Solution Structure of the State 1 Conformer of GTP-bound H-Ras Protein and Distinct Dynamic Properties between the State 1 and State 2 Conformers*

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTP-bound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and 31P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an H-RasT35S mutant in complex with guanosine 5′-(β, γ-imido)triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone 1H,15N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras·GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.

Small GTPases H-Ras, K-Ras, and N-Ras, collectively called Ras, are the products of the ras proto-oncogenes and function as molecular switches by cycling between the GTP-bound active and the GDP-bound inactive forms in intracellular signaling pathways controlling proliferation, differentiation, and apoptosis of cells. GTP hydrolysis on Ras is markedly stimulated by GTPase-activating proteins, whereas conversion from the GDP-bound form to the GTP-bound form is promoted by guanine nucleotide exchange factors (1,2). Ras comprise the Ras family of small GTPases together with a number of its relatives including Rap1, Rap2, R-Ras, R-Ras2/TCL, M-Ras/R-Ras3, RalA, RalB, etc. (3). Structural studies of Ras showed that structural differences between the GDP-and GTP-bound forms universally exist in two flexible regions, called switch I (residues 32-38 in H-Ras) and switch II (residues 60 -75 in H-Ras) (1). GTP-sensitive orientation of the switch regions enables Ras to interact with their effectors such as Raf kinases and phosphoinositide 3-kinases (2).
Recent 31 P NMR studies suggested that H-Ras in the nucleoside triphosphate form exists in equilibrium between two kinds of conformational states, state 1 and state 2, around the phosphate groups of GTP or its non-hydrolyzable analogues, GppNHp 3 and GTP␥S, bound to the protein (4 -6). This conformational heterogeneity has been commonly observed in a number of Ras homologues (7,8). Because binding to the various effectors, such as c-Raf-1, shifted the equilibrium toward state 2 (5,9), state 1 and state 2 were regarded as the "inactive" and "active" conformations, respectively. The x-ray structures of H-Ras⅐GppNHp by itself or in complex with its effectors revealed the state 2 conformation in which the switch I and switch II regions are fixed by hydrogen bonds of the backbone amides of Thr-35 and Gly-60, respectively, with the ␥-phosphate oxygen atoms of the nucleoside triphosphate (1, 10 -12). On the other hand, x-ray structures corresponding to state 1 were recently determined by using H-Ras mutants, H-RasT35S⅐GppNHp (9,13) and H-RasG60A⅐GppNHp (14), or M-Ras⅐GppNHp (15), all of which predominantly adopted state 1, whereas that of H-Ras⅐GppNHp has remained unsolved. In these state 1 structures, Thr-45 (corresponding to Thr-35 in H-Ras) of M-Ras and Ser-35 of H-RasT35S are not capable of interacting with the guanine nucleotide and magnesium ion, causing marked deviation of the switch I loop from the nucleotide (13,15). This structural feature of state 1 results in greater exposure of the nucleotide to the solvent and allows faster association and dissociation of GTP compared with state 2 (7). A similar switch I loop deviation was observed in the conformation of nucleotide-free H-Ras in complex with Sos, a guanine nucleotide exchange factor for Ras. In the structure of the H-Ras-Sos complex, the helical hairpin segment of Sos opens wide the nucleotide-binding site, causing deviation of the switch I loop of H-Ras further away from this site (16). The results suggested that state 1 might play a role in the guanine nucleotide cycle involving guanine nucleotide exchange factors, although its function remained to be clarified (14,17). So far studies on the state transition of the GTP-bound Ras have been based on static crystal structures or 31 P NMR spectra, which are in principle unsuitable for studying the dynamic aspects of conformational transition.
Heteronuclear NMR spectroscopy is the most suitable technique to examine detailed conformational dynamics of proteins in solution. Nevertheless, structural studies on the GTP-bound Ras using this technique have been hampered by chemical exchange processes at intermediate rates on the NMR time scale that result in broadening or even disappearance of the resonance signals (18 -20). Although the solution structure of RalB⅐GppNHp in state 1 was recently determined by this technique, backbone amide resonances were not observed for several residues in the switch regions. In the case of H-Ras in complex with GppNHp or GTP␥S, backbone amide 1 H, 15 N HSQC cross-peaks underwent extreme broadening for most of the residues in the P-loop (residues 10 -17) and switch I and switch II regions (19). Also, in the physiological GTP-bound form, the cross-peaks from these regions were two to four times broader than the normal ones (19), although they dominantly existed as state 2 as revealed by 31 P NMR (4,5). These results imply that the exchange processes are an intrinsic property of H-Ras in the nucleoside triphosphate form. The analysis of 15 N spin relaxation relaxation-compensated Carr-Purcell-Meiboom-Gill measurements showed that the exchange processes involve a major part of the H-Ras structure (21). However, conformational species representing the exchange processes have not been characterized.
In this study, we report successful assignments of the backbone resonances for state 1 and state 2 by 1 H and 15 N NMR analysis of H-RasT35S⅐GppNHp and H-Ras⅐GppNHp in complex with c-Raf-1 RBD, respectively. Moreover, the backbone resonances for almost all the residues of H-Ras⅐GppNHp are successfully identified by measurement at low temperature. Comparison of these resonance data proves that the chemical exchange process observed in 1 H and 15 N NMR of H-Ras⅐GppNHp corresponds to the interconversion between state 1 and state 2. Also, analysis of the backbone dynamics by measuring 15 N relaxation times and heteronuclear NOEs reveals a significant difference in rapid internal motions of the switch regions between state 1 and state 2. Furthermore, the solution structure of state 1 is solved with H-RasT35S⅐GppNHp for the first time. It shows unique conformations of the switch regions, some of which are very similar to those of the nucleotide-free form found in the H-Ras-Sos complex. These structural features characteristic of state 1 will be discussed in relation to results of H-Ras/Sos binding experiments.

EXPERIMENTAL PROCEDURES
Protein Expression and Purification-Residues 1-166 of human H-Ras and H-RasT35S were expressed as fusions with glutathione S-transferase in Escherichia coli using pGEX-6P-1 vector (GE Healthcare). M9 minimal media containing 15 NH 4 Cl and [ 13 C]glucose/ 15 NH 4 Cl were used to express the uniformly 15 N-labeled and 15 N/ 13 C-labeled proteins, respectively. Proteins were immobilized on glutathione-agarose and eluted by cleavage with PreScission protease (GE Healthcare). After further purification by ion exchange chromatography to a final purity of Ͼ95% as measured by SDS-PAGE, they were loaded with GppNHp (Sigma). Human c-Raf-1 RBD consisting of residues 51-130 was purified as described (7).
NMR Spectroscopy-Each protein sample was concentrated and dissolved in a buffer containing 25 mM sodium phosphate, pH 6.8, 150 mM NaCl, and 10 mM MgCl 2 in 90% 1 H 2 O and 10% 2 H 2 O using a centrifugal filter unit. For the 100% 2 H 2 O samples, the protein solutions were incubated at 34°C for 4 days after buffer displacement and filtration with a filter (0.2 m pore size). Protein concentrations were determined by absorbance measurements as described (22). NMR measurements were performed on a Bruker DMX-750 spectrometer on protein samples of 1.0 -2.0 mM concentration at 25°C unless stated otherwise. The following spectra were acquired on 13 C, 15  Backbone Dynamics-All experiments were carried out at 25°C on 1.5 mM 15 N-labeled H-RasT35S⅐GppNHp. 15 N T 1 , T 2 , and heteronuclear NOE measurements were performed using the pulse sequence previously reported (24). T 1 experiments were recorded with time delays of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 s, in which 1 H 180°pulses were applied every 5 ms to eliminate the effects of cross-correlation between 1 H, 15 N dipolar and 15 N chemical shift anisotropy relaxation mechanisms (25). T 2 experiments were recorded with time delays of 0.0170, 0.0339, 0.0509, 0.0678, 0.0848, 0.1018, and 0.1187 s in which 15 N 180°pulses at a field strength of 3.1 kHz were applied every 0.9 ms, and 1 H 180°pulses were applied every 8.4 ms to suppress cross-correlated relaxation. The intensity decays were fitted using an equation I(t) ϭ I 0 exp(Ϫt/T 1,2 ). 1 H, 15 N steadystate heteronuclear NOE measurements were carried out by recording a pair of spectra with and without proton saturation using a total recycle delay of 3.2 s in which proton saturation of 3 s was achieved by applying 120°proton pulses every 5 ms. The NOE values were analyzed by calculating the peak height ratios obtained from reference and saturated experiments (I sat /I ref ). The series of experiments was also performed on a mixture containing 1.5 mM 15 N-labeled H-Ras⅐GppNHp and 1.8 mM unlabeled c-Raf-1 RBD in which state 2 was predominantly populated (5).
Structure Calculations of H-RasT35S⅐GppNHp-In the N-terminal sequence (GPLGSD) corresponding to remainder of the cleavage site by PreScission protease, intense NMR signals indicated that this region is unstructured with high mobility. Interproton distance restraints were obtained from the 13 Cseparated NOESY-HSQC, 15 N-separated NOESY-HSQC, and two-dimensional homonuclear 1 H NOESY spectra recorded at 25°C. Standard pseudo atom distances were used when they were needed. Structures were calculated by using the program CYANA 2.1 (26) and CNS 1.2 (27). A large number of ambiguous NOE peaks were identified by iterative calculations using structures computed from unambiguously assigned peaks, whereas 568 NOEs were excluded from the calculation because of overlapping with other signals or difficulty to remove ambiguity. A total of 3116 meaningful NOE upper distance restraints were finally obtained by CYANA, including 1021 long range distances. Backbone torsion angle restraints and were estimated from the C ␣ , C ␤ , CЈ, H N , N, and H ␣ chemical shifts using the program TALOS (28). The error values were set to twice the standard deviation of the TALOS prediction. The GppNHp nucleotide was modeled by adding distance restraints to coordinate the Mg 2ϩ ion to three water molecules, the O 2␤ and O 2␥ of GppNHp and the side-chain oxygen atom of Ser-17 as shown in the x-ray structure of H-RasT35S⅐GppNHp (13). A total of 100 structures was generated by using CYANA, then the 20 structures with the lowest target function that had no NOE violations more than 0.2 Å and no dihedral angle violations more than 5°were refined using CNS. The lowest energy structures finally obtained were selected to represent three-dimensional structure and analyzed using PROCHECK-NMR software (29). The atomic coordinates have been deposited in the Protein Data Bank (PDB code 2LCF).

Assignments of the Backbone Resonances for State 1 and State 2-
In the two-dimensional 1 H, 15 N HSQC spectrum for H-Ras⅐GppNHp, it was previously reported that the backbone amide 1 H, 15 N cross-peaks could not be detected for residues 10 -13 in the P-loop, 31-39 in or near switch I, and 57-64 and 71 in or near switch II (19). Its temperature and magnetic field dependencies indicated that these regions undergo the structural conversion between two or more stable conformations, named as "polysterism" (19). The structural basis for this polysterism has been unknown even though it looks in parallel with the interconversion between state 1 and state 2 observed in the 31 P NMR spectrum of H-Ras⅐GppNHp (5). On the other hand, H-RasT35S⅐GppNHp exists almost exclusively in state 1 as revealed by its 31 P NMR spectrum, showing that each phosphate resonance, especially that of the ␥-phosphate, yields a single peak equivalent to the state 1 peak of H-Ras⅐GppNHp (9). Hence, we investigated this mutant protein by heteronuclear NMR spectroscopy. In contrast to the case of H-Ras⅐GppNHp, complete assignments of the backbone 1 H, 13 C, and 15 N resonances were achieved for all the 166 residues of H-RasT35S⅐GppNHp by analysis of the seven triple-resonance NMR spectra (see "Experimental Procedures" for details). The assigned resonances were expected to represent the chemical shift positions for state 1. 31 P NMR studies showed that binding to c-Raf-1 RBD shifts the conformational equilibrium of H-Ras⅐GppNHp toward state 2 (5,9). In the 1 H, 15 N HSQC spectra of H-Ras⅐GppNHp, the addition of c-Raf-1 RBD generated cross-peaks corresponding to the P-loop and the switch regions, which were missing in its free form (19). We thus attempted to assign the complete backbone resonances of 13 C, 15 N -labeled H-Ras⅐GppNHp in complex with unlabeled c-Raf-1 RBD. However, the backbone 1 H N and 15 N resonances, which are correlated with the intraresidue and sequential 13 C ␣ resonances, were observed for only 134 residues through the HNCA and HN(CO)CA spectra because the sensitivity of the triple-resonance three-dimensional NMR spectra became notably lower in the presence of c-Raf-1 RBD. Therefore, we assigned all the backbone 1 H N and 15 N resonances excluding Glu-31 by combining the analysis of 15 N-separated NOESY-HSQC spectrum, which gives especially intense cross-peaks correlating 1 H N of one residue to 1 H ␣ and 1 H ␤ of the intra-and preceding residues. Nonetheless, ambiguities inherent in the assignments for the residues 30 -36 could not be removed as NOEs or 13 C ␣ , 1 H N correlations were detected partially for these residues concomitantly with broadening of the 1 H, 15  Assignment of the Backbone Resonances of H-Ras⅐GppNHp-We assigned the backbone 1 H, 13 C, and 15 N resonances for most of the 166 residues of H-Ras⅐GppNHp through analysis of the triple-resonance NMR spectra by referring to the previous assignments (19). We failed to detect the 1 H, 15 N cross-peaks for most of the residues in the P-loop and the switch regions when the measurements were done at 25°C as reported (19). However, the spectrum measured at 5°C yielded 20 additional cross-peaks, some of which are indicated by solid boxes in Fig. 1. When these new peaks were compared with the state 1 and state 2 peaks, measured at 5°C on H-RasT35S⅐GppNHp and the H-Ras⅐GppNHp⅐c-Raf-1 RBD complex, respectively, most of them were found overlapped with state 1 or state 2 peaks of the residues whose signals were undetectable upon the measurement of H-Ras⅐GppNHp at 25°C. For example, two new peaks of H-Ras⅐GppNHp indicated by ‡ and † in Fig. 1 coincided with the state 1 (H-RasT35S⅐GppNHp) and state 2 (H-Ras⅐GppNHp⅐c-Raf-1 RBD complex) peaks of Gly13, respectively. Similarly, two separate peaks corresponding to both state 1 and state 2 were identified for Gly-10, Gly-12, and Gly-60 ( Fig. 1). For most of the residues in the switch regions, one or both of the peaks for the two states were overlapped with other peaks, preventing accurate identification. Furthermore, the measurement at 5°C caused splitting of several cross-peaks as indicated by boxes drawn by dotted lines in Fig. 1. In most of these cases, one of the split peaks overlapped with the state 1 peak, whereas the other was located near the state 2 peak. For example, Gly-15 gave a single peak at 25°C, whereas it was split into two peaks at 5°C as indicated by * and # in Fig. 1, which coincided with the state 1 and state 2 peaks, respectively. The new results obtained for the H-Ras⅐GppNHp spectra at 5°C could be ascribable to the difference in the static magnetic field strength, i.e. 17.6T compared with 9.4 (14.1)T in the previous The dotted and solid boxes highlight the splitting cross-peaks for the residues that exhibited single and undetectable peaks, respectively, at 25°C. ‡ and † represent the cross-peaks for Gly-13 corresponding to state 1 and state 2, respectively. * and # represent the cross-peaks for Gly-15 corresponding to state 1 and state 2, respectively. NOVEMBER 11, 2011 • VOLUME 286 • NUMBER 45 work (19). Intensity ratios and chemical shift differences of the split peaks unambiguously assigned were plotted against the amino acid sequence (Fig. 2, A and B). Most of the intensity ratios (I state1 /I state2 ) were distributed in the range of 0.3-0.8 with the mean value of 0.53, which was close to the equilibrium constant between state 1 and state 2 (1/K 12 ) with the value of 0.6, derived from the 31 P NMR (8). In the tertiary structure of H-Ras⅐GppNHp, the residues exhibiting the peak splitting are distributed in the P-loop, switch II, and the rigidly structured regions (Fig. 2C).

Solution Structures and Dynamics of GTP-bound H-Ras
Solution Structure of H-RasT35S⅐GppNHp and Conformational Assessment of Its Switch Regions-The chemical shift resonances were assigned for all the side chains of H-RasT35S⅐GppNHp, except for the 1 H and 13 C resonances derived from ␥-⑀ positions of Lys-16, ⑀positions of Phe-82, and ␥-⑀ positions of Lys-117. The H1 and H8 chemical shifts for the purine ring and the H1Ј-H3Ј shifts for the ribose ring of GppNHp were assigned from two-dimensional 1 H NOESY and TOCSY spectra. A total of 5636 NOEs were translated by CYANA into meaningful 3116 upper distance restraints. The final structural calculation was performed with these distance restraints in addition to 114 and 116 backbone dihedral angle restraints ( Table 1). The overall NMR structure of H-RasT35S⅐GppNHp was well defined excluding pre-switch I (residues 28 -31), switch I, and switch II regions, with the r.m.s.d. value of 0.48 Ϯ 0.05 Å (Fig. 3A and Table 1). The structure was consistent with typical guanine nucleotide-binding proteins, which consist of a six-stranded ␤-sheet (␤1-6) surrounded by five ␣-helices (␣1-5) (1) (Fig. 3B). A profile of residual dipolar couplings of the rigidly structured regions in H-RasT35S⅐GppNHp provided a good correlation between experimentally measured and theoretical residual dipolar couplings calculated from the mean structure of the 20 structures we determined, supporting validation of the structural model encompassing these regions (supplemental Fig. 1). On the other hand, residues 28 -38 and 59 -64 constituting the switch I and switch II loops, respectively, were poorly defined (Fig. 3A). Also, multiple residues in the switch loops exhibit residual dipolar coupling outliers with smaller experimental values, suggesting flexibilities of these loop regions (supplemental Fig. 1).

TABLE 1 Structural statistics for the 20 lowest energy structures of H-RasT35S⅐GppNHp
Ramachandran analysis was evaluated by using the program PROCHECK (29). In Fig. 4, the backbone structure of the two switch regions of H-RasT35S⅐GppNHp (state 1) (blue) was compared with those of the x-ray structures of H-Ras⅐GppNHp (state 2, PDB code 5P21 (10)) (black) and the nucleotide-free form of H-Ras (PDB code 1BKD (16)) (green) and of the NMR structure of H-Ras-GDP (PDB code 1CRP (30)) (brown). Ser-35 in switch I was located too far from the nucleotide to form a direct hydrogen bond with the ␥-phosphate as observed for the corresponding residues in the state 1 structures, such as H-RasT35S⅐GppNHp form 1 and form 2, H-RasG60A⅐GppNHp, M-Ras⅐GppNHp, and RalB⅐GppNHp (13)(14)(15)20). Intriguingly, some of the 20 switch I structures resembled that of the nucleotide-free H-Ras in complex with Sos (Fig. 4A), whereas one of them resembled that of H-RasGppNHp state 2. These results indicated that in solution H-RasT35S⅐GppNHp possesses a wide range of structural variations in switch I, the extent of which was further compared with those found on other NMR structures, H-Ras-GDP and RalB⅐GppNHp (state 1, PDB code 2KE5 (20)). By using CYANA, the backbone r.m.s.d. for switch I (residues 32-38) of H-RasT35S⅐GppNHp is calculated to be 0.89 Ϯ 0.18 Å, which is notably larger than 0.58 Ϯ 0.16 and 0.48 Ϯ 0.17 Å, respectively, for the corresponding regions of H-Ras-GDP (residues 32-38) and RalB⅐GppNHp (residues 43-49) (Fig. 4A). The differences in the r.m.s.d. values could be accounted for as follows. The NOESY spectra of RalB⅐GppNHp gave unambiguous crosspeaks that correlated the methyl group of the Thr-46 (corresponding to Thr-35 in H-Ras) side chain to the Phe-82 ring protons and the Leu-67 side chain showing interactions between switch I and switch II (20). In the H-Ras-GDP structure, the phenol ring of Tyr-32 was located near the magnesium ion and the phenol ring of Tyr-40, which was consistent with the nearly normal values of the heteronuclear NOE, T 1 , and T 2 in the residues 33-40 (30). These results indicated that the switch I regions of H-Ras-GDP and RalB⅐GppNHp are constrained by some non-local interactions. On the other hand, in the H-RasT35S⅐GppNHp structure, any long range NOEs (͉i Ϫ j͉ Ͼ 4) involving the residues 30 -36 could not be observed, whereas sequential and medium range NOEs including Y32C ␦ H-I36C ␦ H and D33C ␤ H-I36C ␥ H were present. Thus, a large portion of switch I of H-RasT35S⅐GppNHp is stabilized only by local interactions, resulting in the markedly large r.m.s.d. value.

Number of distance restraints
In contrast, the backbone r.m.s.d. values for switch II were calculated to be 0.77 Ϯ 0.33 and 0.75 Ϯ 0.18 Å, respectively, for H-RasT35S⅐GppNHp (residues 60 -75) and RalB⅐GppNHp (residues 71-86), both of which were significantly smaller than 1.21 Ϯ 0.23 Å for H-Ras-GDP (residues 60 -75) (Fig. 4B). This was consistent with the presence of a small number of long range NOEs including A59C ␤ H-Y64C ␦ H, G12NH-G60C ␣ H, and S65C ␣ H-Q99N ⑀ H in the residues 59 -65 of H-RasT35S⅐GppNHp, whereas the corresponding region of H-Ras-GDP was remarkably ill-defined due probably to lack of long range NOEs. Many of the 20 structures of H-RasT35S⅐GppNHp showed that the backbone amide proton of Gly-60 is not located too far to form a hydrogen bond with the ␥-phosphate oxygen of GppNHp (data not shown). Moreover,   NOVEMBER 11, 2011 • VOLUME 286 • NUMBER 45 the backbone trajectories of this residue were moderately biased and similar to the orientation of H-Ras⅐GppNHp state 2 (Fig. 4C), suggesting a high probability of forming the Gly-60-␥-phosphate hydrogen bond in the solution structure of H-RasT35S⅐GppNHp. This gained a strong support from the fact that the formation of the Gly-60-␥-phosphate hydrogen bond was actually observed in the x-ray structure, H-RasT35S⅐GppNHp form 2 (13). The results collectively indicated that switch I of H-RasT35S⅐GppNHp exhibits a wide range of structural variations, whereas switch II is moderately constrained by some non-local interactions including the hydrogen bond with GppNHp.

Solution Structures and Dynamics of GTP-bound H-Ras
Backbone Amide Dynamics-To assess the mobility of the poorly defined loops in H-RasT35S⅐GppNHp more rigorously, we studied the backbone dynamics by measuring 15 N relaxation times and heteronuclear NOEs. For the study of protein dynamics, T 1 values and heteronuclear NOEs depend on internal motions occurring at high frequencies of 10 8 -10 12 s Ϫ1 , whereas T 2 values are also sensitive to much lower frequency motions (10 3 -10 6 s Ϫ1 ) (31). Plots of these relaxation parameters for H-RasT35S⅐GppNHp clearly showed that the regions comprising the residues 31-42, 61-75, 107-109, and 121-123 exhibit significant rapid internal motions on the picosecond to nanosecond time scale, i.e. lower values of T 1 and heteronuclear NOE in contrast to higher T 2 values (Fig. 5A). The backbone dynamics and structural orientations in the residues 107-109 (L7 loop between ␣3 and ␤5) and 121-123 (L8 loop between ␤5 and ␣4) were almost identical to those of H-Ras-GDP (data not shown) (30), indicating that the motions of these regions occur irrespective of the nucleotide type. Both switch I and switch II of state 1 were shown to be flexible as predicted from the x-ray analyses. This implied that the genuine mobilities, not the lack of experimental restraints, are the reason why the two switch regions are ill-defined in the solution structures of H-RasT35S⅐GppNHp. The relaxation data of H-RasT35S⅐GppNHp were drastically different from those of H-Ras⅐GppNHp, of which 15 N T 2 values in the switch regions are extremely short due to the slow internal motions on the millisecond time scale (19). Therefore, the mobilities of the switch regions of H-RasT35S⅐GppNHp are close to those of H-Ras-GDP rather than those of H-Ras⅐GppNHp. Nevertheless, it is likely that the chemical exchange contribution partially remains as the 15 N T 2 values of Asp-33, Ser-35, and Glu-37 are significantly shorter than those of the other residues (Fig. 5A). Next we examined how binding to c-Raf-1 RBD affects the internal motions of H-Ras⅐GppNHp. In the presence of c-Raf-1 RBD, 15 N T 1 , T 2 , and heteronuclear NOE values for most of the residues in the P-loop and the switch regions reached near the average value (Fig. 5B). However, the 15 N T 2 values for Asp-30, Thr-35, and Tyr-40 in or near switch I and Gly-60 in switch II were significantly shorter than the average, suggesting that the exchange contribution was not completely excluded even in the presence of 1.2-fold excess of the effector.

Slow Conformational Dynamics of the State Transition and Rapid Internal Motions Inherent in Each
State-In the backbone amide 1 H, 15 N HSQC spectra of H-Ras⅐GppNHp, the measurements at 5°C result in the generation of two new signals for several individual residues that are unobservable at 25°C and induction of peak splitting for other several residues that yield single peaks at 25°C. These phenomena are typical of a two-site chemical exchange with a temperature-dependent decrease in the exchange rate. In either case, the two resonance peaks coincide very well with the peaks of state 1 and state 2, which are separately identified by using H-RasT35S⅐GppNHp and the H-Ras⅐GppNHp⅐c-Raf-1 RBD complex, respectively. Moreover, the mean value of the intensity ratios of the split peaks is close to the equilibrium constant between state 1 and state 2. These results clearly indicate that the slow exchange process observed in 1 H and 15 N NMR of H-Ras⅐GppNHp does correspond to the interconversion between state 1 and state 2.
The spatial distribution of the dynamic residues indicates that the state transition involves global conformational rearrangement centered around the effector interface (Fig. 2C). Global conformational dynamics of the GTP-bound H-Ras in the millisecond time scale was previously suggested from the analysis of the backbone amide 15 N spin relaxation relaxationcompensated Carr-Purcell-Meiboom-Gill measurements (21), which identified Ser-17 in the P-loop, Gly-75 in switch II, and several residues in the other regions as the residues involved. Our study, which deals with the residues showing peak splitting in the backbone amide 1 H, 15 N HSQC spectra, is capable of detecting particularly dynamic residues upon the state transition. First, we reveal the dynamics of the residues whose signals were previously unobservable: Gly-10, Gly-12, and Gly-13 in the P-loop, Val-29 in the switch I-flanking region, Gly-60 in switch II, and Ile-21 and Asp-54 in the structured regions. Furthermore, we extend our analysis to the residues whose amide protons, 1 H N , undergo the slow exchange process: Gly-15 in the P-loop, Thr-148 in the other loops, and Val-8, Gln-25, and Leu-79 in the rigidly structured regions. A chemical shift difference value between the split peaks, ⌬␦, reflects a chemical environmental change upon the state transition. We observe notable differences in multiple residues, Gly-10, Gly-12, Gly-13, and Gly-15, in the P-loop in addition to the residues in the switch II loop and the regions flanking the switch I and switch II (Fig. 2B). This is rather unexpected because the P-loop does not display a significant conformational difference between the x-ray structures of H-RasT35S⅐GppNHp (state 1) and H-Ras⅐GppNHp (state 2) (data not shown) and because the backbone 15 N dynamics of this loop fails to show any conformational flexibil-ity in both state 1 and state 2 (Fig. 5, A and B). We speculate that the large chemical shift differences of the P-loop residues may be caused by the effect of the conformational change of their neighboring regions such as switch II rather than their intrinsic conformational changes. Furthermore, we detect Gln-95 in the ␣3-helix as a residue showing peak splitting, suggesting that the ␣3-helix undergoes a conformational change upon the state transition. This is supported by the result of the 15 N spin relaxation relaxation-compensated Carr-Purcell-Meiboom-Gill measurements, showing that the residues 89 -90, 93-96, and 98 in the ␣3-helix are involved in the slow dynamics (21). Because the ␣3-helix is also located adjacent to switch II, these residues may be sensitive to the conformational change of switch II. It was reported that certain amino acid substitutions in the P-loop (8) and the ␣3-helix (32) caused substantial shifts of the conformational equilibrium between state 1 and state 2, supporting our notion that both the P-loop and the ␣3-helix interact with switch II and facilitate the transition of state 1 to state 2.
Analysis of the backbone dynamics involving rapid internal motions of the GTP-bound H-Ras has been hampered by the existence of the slow conformational exchange process, namely the state transition. In this study the effect of the state transition is mostly excluded by using a T35S mutant and a complex with c-Raf-1 RBD for extraction of the dynamics intrinsic to state 1 and state 2, respectively. The 15 N T 1 , T 2 , and heteronuclear NOE values of H-RasT35S⅐GppNHp clearly show that the switch regions exhibit significant rapid internal motions on the picosecond to nanosecond time scale, which provide state 1 with further flexibility. It is intriguing that T35S replacement in switch I, which weakens interactions with the nucleotide (9, 13), makes both the switch regions flexible. On the other hand, the relaxation data of the H-Ras⅐GppNHp in complex with c-Raf-1 RBD indicate that most of the residues in the switch regions in state 2 are as rigid as the other structured regions of the protein. This result is supported from the state 2-specific conformational feature, in which guanine nucleotide-mediated interactions between switch I and switch II fix these regions (13). Consequently, the backbone dynamics of the two states suggest that the immobilization or folding of the two switch regions through these interactions is a cooperative process. Interestingly, another signaling protein, NtrC r , has similar conformational features in its active and inactive states (33), i.e. multi-conformations exist in the inactive state, whereas these are mostly deselected upon activation, although NtrC r is activated by phosphorylation of its receiver domain. In the case of Ras, because the effector binding stabilizes state 2 and eliminates backbone conformations inherent in the highly mobile state 1 conformer, it would appear to have a large entropy cost in addition to the decrease in the rotational and translational entropy associated with binding of two protein molecules (34). This unfavorable entropy loss would be compensated mainly by a favorable enthalpy effect (35). Furthermore, the fast sidechain dynamics in Ras may also contribute to free energy of the effector binding (36). For example, other signaling proteins, calmodulin (37) and Cdc42Hs (38), reportedly undergo widespread redistributions in side-chain dynamics upon binding to their target proteins or peptides, suggesting the importance of side-chain entropy in extensive regions centered around the protein-protein interfaces. Therefore, in addition to the dynamic behavior of the main chain in proteins, which is characterized in our experiments, residual conformational entropy arising from changes in the fast side-chain dynamics might be significantly connected to target binding of signaling proteins.  (Fig. 4B). In sharp contrast, the switch I loop of H-RasT35S⅐GppNHp exhibits a marked fluctuation compared with that of H-Ras-GDP. The large fluctuation resulting from the loss of the Thr-35-␥-phosphate interaction is supported by a structural study of H-Ras in state 1 using molecular dynamics simulations (39). Furthermore, some of the 20 backbone trajectories of switch I extend toward the structure of the nucleotide-free form in complex with Sos (Fig. 4A). X-ray structure analyses of the H-Ras-Sos complex showed the conformational features of the nucleotide-free H-Ras (16,40); 1) the switch I loop is further pulled away from the nucleotide-binding site by the insertion of the helical hairpin segment of Sos, and 2) switch II is held very tightly by Sos, and large interfaces involving switch II are formed through numerous side-chain interactions in the complex.

State 1-specific Conformation of the Switch Regions and Its
We reason that the structural feature of H-RasT35S⅐GppNHp, capable of moving the switch I loop away from the nucleotide to an extent similar to that of the nucleotide-free form, may give an energetic advantage for stabilization of the H-Ras-Sos complex. To test our hypothesis, H-Ras/Sos binding was measured by in vitro binding assay and isothermal titration calorimetry using mSos1W729E possessing the intact guanine nucleotide exchange factor catalytic site, whereas its distal Ras-binding site is inactivated. As a result, H-RasT35S⅐GppNHp exhibits a stronger Sos binding activity than that of H-Ras⅐GppNHp (supplemental Fig. 2 and 3), supporting our hypothesis. However, its binding activity is weaker than that of H-Ras-GDP (supplemental Fig. 2 and 3), which appears to contradict the conformational similarity of the switch I loop with the nucleotide-free form. We speculate that this discrepancy may be accounted for by the difference in the mobility of the switch II loop, which is constrained presumably by the Gly-60-␥-phosphate interaction in H-RasT35S⅐GppNHp but not in H-Ras-GDP (Fig. 4, B and C). The structures of Ala-59 and Gln-61 neighboring Gly-60 are also significantly altered in H-RasT35S⅐GppNHp; the side chain of Ala-59 orients away from the nucleotide, whereas that of Gln-61 orients toward the nucleotide (Fig.  4C). The side-chain orientations of Ala-59 and Gln-61 in H-RasT35S⅐GppNHp include those of the corresponding residues in the state 2 structure of H-Ras⅐GppNHp and exhibit a nearly opposite orientation to those in the nucleotide-free H-Ras in the H-Ras-Sos complex (Fig. 4C). In contrast, the side-chain orientations of Ala-59 and Gln-61 of H-Ras-GDP include those similar to the nucleotide-free form. Thus, it is likely that the structural features of the switch II loop of H-RasT35S⅐GppNHp would make a substantial disadvantage for the interaction with Sos, accounting for its weaker binding activity to Sos than H-Ras-GDP.
In conclusion, our study has successfully determined the solution structure and dynamics of state 1 by NMR analysis of H-RasT35S⅐GppNHp, which faithfully reflects those of H-Ras⅐GppNHp as proved by the coincidence of the assigned signals between the two proteins. The structural information on state 1 of wild-type Ras, unveiled for the first time in this study, will provide an invaluable tool for the structure-based drug design of Ras inhibitors.