The protonation states of GTP and GppNHp in Ras proteins.

The small GTPase Ras transmits signals in a variety of cellular signaling pathways, most prominently in cell proliferation. GTP hydrolysis in the active center of Ras acts as a prototype for many GTPases and is the key to the understanding of several diseases, including cancer. Therefore, Ras has been the focus of intense research over the last decades. A recent neutron diffraction crystal structure of Ras indicated a protonated γ-guanylyl imidodiphosphate (γ-GppNHp) group, which has put the protonation state of GTP in question. A possible protonation of GTP was not considered in previously published mechanistic studies. To determine the detailed prehydrolysis state of Ras, we calculated infrared and NMR spectra from quantum mechanics/molecular mechanics (QM/MM) simulations and compared them with those from previous studies. Furthermore, we measured infrared spectra of GTP and several GTP analogs bound to lipidated Ras on a membrane system under near-native conditions. Our findings unify results from previous studies and indicate a structural model confirming the hypothesis that γ-GTP is fully deprotonated in the prehydrolysis state of Ras.

A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys.).

The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu ("EF-Pyl"). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg(2+), MM1309·GDP·Mg(2+), and MM1309·GMPPNP·Mg(2+) provided dissociation constants of 0.43, 26.2, and 222.2 µM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain-domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNA(Cys) and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNA(Cys).

Structures of Drosophila melanogaster Rab2 and Rab3 bound to GMPPNP.

Rab GTPases belong to the large family of Ras proteins. They act as key regulators of membrane organization and intracellular trafficking. Functionally, they act as switches. In the active GTP-bound form they can bind to effector proteins to facilitate the delivery of transport vesicles. Upon stimulation, the GTP is hydrolyzed and the Rab proteins undergo conformational changes in their switch regions. This study focuses on Rab2 and Rab3 from Drosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+ are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function.

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase.

Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Solution structure and dynamics of the small GTPase RalB in its active conformation: significance for effector protein binding.

The small G proteins RalA/B have a crucial function in the regulatory network that couples extracellular signals with appropriate cellular responses. RalA/B are an important component of the Ras signaling pathway and, in addition to their role in membrane trafficking, are implicated in the initiation and maintenance of tumorigenic transformation of human cells. RalA and RalB share 85% sequence identity and collaborate in supporting cancer cell proliferation but have markedly different effects. RalA is important in mediating proliferation, while depletion of RalB results in transformed cells undergoing apoptosis. Crystal structures of RalA in the free form and in complex with its effectors, Sec5 and Exo84, have been solved. Here we have determined the solution structure of free RalB bound to the GTP analogue GMPPNP to an RMSD of 0.6 A. We show that, while the overall architecture of RalB is very similar to the crystal structure of RalA, differences exist in the switch regions, which are sensitive to the bound nucleotide. Backbone 15N dynamics suggest that there are four regions of disorder in RalB: the P-loop, switch I, switch II, and the loop comprising residues 116-121, which has a single residue insertion compared to RalA. 31P NMR data and the structure of RalB.GMPPNP show that the switch regions predominantly adopt state 1 (Ras nomenclature) in the unbound form, which in Ras is not competent to bind effectors. In contrast, 31P NMR analysis of RalB.GTP reveals that conformations corresponding to states 1 and 2 are both sampled in solution and that addition of an effector protein only partially stabilizes state 2.

Purification and characterization of transducin from capybara Hydrochoerus hydrochaeris.

Polypeptides of approximately 39, 36 and

X-ray crystal structures reveal two activated states for RhoC.

RhoC is a member of the Rho family of Ras-related (small) GTPases and shares significant sequence similarity with the founding member of the family, RhoA. However, despite their similarity, RhoA and RhoC exhibit different binding preferences for effector proteins and give rise to distinct cellular outcomes, with RhoC being directly implicated in the invasiveness of cancer cells and the development of metastasis. While the structural analyses of the signaling-active and -inactive states of RhoA have been performed, thus far, the work on RhoC has been limited to an X-ray structure for its complex with the effector protein, mDia1 (for mammalian Diaphanous 1). Therefore, in order to gain insights into the molecular basis for RhoC activation, as well as clues regarding how it mediates distinct cellular responses relative to those induced by RhoA, we have undertaken a structural comparison of RhoC in its GDP-bound (signaling-inactive) state versus its GTP-bound (signaling-active) state as induced by the nonhydrolyzable GTP analogues, guanosine 5'-(beta,gamma-iminotriphosphate) (GppNHp) and guanosine 5'-(3-O-thiotriphosphate) (GTPgammaS). Interestingly, we find that GppNHp-bound RhoC only shows differences in its switch II domain, relative to GDP-bound RhoC, whereas GTPgammaS-bound RhoC exhibits differences in both its switch I and switch II domains. Given that each of the nonhydrolyzable GTP analogues is able to promote the binding of RhoC to effector proteins, these results suggest that RhoC can undergo at least two conformational transitions during its conversion from a signaling-inactive to a signaling-active state, similar to what has recently been proposed for the H-Ras and M-Ras proteins. In contrast, the available X-ray structures for RhoA suggest that it undergoes only a single conformational transition to a signaling-active state. These and other differences regarding the changes in the switch domains accompanying the activation of RhoA and RhoC provide plausible explanations for the functional specificity exhibited by the two GTPases.

High-frequency 94 GHz ENDOR characterization of the metal binding site in wild-type Ras x GDP and its oncogenic mutant G12V in frozen solution.

The guanine nucleotide binding protein Ras plays a central role as molecular switch in cellular signal transduction. Ras cycles between a GDP-bound "off" state and a GTP-bound "on" state. Specific oncogenic mutations in the Ras protein are found in up to 30% of all human tumors. Previous 31P NMR studies had demonstrated that in liquid solution different conformational states in the GDP-bound as well as in the GTP-bound form coexist. High-field EPR spectroscopy of the GDP complexes in solution displayed differences in the ligand sphere of the wild-type complex as compared to its oncogenic mutant Ras(G12V). Only three water ligands were found in the former with respect to four in the G12V mutant [Rohrer, M. et al. (2001) Biochemistry 40, 1884-1889]. These differences were not detected in previous X-ray structures in the crystalline state. In this paper, we employ high-frequency electron nuclear double resonance (ENDOR) spectroscopy to probe the ligand sphere of the metal ion in the GDP-bound state. This technique in combination with selective isotope labeling has enabled us to detect the resonances of nuclei in the first ligand sphere of the ion with high spectral resolution. We have observed the 17O ENDOR spectra of the water ligands, and we have accurately determined the 17O hyperfine coupling with a(iso) = -0.276 mT, supporting the results of previous line shape analysis in solution. Further, the distinct resonances of the alpha-, beta-, and gamma-phosphorus of the bound nucleotides are illustrated in the 31P ENDOR spectra, and their hyperfine tensors lead to distances in agreement with the X-ray structures. Finally, 13C ENDOR spectra of uniformly 13C-labeled Ras(wt) x GDP and Ras(G12V) x GDP complexes as well as of the Ras(wt) x GppNHp and the selectively 1,4-13C-Asp labeled Ras(wt) x GDP complexes have revealed that in frozen solution only one amino acid is ligated to the ion in the GDP state, whereas two are bound in the GppNHp complex. Our results suggest that a second conformational state of the protein, if correlated with a different ligand sphere of the Mn2+ ion, is not populated in the GDP form of Ras at low temperatures in frozen solution.

Crystal structure of M-Ras reveals a GTP-bound "off" state conformation of Ras family small GTPases.

Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of M-Ras resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the gamma-phosphate of Gpp(NH)p. Previous 31P NMR studies showed that H-Ras-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off" conformation, whose tertiary structure remains unknown. 31P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gp-p(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off" conformation.

Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins.

The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.

Dynamic properties of the Ras switch I region and its importance for binding to effectors.

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

GTP plus water mimic ATP in the active site of protein kinase CK2.

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs that target CK2 or other kinases with this property.

The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion.

BACKGROUND: The G protein p21 ras is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself. However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively 2H beta-threonine and 15N-threonine labeled p21.Mn2+ GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21. RESULTS: A 13C beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 A and a 15N epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 A were determined from ESEEM spectra of the selectively 13C beta-Thr and 15N epsilon-Lys labeled p21.Mn2+ GMPPNP frozen solution structure. The 13C beta-Thr35 to Mn2+ distance is greater than that (3.16 A) observed in the crystal structure. In contrast, the 15N epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 A crystal structure distance. CONCLUSIONS: The 13C beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21.Mg2+ GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution. Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP- and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.

Conformational and dynamic differences between N-ras P21 bound to GTPgammaS and to GMPPNP as studied by NMR.

Heteronuclear-edited proton-detected NMR methods are used to study the nucleotide-dependent conformational changes between the GMPPNP form of human N-ras P21 as compared to GDP and GTPgammaS forms. Full-length N-ras P21 was also compared with protein truncated beyond residue 167, to search for interaction points between the more invariant part of the protein and the variable C-terminal section. In both cases, the reporter was the 15N-H 2D spectrum of aspartate amide groups labeled with 15N. Small truncation-induced changes were seen in the spectrum at the resonances of Asp-54, -108, and -109 which are not far from the C-terminal and, surprisingly, at Asp-57 which is more remote. The spectrum obtained for the GMPPNP-ligated form is similar to that of the GTPgammaS form, except that peaks of several residues are weak at low temperature, and strongly temperature-dependent in their intensity, and a new resonance appears at 15 degrees C and above. The observations are discussed in terms of a two-state model for the GMPPNP-ligated protein, previously proposed by Geyer et al. [(1996) Biochemistry 35, 10308-10320].

Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.

The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs.

The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue.

The X-ray crystal structure of the complex between the Ras-related protein Rap1A in the GTP-analogue (GppNHp) form and the Ras-binding domain (RBD) of the Ras effector molecule c-Raf1, a Ser/Thr-specific protein kinase, has been solved to a resolution of 2.2 A. It shows that RBD has the ubiquitin superfold and that the structure of Rap1A is very similar to that of Ras. The interaction between the two proteins is mediated by an apparent central antiparallel beta-sheet formed by strands B1-B2 from RBD and strands beta 2-beta 3 from Rap1A. Complex formation is mediated by main-chain and side-chain interactions of the so-called effector residues in the switch I region of Rap1A.