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.     

Ras catalyzes GTP hydrolysis by shifting negative charges from gamma- to beta-phosphate as revealed by time-resolved FTIR difference spectroscopy

FTIR difference spectroscopy has been used to determine the molecular GTPase mechanism of the small GTP binding protein Ras at the atomic level. The reaction was initiated by the photolysis of caged GTP bound to Ras. The addition of catalytic amounts of the GTPase activating protein (GAP) reduces the measuring time by 2 orders of magnitude but has no influence on the spectra as compared to the intrinsic reaction. The reduced measuring time improves the quality of the data significantly as compared to previously published data [Cepus, V., Scheidig, A., Goody, R. S., and Gerwert, K. (1998) Biochemistry 37, 10263-10271]. The phosphate vibrations are assigned using 18O-labeled caged GTP. In general, there is excellent agreement with the results of Cepus et al., except in the nu(a)(alpha-PO2-) vibration assignments. The assignments reveal that binding of GTP to Ras induces vibrational uncoupling into mainly individual vibrations of the alpha-, beta-, and gamma-phosphate groups. In contrast, for unbound GTP, the phosphate vibrations are highly coupled and the corresponding absorption bands are broader. This result indicates that binding to Ras forces the flexible GTP molecule into a strained conformation and induces a specific charge distribution different from that in the unbound case. The binding causes an unusual frequency downshift of the GTP beta-PO2- phosphate vibration, whereas the alpha-PO2- and gamma-PO3(2-) phosphate vibrations shift to higher wavenumbers. The frequency downshift indicates a lowering of the bond order of the nonbridged P-O bonds of the beta-phosphate group of GTP and GDP. The bond order changes can be explained by a shift of negative charges from the gamma- to the beta-oxygens. Thereby, the GTP charge distribution becomes more like that in GDP. The charge shift appears to be a key factor contributing to catalysis by Ras in addition to the correct positioning of the attacking water. Ras appears to increase the negative charge at the pro-R beta-oxygen mainly by interaction of Mg(2+) and at the pro-S beta-oxygen mainly by interactions of the backbone NHs of Lys 16, Gly 15, and Val 14. The correct positioning of the backbone NHs of Lys 16, Gly 15, and Val 14, and especially the Lys 16 side chain, of the structural highly conserved phosphate binding loop relative to beta-phosphate therefore seems to be important for the catalysis provided by Ras.     

Fourier transform infrared spectroscopy on the Rap.RapGAP reaction, GTPase activation without an arginine finger

GTPase activating proteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004) Nature 429, 197-201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP(*) conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras.RasGAP reactions. However, the charge shift toward beta-phosphate observed with RasGAP was also observed for RapGAP. A GDP.P(i) intermediate accumulates in the GAP-catalyzed reaction, because the release of P(i) is eight times slower than the cleavage reaction, and significant GTP synthesis from GDP.P(i) was observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap.RapGAP catalytic machinery compensates for the absence of a cis-Gln by a trans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.     

Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPgammaS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.     

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.     

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.     

Role of the arginine finger in Ras.RasGAP revealed by QM/MM calculations

In the Ras.Ras.GAP complex, hydrolysis of guanosine triphosphate is strongly accelerated GAP as compared to Ras alone. This is largely attributed to the arginine finger R789(GAP) pointing to AlF(x) in the transition state analogue. We performed QM/MM simulations where triphosphate was treated using the quantum mechanical method of density functional theory, while the protein complex and water environment were described classically using MD. Compared to Ras, the crucial electron shift, bond stretching and distortion towards an eclipsed gamma-to-beta orientation are much more pronounced. The arginine finger is shown to act by displacing water out of the binding niche. The resulting enhanced electrostatic field catalyses the cleavage step.     

Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.     

gamma-phosphate protonation and pH-dependent unfolding of the Ras.GTP.Mg2+ complex: a vibrational spectroscopy study

The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the gamma-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes. Isotope-edited IR studies of the Ras.GTP.Mg(2+) complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma-phosphate is not protonated at pH > or = 3.3, indicating that the active site decreases the gamma-phosphate pK(a) by at least 1.1 pK(a) units compared with solution. Amide I studies show that the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes (3.7 and 4.8, respectively) reveal that the enzyme-gamma-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras.GTP.Mg(2+) complex provides an alternative interpretation of the data that had been used to support the gamma-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.     

Electrostatic effects of mutations of Ras glutamine 61 measured using vibrational spectroscopy of a thiocyanate probe

Mutations of human oncoprotein p21(Ras) (hereafter Ras) at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that electrostatically stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. We have tested the interactions between amino acids at this position and water by measuring changes in the electrostatic field experienced by a nitrile probe positioned near Ras Q61 using vibrational Stark effect (VSE) spectroscopy. We mutated this glutamine to every amino acid except cysteine and proline and then incubated these mutants with a Ral guanine nucleotide dissociation stimulator (Ral) containing the I18C mutation that was chemically labeled with a thiocyanate vibrational spectroscopic probe. The formation of the docked Ras Q61X-labeled Ral complex was confirmed by measurement of the dissociation constant of the interaction. We measured the absorption energy of this nitrile to determine any differences in electrostatic environment in the immediate vicinity of the thiocyanate probe between wild type and mutants of Ras. For each Ras Q61X mutant, we correlate the change in electrostatic field at position 61 with the solvent accessible surface area of polar components of the mutant side chain determined from a Boltzmann-weighted ensemble of structures, as well as the residue's hydration potential. These results support the hypothesis that the role of Ras Q61 is to stabilize water in or near the active site during GTP hydrolysis. The substantial effect that nonpolar side chains of Ras Q61X have on the absorption energy of the thiocyanate must be investigated with further experiments.     

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).     

Ab initio study of the role of lysine 16 for the molecular switching mechanism of Ras protein p21

Quantum chemical computations using the ab initio molecular orbital (MO) method have been performed to investigate the molecular switching mechanism of Ras protein p21, which has an important role in intracellular signal cascades. Lys(16) was demonstrated to be crucial to the function of Ras p21, and the hydrolysis of GTP to GDP was found to be an one-step reaction. The potential energy barrier of this hydrolysis reaction from GTP to (GDP + P) was calculated to be approximately 42 kcal/mol. The role of GAP (GTPase-activating protein) was also discussed in terms of the delivery of the water molecules required for the hydrolysis.     

Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7

Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.     

Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy

The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras. In the present study we investigated structural changes of H-Ras in its di- and triphosphate form that occur upon glucosylation of the effector domain amino acid threonine-35 by LT. (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides. In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center. In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinct conformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination. This conformation is known to have a low affinity to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

The mechanism of GTP hydrolysis by Ras probed by Fourier transform infrared spectroscopy

Time-resolved Fourier transform infrared spectroscopy (FTIR) in combination with photo-induced release of (18)O-labeled caged nucleotide has been employed to address mechanistic issues of GTP hydrolysis by Ras protein. Infrared spectroscopy of Ras complexes with nitrophenylethyl (NPE)-[alpha-(18)O(2)]GTP, NPE-[beta-(18)O(4)]GTP, or NPE-[gamma-(18)O(3)]GTP upon photolysis or during hydrolysis afforded a substantially improved mode assignment of phosphoryl group absorptions. Photolysis spectra of hydroxyphenylacyl-GTP and hydroxyphenylacyl-GDP bound to Ras and several mutants, Ras(Gly(12))-Mn(2+), Ras(Pro(12)), Ras(Ala(12)), and Ras(Val(12)), were obtained and yielded valuable information about structures of GTP or GDP bound to Ras mutants. IR spectra revealed stronger binding of GDP beta-PO(3)(2-) moiety by Ras mutants with higher activity, suggesting that the transition state is largely GDP-like. Analysis of the photolysis and hydrolysis FTIR spectra of the [beta-nonbridge-(18)O(2), alphabeta-bridge-(18)O]GTP isotopomer allowed us to probe for positional isotope exchange. Such a reaction might signal the existence of metaphosphate as a discrete intermediate, a key species for a dissociative mechanism. No positional isotope exchange was observed. Overall, our results support a concerted mechanism, but the transition state seems to have a considerable amount of dissociative character. This work demonstrates that time-resolved FTIR is highly suitable for monitoring positional isotope exchange and advantageous in many aspects over previously used methods, such as (31)P NMR and mass spectrometry.     

Rap1A antagonizes the ability of Ras and Ras-Gap to inhibit muscarinic K+ channels

Rap1A is a Ras-related GTP binding protein which has an amino acid sequence identical to that of Ras in the putative "effector" domain (amino acids 32-40). The binding of Rap1A to Ras-GTPase activating protein (GAP) through this domain is a potential mechanism for explaining the observation that Rap1A can antagonize the ability of oncogenic Ras to transform cells. It was recently shown (Yatani, A., Okabe, K., Polakis, P., Halenbeck, R., McCormick, F., and Brown, A. M. (1990) Cell 61, 769-776) that the activation of M2-muscarinic receptor-coupled K+ channels in heart is inhibited by the addition of exogenous Ras and Ras-GAP. We have made use of this system in the present paper to show that Rap1A is able to effectively block this inhibitory action of Ras-GAP. We observed that both Rap1A-GDP and Rap1A-guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) were able to block the inhibitory effect of Ras-GAP upon channel activation. This effect occurred at picomolar concentrations of Rap1A, and the GTP gamma S-bound form of the protein was consistently found to be more potent than the GDP form. A Rap1A Thr35----Ala mutation which bound GTP gamma S did not prevent K+ channel inhibition by Ras-GAP, suggesting that the antagonism by wild type Rap1A involves an interaction with GAP in the effector domain. The effectiveness of Rap1A to inhibit Ras-GAP is dependent upon the amount of Ras-GAP present in the assay and can also be overcome by the addition of GTP-bound N-Ras (GC-43), suggesting a competitive mechanism is operative. Finally, a truncated form of Ras-GAP (GAP32) which is no longer dependent upon Ras for inhibition of the M2-activated K+ channel is also no longer sensitive to blockade by added Rap1A. These data support the concept of GAP as an effector of Ras action and indicate that Rap1A can serve as an inhibitor of Ras action in a system distinct from cell transformation by a competitive mechanism involving the GAP binding domain of Rap1A.     

Conformational states of human rat sarcoma (Ras) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.     

Structural analysis of autoinhibition in the Ras activator Son of sevenless

The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that Ras*GTP is an allosteric activator of SOS indicated that the regulation of SOS is more complex than originally envisaged. We now present crystallographic and biochemical analyses of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains and show that the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of Ras*GDP binding, and maximal activity, which requires Ras*GTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low to high activity forms as Ras*GDP is converted to Ras*GTP by SOS.     

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.