in the active site tion to charge neutralization and structural transition-state throughout our simulation. Because the structural reorganiza- stabilization, Arg-789 also plays a role in the initial proton tion took place around 350 ps, the MD results between 250 and transfer that generates the nucleophile. 500 ps were not used (to avoid statistical corruption). The analysis was performed separately for the first 250-ps and for the Discussion last 500-ps parts of the run. For each snapshot of the MD Based on an impressive body of experimental data (23, 24, 26, 36, trajectory, a bitmap was generated showing which of the seven 37), an ‘‘arginine finger’’ hypothesis has emerged to explain how key hydrogen bonds existed at that time. All of the equivalent RasGAPs increase the GTPase activity of p21ras by factors as ␥ sites (for example, three oxygens of GTP -phosphate or two large as 105. Recent theoretical work (25) has shed considerable water hydrogens), were included in deciding whether a particular light on the mechanisms by which an arginine residue in the hydrogen bond was present. For seven bonds, there are a total active site can influence the energetics of GTP hydrolysis. 7 ϭ of 2 128 possible bonding patterns. Patterns observed in more Charge neutralization of the active site groups is clearly one of †† than 5% of the conformations sampled from the MD trajectory the ways that Arg-789 participates in the catalytic process. are shown in Table 1. The five configurations that predominate during the first 250 ps of the simulation (Table 1) are slight variants of each other. Note that hydrogen bonds 1 and 4 are always present whereas 2 and 5 are never seen. Hydrogen bond 6 is always present except in configuration 3. In all cases, crystallographic water W230 is forming a bridge between the ␥-phosphate of GTP and the side-chain carbonyl of the Gln-61. Fig. 4 shows the spatial arrangement of the active site corresponding to hydrogen-bond pattern no. 1. Table 1 shows that the local structure of the catalytic site is dominated by a single configuration during the last 500 ps of our MD simulation. The dominant configuration, illustrated by Fig. 5, has the water molecule in the active site interacting with the ␥ GTP -phosphate group, the NH2 group of Gln-61, and the backbone of Arg-789. The strong interactions of the nucleophilic water with Arg-789 and Gln-61 orient it toward the ␥-phosphate so that this water molecule can be said to bridge all three groups. The other configurations in Table 1, which collectively occur with a probability of 37%, are mainly slight variations from the dominant configuration. Configuration 4 has a direct interaction ␥ between Gln-61 NH2 and the -phosphate, as well as with the

Fig. 4. Conformation of the active site corresponding to the most probable ††Conformations for H-bond analysis were saved every 10 ps during the first 250 ps of the hydrogen-bonding pattern during the first 250 ps of the MD simulation simulation and every 1 ps during the final 500 ps. (configuration 1 in Table 1).

6036 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.091506998 Resat et al. Downloaded by guest on September 29, 2021 relative to the ␥-phosphate (see for example figure 5C of ref. 26). However, these arginine residues point toward the GTP from different directions in the two systems. The orientation of the backbone of the arginine finger in the p21ras–p120GAP complex allows it to interact with the nucleophilic water, which is not ␣ possible for the orientation in G i1. Having the same residue participate in both the activation and bond-cleavage steps could increase the efficiency of the overall reaction, thereby making the hydrolysis rate by the Ras–RasGAP complex higher than that of the G␣ proteins. The role of Gln-61 in GAP-stimulated hydrolysis of GTP, suggested by the insensitivity of Gln-61 mutants, remains the greatest area of uncertainty in the catalytic mechanism. The proximity of Gln-61 to the ␥-phosphate in the p21ras-p120GAP complex raises the possibility that it could participate in stabilization of the transition state; however, recent work by Glennon et al. (25) appears to rule out stabilization by direct electrostatic interactions. Alternatively, Gln-61 might contribute significantly to stabilizing a conformation of Ras that is favorable for catalysis of the hydrolysis reaction by the inserted arginine finger (25). The configuration that we observe in the last 500 ps of our MD Fig. 5. Conformation of the active site in the dominant hydrogen-bonding simulation, where the nucleophilic water is linking both Gln-61 pattern during the last 500 ps of the MD simulation (conﬁguration 1 in and Arg-789 to the ␥-phosphate, might be important in this Table 1). regard. The prediction by Schweins et al. (22) that the ␥-phosphate of 31 GTP in p21ras has a pKa around 3 was confirmed by P-NMR ␥ During our MD simulation, one of the NH2 moieties of the experiments (18). Our pKa-shift calculations for the -phosphate Arg-789 guanidinium group formed strong hydrogen bonds with oxygens of GTP in p21ras alone and in the complex with ␥ ␣ the - and the -phosphate groups. This bridged configuration, p120GAP suggest that complex formation decreases the proton seen in Figs. 4 and 5, was extremely stable and stayed intact affinity of the ␥-phosphate oxygen because of the presence of throughout the production run. The interaction of the other NH2 additional positive charge in the active site. The configuration group of Arg-789 was less specific; it interacted with GTP that dominates the later part of our MD simulation might be ␣-phosphate group, with the backbone of Thr-785, and with the optimal for proton transfer when Arg-789 is inserted to stabilize Glu-31 carbonyl group. the bond-cleavage transition state. Gln-61 could be essential in Allosteric effects of complex formation are another important such a mechanism because its shape and electrostatic properties mechanism in the stimulation of GTPase activity by RasGAPs. complement Arg-789. Because Gln-61 is not charged, it would BIOCHEMISTRY Glennon et al. (25) showed that the conformation of Ras in the not destabilize the transition state by interacting with Arg-789 p21ras-p120GAP-GTP complex (26) was more effective in and GTP too strongly; however, Gln-61 has enough polarity and product stabilization than the conformation observed in x-ray the correct size to help orient the nucleophilic water. Clearly, structures of p21ras alone (19, 21). Our simulations show a additional work is needed to investigate these speculations about dramatic effect of complex formation on the configuration of the role of active-site conformations in the catalytic process. Gln-61 and the nucleophilic water in the active site. That After our study was submitted, Farrar et al. (38) reported that RasGAP binding moves Gln-61 closer to the ␥-phosphate of the association of p120GAP induces a conformational change GTP is clear from structural data. Sondek et al. (23) proposed near the metal ion of the active site. By using a combination of that this allosteric effect allowed Gln-61 to act as a proton shuttle electron spin-echo envelope modulation (ESEEM) experiments from the nucleophilic water to the ␥-phosphate. During the and short-time MD simulations (50 ps), they observed that the second half of our 1-ns MD simulation, crystallographic water residues, particularly the Gly-13 and -60, which are implicated in W230 resided very close to Arg-789 and interacted strongly with oncogenic mutants, in the active-site region can rearrange upon its backbone carbonyl. This configuration, which has not been complex formation with the GAP. Conclusions of the study by reported previously, suggests that Arg-789 plays direct role in Farrar et al. and of our study are complementary and support activating this water molecule for nucleophilic attack on the each other in the finding that association of p120GAP can induce ␥-phosphate. a conformation change in the active site that is necessary in the The possibility for the same residue to participate in formation GAP-dependent GTPase reaction. of the nucleophile as well as to stabilize the transition state for the subsequent bond cleavage may help explain why insertion of This research was performed at the W. R. Wiley Environmental Mo- an arginine finger enhances GTPase activity of Ras to such a lecular Sciences Laboratory, a national scientific user facility sponsored large extent. It is known that the GTP hydrolysis rate of closely by the U.S. Department of Energy Office of Biological and Environ- related G␣ proteins is higher than the intrinsic rate for p21ras but mental Research and located at Pacific Northwest National Laboratory, which is operated for the Department of Energy by Battelle. The lower than the RasGAP-stimulated rate (8–12). Structural com- ␣ computations were performed by using the supercomputing resources at parison of Arg-178 in G i1 and Arg-789 in the p21ras–p120GAP the National Energy Research Scientific Computer Center (NERSC) complex shows that the terminal groups on the side chains of and in the Molecular Sciences Computing Facility in the Environmental these arginine residues are located at almost the same position Molecular Sciences Laboratory.

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