Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.

The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.     

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins

BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.     

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.     

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 structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

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.     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.     

Studies on the structure and mechanism of H-ras p21.

Current knowledge of the structure of H-ras p21 is reviewed with particular emphasis on the interaction between guanine nucleotides and the active site of the protein. The nature of the conformational change induced by GTP hydrolysis is discussed. The major change is seen in the region known as the effector loop (loop 2), with significant but less well-defined changes occurring in loop 4, which is implicated in the GTPase reaction. Other evidence concerning the mechanism of GTP hydrolysis and its activation by GAP (GTPase-activating protein) is also discussed. Evidence regarding the rate limiting step in the p21 GTPase reaction, and the manner in which this and possibly other steps are accelerated by GAP, is inconclusive.     

Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules

The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12----Arg, Gly-12----Val, Gln-61----His, Gln-61----Leu, which are all oncogenic, and the effector region mutant Asp-38----Glu. The resolutions of the crystal structures range from 2.0 to 2.6 A. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the gamma-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.     

Comparison of the computed structures for the phosphate-binding loop of the p21 protein containing the oncogenic site Gly 12 with the X-ray crystallographic structures for this region in the p21 protein and EFtu. A model for the structure of the p21 protein in its oncogenic form

The GTP-binding p21 protein encoded by the ras-oncogene can be activated to cause malignant transformation of cells by substitution of a single amino acid at critical positions along the polypeptide chain. Substitution of any non-cyclic L-amino acid for Gly 12 in the normal protein results in a transforming protein. This substitution occurs in a hydrophobic sequence (residues 6-15) which is known to be involved in binding the phosphate moities of GTP (and GDP). We find, using conformational energy calculations, that the 6-15 segment of the normal protein (with Gly 12) adopts structures that contain a bend at residues 11 and 12 with the Gly in the D* conformation, not allowed energetically for L-amino acids. Substitution of non-cyclic L-amino acids for Gly 12 results in shifting this bend to residues 12 and 13. We show that many computed structures for the Gly 12-containing phosphate binding loop, segment 9-15, are superimposable on the corresponding segment of the recently determined X-ray crystallographic structure for residues 1-171 of the p21 protein. All such structures contain bends at residues 11 and 12 and most of these contain Gly 12 in the C* or D* conformational state. Other computed conformations for the 9-15 segment were superimposable on the structure of the corresponding 18-23 segment of EFtu, the bacterial chain elongation factor having structural similarities to the p21 protein in the phosphate-binding regions. This segment contains a Val residue where a Gly occurs in the p21 protein. As previously predicted, all of these superimposable conformations contain a bend at positions 12 and 13, not 11 and 12. If these structures that are superimposable on EFtu are introduced into the p21 protein structure, bad contacts occur between the sidechain of the residue (here Val) at position 12 and another phosphate binding loop region around position 61. These bad contacts between the two segments can be removed by changing the conformation of the 61 region in the p21 protein to the corresponding position of the homologous region in EFtu. In this new conformation, a large site becomes available for the binding of phosphate residues. In addition, such phenomena as autophosphorylation of the p21 protein by GTP can be explained with this new model structure for the activated protein which cannot be explained by the structure for the non-activated protein.     

Interactions of GTP with the ATP-grasp domain of GTP-specific succinyl-CoA synthetase

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 A resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20beta) and with backbone atoms provide the specificity. The gamma-phosphate interacts with the side chain of an arginine residue (Arg-54beta) and with backbone amide nitrogen atoms, leading to tight interactions between the gamma-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the gamma-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the alpha- and beta-phosphates allow the gamma-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the beta-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.     

Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide gamma-phosphate

The effect of bound nucleotide on the conformation of cell division protein FtsZ from Methanococcus jannaschii has been investigated using molecular dynamics and site-directed mutagenesis. The molecular dynamics indicate that the gamma-phosphate of GTP induces a conformational perturbation in loop T3 (Gly88-Gly99 segment), in a position structurally equivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state, loop T3 is pulled by the gamma-phosphate into a more compact conformation than with GDP, related to that observed in the homologous proteins alpha- and beta-tubulin. The existence of a nucleotide-induced structural change in loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan (12 A away from gamma-phosphate) shows large differences in fluorescence emission, depending on which nucleotide is bound to FtsZ monomers. Loop T3 is located at a side of the contact interface between two FtsZ monomers in the current model of FtsZ filament. Such a structural change may bend the GDP filament upon hydrolysis by pushing against helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in tubulin activation.     

GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.

Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.     

Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.     

The homologous pairing domain of RecA also mediates the allosteric regulation of DNA binding and ATP hydrolysis: a remarkable concentration of functional residues

Switching between the active (ATP and DNA bound) and inactive conformations of the homologous recombination RecA protein is regulated by ATP hydrolysis. First, we use the homologous pairing domain of RecA derived from its mobile loop L2 to show that the interaction of this random coil peptide with the gamma-phosphate of ATP results in a peptide beta-conformation similar to that previously shown to be induced by DNA binding. Next, we show that in the whole RecA protein two residues in this L2 domain, Gln194 and Arg196, are catalytic amino acid residues for ATP hydrolysis and functionally resemble the corresponding residues engaged in GTP hydrolysis by two distinct classes of G proteins. Finally, we show that the role of DNA and high salt in the stimulation of the ATPase of RecA is to stabilize this highly mobile region involved in hydrolysis. This is a role similar to that described for RGSs in the activation of the GTPase of heterotrimeric G proteins. Therefore, (i) a prototypical DNA-dependent ATPase and ATP-stimulated DNA-binding protein, RecA, and eukaryotic signaling proteins share common stereochemical regulatory mechanisms; and (ii) in a remarkable example of parsimony, loop L2 is a molecular switch that controls both ATP promoted DNA binding and pairing reactions and DNA stimulated ATP hydrolysis.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

On the mechanism of guanosine triphosphate hydrolysis in ras p21 proteins.

The residue Gln61 is assumed to play a major role in the mechanism of ras p21, and mutations of this residue are often found in human tumors. Such mutations lead to a major reduction in the rate of GTP hydrolysis by the complex of ras p21 and the GTPase activating protein (GAP) and lock the protein in a growth-promoting state. This work examines the role of Gln61 in ras p21 by using computer simulation approaches to correlate the structure and energetics of this system. Free energy perturbation calculations and simpler electrostatic considerations demonstrate that Gln61 is unlikely to serve as the general base in the intrinsic GAP-independent reaction of p21. Glutamine is already a very weak base in water, and surprisingly the GlnH+ OH-reaction intermediate is even less stable in the protein active site than in the corresponding reaction in water. The electrostatic field of Glu63, which could in principle stabilize the protonated Gln61, is found to be largely shielded by the surrounding solvent. However, it is still possible that Gln61 is a general base in the GAP/ras p21 complex since this system could enhance the electrostatic effect of Glu63. It is also possible that the gamma-phosphate acts as general base and that Gln61 accelerates the reaction by stabilizing the OH- nucleophile. If such a mechanism is operative, then GAP may enhance the effect of Gln61 by preorienting its hydrogen bonds in the transition-state configuration.     

Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.     

Theoretical IR spectroscopy based on QM/MM calculations provides changes in charge distribution, bond lengths, and bond angles of the GTP ligand induced by the Ras-protein

The GTPase Ras p21 is a crucial switch in cellular signal transduction. Fourier transform infrared (FTIR) spectra of the substrate guanosine triphosphate (GTP) show remarkable changes when it binds to the enzyme. The reduced band widths indicate that the flexible GTP molecule is guided by the protein into a preferred conformation. The delocalized phosphate vibrations of unbound GTP become localized. The frequency shifts show an electron movement toward beta-phosphate, which probably contributes to catalysis by reducing the free activation energy. To quantify these qualitative observations we performed QM/MM molecular dynamics simulations of Ras.GTP and GTP in water. The triphosphate part of GTP was treated quantum mechanically using density functional theory (DFT). Vibrational spectra were calculated in harmonic approximation with an average deviation of 3% from the experimental frequencies. This provides a high confidence in the computational results as vibrational spectra are highly sensitive to conformation and charge distribution. As compared to GTP in water, Ras-bound GTP shows a shift of negative charge of approximately 0.2 e toward the beta-phosphate from gamma-phosphate and from alpha-phosphate due to the positive charge of the magnesium ion, to a lesser extent of Lys-16, and surprisingly without any effect of the P-loop backbone. Magnesium and Gly-13 twist and bend the gamma-O-beta bonds such that the crucial bond is stretched before cleaving.