Báo cáo khoa học: Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPcS pot

Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPcS Michael Spoerner 1 , Andrea Nuehs 1 , Christian Herrmann 2 , Guido Steiner 1 and Hans Robert Kalbitzer 1 1 Universita ¨ t Regensburg, Institut fu ¨ r Biophysik und physikalische Biochemie, Germany 2 Ruhr Universita ¨ t Bochum, Physikalische Chemie I, Germany Guanine nucleotide-binding proteins of the Ras super- family function as molecular switches, cycling between a GDP-bound ‘off’ and a GTP-bound ‘on’ state. They regulate a diverse array of signal transduction and transport processes. It has been shown using 31 P NMR spectroscopy that Ras (rat sarcoma) protein occurs in two con- formational states (state 1 and 2) when complexed with the GTP analogues guanosine-5¢-(b,c-imido)tri- phosphate (GppNHp) [1] or guanosine-5¢-(b,c-methy- leno)triphosphate (GppCH 2 p) [2]. These two states interconvert with rate constants in the millisecond time scale. They are characterized by typical 31 P NMR chemical shifts, with shift differences up to 0.7 p.p.m. NMR structural studies have shown that this dynamic equilibrium comprises two regions of Keywords conformational equilibria; GTP analog; GTPcS; Ras Correspondence H. R. Kalbitzer, Institut fu ¨ r Biophysik und physikalische Biochemie, Universita ¨ tsstraße 31, Regensburg, D-93040, Germany Fax: +49 941 943 2479 Tel: +49 941 943 2595 E-mail: hans-robert.kalbitzer@biologie. uni-regensburg.de (Received 28 July 2006, revised 13 Novem- ber 2006, accepted 8 January 2007) doi:10.1111/j.1742-4658.2007.05681.x 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¢-(b,c-imido)tri- phosphate or guanosine-5¢-(b,c-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) (GTPcS) leads to 31 P NMR spectra that indicate the prevalence of only one con- formational 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 GTPcS 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 tempera- tures an activation energy DH | 1a1b of 61 kJÆmol )1 and an activation entropy DS | 1a1b of 65 JÆ K )1 Æmol )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 com- pared to the wild-type with the strongest reduction observed for the GTPcS complex. Abbreviations GppCH 2 p, guanosine-5¢-(b,c-methyleno)triphosphate; GppNHp, guanosine-5¢-(b,c-imido)triphosphate; GTPcS, guanosine-5¢-O-(3- thiotriphosphate); ITC, isothermal titration calorimetry; Raf-RBD, Ras-binding domain of Raf-kinase; Ras, protein product of the proto oncogene ras (rat sarcoma). FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1419 the protein called switch I and switch II [1,3,4]. Solid- state NMR shows that even in single crystals or crys- tal powders of Ras(wt)•Mg 2+ •GppNHp the two conformational states can be observed to be in dyna- mic equilibrium at ambient temperatures [5,6]. A threonine residue located in the effector loop (Thr35 in Ras) is conserved in all members of the Ras superfamily and seems to play a pivotal role in the conformational equilibrium. It is involved, via its side-chain hydroxyl, in the coordination of the diva- lent metal ion and, via its main-chain amide, in a hydrogen bond with the c-phosphate of the nucleo- tide when complexed to the effector [7,8]. The same coordination pattern is most probably preserved in state 2 of free Ras. Replacing this threonine in Ras with an alanine or serine residue leads to a complete shift of the equilibrium towards state 1 in solution, when Ras is bound to the GTP analogues GppNHp [9] or GppCH 2 p [2]. These Ras variants, previously used as partial loss-of-function mutants in cell-based assays, show a reduced affinity between Ras and effector proteins without Thr35 being involved in any interaction. X-Ray crystallography [9] on Ras(T35S)•Mg 2+ •GppNHp and EPR investigations [10] suggest that switch I and switch II exhibit high mobility in state 1. Recently, X-ray structures of M-Ras [11] and of the G60A mutant of human H-Ras [12], both in the GppNHp-bound form, were published. These Ras variants seem to exist in conformational state 1, as shown using 31 P NMR spectroscopy. In the X-ray structure the contacts of Thr35 (Thr45 in M-Ras) with the metal ion and the c-phosphate group do not exist. 31 P NMR data indi- cate that state 2 corresponds to the conformation of Ras found in complex with the effectors. State 1, characteristic of the mutants Ras(T35S) and Ras(T35A) in the GppNHp form, represents a weak- binding state of the protein [9,13]. Upon addition of the Ras effector Raf-kinase, the 31 P NMR lines of Ras(T35S) but not Ras(T35A) shift to positions cor- responding to the strong binding conformation of the protein [9]. A conformational equilibrium in the interaction site with effectors seems to be a general property of Ras and other small GTPases [14]. The equilibrium is influ- enced not only by specific mutations but also by the nature of the GTP analogue bound (GppNHp or GppCH 2 p). In this study we investigate the dynamic behaviour of Ras in complex with guanosine-5¢-O-(3- thiotriphosphate) (GTPcS), another commonly used GTP analogue that is hydrolysed slowly to find more evidence for the biological importance of the conform- ational equilibria. Results Chemical shifts of the nucleotide analogue GTPcS in the absence and in the presence of magnesium ions Chemical shift values for the phosphates and the thio- phosphate group of the nucleotide depend strongly on the degree of protonation of their oxygens. Further- more, chemical shifts and pK values are influenced by Mg 2+ binding to the protein–nucleotide complex. For a better interpretation of the chemical shifts of the protein-bound nucleotide analogue we first studied GTPcS in the presence and absence of Mg 2+ ions within a pH range of 2–13. The rate of exchange between Mg 2+ and the nucleoside triphosphate is slow enough to observe the resonances of the metal-free form separately from the metal-complexed form at lower temperatures. Therefore, experiments were per- formed at 278 K to ensure that over the whole pH range a significant contribution of metal-free nucleo- tide, if existing, could be directly detected by addi- tional resonance lines. At a magnesium concentration of 3 mm the nucleotide is completely saturated with the divalent ion in the pH range studied since further increase of the Mg 2+ concentration does not influence the observed chemical shifts (also see Experimental procedures). Figure 1 shows the titration curves for GTPcSin the absence and presence of Mg 2+ . Separation of the three phosphate signals by more than 60 p.p.m. is rather large. Particularly in case of the c-phosphorus (Fig. 1A,B) two pK values are necessary in order to describe the observed dependence of chemical shifts in the pH range studied. The corresponding pK values and chemical shifts are summarized in Table 1 together with the data for the analogues GppNHp and GppCH 2 p [2]. As expected, the apparent pK values decrease substantially in the presence of the metal ion. By far the largest effect on the chemical shifts is found for the b- and c-phosphate group, but a slight shift of 0.6 p.p.m. is also seen for the a-phosphorus resonance in the Mg 2+ •GTPcS complex. In agreement with pre- vious studies on ATP [15], our data suggest a mixture of different metal complexes in solution with a high population of complexes where the b- and c-phosphate is involved, as shown previously for the GTP ana- logues GppNHp and GppCH 2 p [2]. The pK 3 values in GTPcS are much smaller than those reported for GppNHp and GppCH 2 p. The value of pK 2 does not depend much on the analogue when a relatively large error is taken into consideration. pK 2 and pK 3 are usually associated with the first and the second Conformational dynamics of Ras bound to GTPcS M. Spoerner et al. 1420 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS deprotonation step at the c-phosphate group of the nucleotide for the transition from the threefold negat- ively charged state to the fourfold negatively charged state. In line with this suggestion the largest shifts are observed for the c-phosphate group for the first deprotonation step for the three analogues. However, the second deprotonation step is associated with larger changes in the b-phosphate shifts in GppNHp and GppCH 2 p, indicating a more complex pH perturbation of the electronic system in these analogues. Fig. 1. Titration curves of free and Mg 2+ bound GTPcS. (A,C) 31 P chemical shift val- ues of the a-, b- and c-phosphate groups were determined on a 2.5 mL of a 1 m M GTPcS solution in 100 mM Tris, 95% H 2 O and 5% D 2 O containing 0.1 mM 2,2-dimeth- yl-2-silapentane-5-sulfonate for indirect refer- encing. The pH was adjusted by adding HCl or NaOH. Measurements were performed in a 10-mm sample tube at 278 K. (B,D) Meas- urements on the Mg 2+ complexes were per- formed in the presence of 3 m M MgCl 2 . The dependence of chemical shifts on the pH values was fitted to Eqn (7). The 31 P reso- nances were assigned by selective 1 H- and 31 P-decoupling experiments. Table 1. pH dependence of chemical shifts of different GTP analogues. Data were recorded at 278 K in solutions of 1 mM nucleotide in the absence or presence of 3 m M MgCl 2 in 95% H 2 O ⁄ 5% D 2 O. In a first approximation d 2 , d 3 , and d 4 correspond to the chemical shifts of two-, three-, and fourfold negatively charged nucleotide. pK 2 and pK 3 are the corresponding pK a values of the three phosphates of the nucleotide. d 2 values are given in parentheses the titration up to pH 1.5 does not allow the precise estimation of this value. For d 3 and d 4 the estimated error is ± 0.05 p.p.m. Nucleotide Phosphate group d 2 ⁄ p.p.m. pK 2 d 3 ⁄ p.p.m. pK 3 d 4 ⁄ p.p.m. GTPcS a ()11.3) ) 11.30 ) 11.04 b ()24.0) 2.8 ± 0.1 ) 24.0 5.78 ± 0.02 ) 23.06 c (40.8) 39.70 33.91 Mg 2+ •GTPcS a ()11.2) ) 11.27 ) 10.67 b ()24.2) 1.7 ± 0.5 ) 23.78 ) 20.51 c (41.6) 40.38 4.11 ± 0.02 36.85 GppCH 2 p a a ()10.86) ) 10.93 ) 10.82 b (7.14) 3.2 ± 0.15 8.74 8.96 ± 0.02 13.22 c (17.85) 14.63 6.57 ± 0.02 11.23 Mg•GppCH 2 p a a ()10.83) ) 10.47 ) 10.33 b (9.50) 2.3 ± 1.5 9.93 14.93 c (16.98) 14.29 11.46 GppNHp a a ()10.95) ) 10.80 8.79 ± 0.02 ) 10.55 b ()12.27) 3.4 ± 0.04 ) 10.91 ) 7.76 c (0.20) ) 1.64 ) 0.91 Mg•GppNHp a a ()11.17) ) 10.34 6.56 ± 0.02 ) 10.01 b ()9.36) 2.0 ± 0.8 ) 8.95 ) 5.46 c ()1.38) ) 2.16 ) 1.02 a Data from Spoerner et al. [2]. M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1421 Conformational states of Ras complexed with Mg 2+ •GTPcS Figure 2 shows 31 P NMR spectra of Ras(wt) in com- plex with the slowly hydrolysable GTP analogue GTPcS at various temperatures. Assignment of the res- onance lines was confirmed by a 2D 31 P– 31 P NOESY experiment on Ras(wt)•Mg 2+ •GTPcS (data not shown). Binding of GTPcS to the Ras protein leads to rather large chemical shift changes. In contrast to the observations made for the GTP analogues GppNHp and GppCH 2 p [1,2] only one set of resonances can be observed for the wild-type protein in the temperature range 278–308 K (Fig. 2). This most probably means that wild-type Ras occurs predominantly in one state when GTPcS is bound. It is reasonable to assume that a second structural state also exists and is character- ized by different chemical shift values, as observed in the GppNHp and GppCH 2 p complexes [1,2]. When this second state has clearly different chemical shifts compared with the first state then two scenarios are consistent with the observed spectrum. If fast exchange conditions prevail over the whole temperature range, then only one averaged resonance signal per phosphate group would be observed. If slow exchange conditions prevail, a second conformational state, characterized by clearly different chemical shifts, must have a rather low population because no signals can be detected above noise level. In this case, from the signal-to-noise ratio the equilibrium constant for the two states can be estimated to be > 10. Analysing the temperature dependence of the line width, particularly of the c-phosphorus resonance, slow exchange conditions are more likely. At lower temperatures the line width decreases with increasing temperature due to the decrease of the rotational correlation time. At higher temperatures the line width increases again (51 Hz at 298 K, 57 Hz at 303 K). Chemical shift also changes within the temperature range of 278–308 K by +0.26 p.p.m. At higher temperatures, the GTP ana- logue hydrolyses, and resonances of Ras-bound GDP are thus detected. In principle, one would expect to observe thiophosphate and Ras-bound GDP as result of GTPcS hydrolysis. In contrast, with all the meas- urements performed in this study, inorganic phosphate could be observed only using 31 P NMR. In addition, H 2 S could be detected by its smell after a time. The exact mechanism of thio phosphate decay could not be clarified. It is dependent on the presence of Ras, but may be also due to other protein impurities occurring in low concentrations in the Ras preparations. In con- trast to the situation observed for wild-type protein in the complex of GTP cS with the mutant Ras(T35S) or Ras(T35A), additional 31 P NMR lines are found at low temperature (Fig. 3A). With increasing tempera- ture, the lines initially become broader before coales- cing again at higher temperature (Fig. 4A). From our studies with GppNHp and GppCH 2 p we expect that the effector interaction state 2 becomes destabilized by replacing Thr35 with a serine or an alanine residue, and therefore at least one of the new lines seen in the mutant is likely to correspond to state 1. Because no component of the two sets of resonances of Ras(T35S) and Ras(T35A) has a chemical shift that corresponds to that of Ras(wt) it is not clear whether the two sets of resonance lines correspond to state 1 and state 2 or if they represent two substates of state 1 (see below). In the following, we call them state 1a and state 1b. The equilibrium constant K 1a1b ¼ [1b] ⁄ [1a] between these two states is 0.5. In the case of the serine mutant, a weak third line of the c-phosphorus signal with a similar chemical shift to the resonance of wild-type Ras seems to exist (Fig. 3A); this is not visible in the spectrum of the T35A mutant. The chemical shifts are summarized in Table 2. With knowledge of the resonance positions corres- ponding to state 1a and 1b, we investigated whether these states also exist in wild-type Ras bound to GTPcS. Separation of the chemical shift values between state 1b and state 2 of more than 4 p.p.m. allowed us to perform a saturation-transfer experiment with presaturation at frequencies around the signal corresponding to state 1b. If exchange occurs over a Fig. 2. 31 P NMR spectra of wild-type Ras complexed with Mg 2+ •GTPcS at various temperatures. The samples contained 1m M Ras(wt)•Mg 2+ •GTPcSin40mM Hepes ⁄ NaOH pH 7.4, 10 m M MgCl 2 , 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate in 5% D 2 O, 95% H 2 O, respectively. The absolute temperature was controlled by immer- sing a capillary with ethylene glycol and measuring the hydroxyl- methylene shift difference [28]. Conformational dynamics of Ras bound to GTPcS M. Spoerner et al. 1422 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS timescale < T 1 a decrease in the integral of the reson- ance corresponding to state 2 should be observed, even when state 1 is too sparsely populated to be detectable directly. Some results are shown in Fig. 3B. A mini- mum of the resonance integral of state 2 is obtained at a presaturation frequency of 32.7 p.p.m., which corres- AB Fig. 3. Conformational equilibria of wild-type Ras and Ras mutants complexed with Mg 2+ •GTPcS. (A) The sample contained 1 mM Ras(wt)•Mg 2+ •GTPcS (lower), 1.2 mM Ras(T35S)•Mg 2+ •GTPcS (middle), and 1 mM Ras(T35A)•Mg 2+ •GTPcS (upper) in 40 mM Hepes ⁄ NaOH pH 7.4, 10 m M MgCl 2 , 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate in 5% D 2 O, 95% H 2 O, respectively. Data were recorded at 278 K. The assignment was determined by a 31 P– 31 P NOESY experiment on Ras(wt)•Mg 2+ •GTPcS. 31 P resonances assigned to Ras–nucleotide complex in conformation of state 1a or state 1b are coloured in red, the resonances assigned to state 2 are coloured green. (B) 31 P NMR saturation transfer experiment on Ras(wt)•Mg 2+ •GTPcS. The integrals of the resonance correspond- ing to the c-thiophosphate group in state 2 of Ras(wt) are given in dependence of the frequency of presaturation d. For presaturation a weak rectangular pulse of 1 s duration and a B 1 -field of 18 Hz were used. A Lorentzian function was fitted to the data. The integral of the c-phos- phorus signal without presaturation is set to 100%. Fig. 4. Experimental and simulated 31 P NMR data of Ras(T35S)•Mg 2+ •GTPcS at different temperatures. The sample contained 1.2 mM Ras•Mg 2+ •GTPcSin40mM Hepes ⁄ NaOH pH 7.4, 10 mM MgCl 2 ,2mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate in 5% D 2 O, 95% H 2 O. The absolute temperature was controlled by immersing a capillary with ethylene glycol and measuring the hydroxyl– methylene shift difference [28]. (A) Experimental spectra; (B) simulated spectra. Experimental data were filtered by an exponential filter lead- ing to an additional line broadening of 5 Hz. Total number of scans per spectrum were 1600–5400. The rate constant for the transition state 1a to state 1b are indicated. Data were simulated as described in Experimental procedures. The transverse relaxation rates 1 ⁄ T 2 at 278 K (in the absence of exchange) obtained from the data analysis are 251 s )1 for both state 1a and state 1b of the a-phosphate group of bound GTPcS, 236 s )1 and 204 s )1 for the b-phosphate group of bound GTPcS in state 1a and state 1b, respectively, and 189 s )1 for state 1a and 1b of the bound c-thiophosphate group (values are given with an estimated error of ± 15 s )1 ). M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1423 ponds to the frequency of state 1b detected for the two Thr35 mutants. These results indicate the existence of state 1b in wild-type Ras, but with a very sparse popu- lation. A more detailed analysis including calculation of exchange rates was not possible because of the lim- ited signal-to-noise. Dynamics of the conformational exchange By analysing the temperature dependence of the 31 P NMR data from Ras(T35S)•Mg 2+ •GTPcS (Fig. 4B) for the transition between substates 1a and 1b the Gibb’s free activation energy DG | , the activation enthalpy DH | and the activation entropy DS | can be determined (Table 3) using a full-density matrix analysis. The exchange rates obtained are somewhat higher than that found between states 1 and 2 of Ras(wt)•Mg 2+ •GppNHp or Ras(wt)•Mg 2+ •GppCH 2 p. Whereas DG | of the exchange in Ras(T35S)•Mg 2+ •GTPcS is equal to that obtained for the other com- plexes, both DH | , and DS | are somewhat lower. For the other nucleotides studied, relaxation times T 2 at 278 K for the a- and c-phosphate group were quite different for the two conformational states 1 and 2. We did not find such large differences between the corresponding T 2 relaxation times for the conformational states 1a and 1b of Ras(T35S)•Mg 2+ •GTPcS. Complex of Ras•Mg 2+ •GTPcS with the Ras-binding domain of Raf-kinase Addition of the Ras-binding domain of Raf-kinase (Raf-RBD) to Ras(wt)•Mg 2+ •GTPcS leads to line broadening of the resonances (Fig. 5, Table 2), but only to very small changes in the chemical shifts (|Dd| £ 0.16 p.p.m). This is in line with the assumption that the wild-type protein occurs mainly in conforma- tional state 2 when the GTP analogue GTPcSis bound. Correspondingly, in Ras(T35S)•Mg 2+ •GTPcS, lines preliminary assigned to states 1a and 1b decrease in intensity when Raf-RBD is bound, whereas the intensity of lines located close to those assigned in wild-type Ras to state 2 increases (Fig. 5, Table 2). The changes in chemical shift induced by Raf binding are rather large in the mutant, suggesting that none of the states visible in the spectrum of Ras(T35S)•Mg 2+ •GTPcS corresponds to state 2 found in the wild-type protein. Complex formation between Raf-RBD and Ras(T35A)•Mg 2+ •GTPcS (Fig. 5, Table 2) leads only to a line broadening of the two lines of the c-phosphate group, and not to significant changes in chemical shift or the relative populations of the resonances. In particular, the relative intensity of the downfield-shifted c-phosphorus resonance is not increased in the presence of the effector as would be expected if it corresponded to effector binding state 2. Influence of the GTP analogue on the affinity between Raf-RBD and Ras The affinities of wild-type and (T35S)Ras complexed with the different GTP analogues GppNHp, GppCH 2 p and GTPcS to Raf-RBD were determined using isother- mal titration calorimetry (ITC) at 298 K in a buffer identical to that used in the NMR spectroscopy experiments. Within the limits of error, the effective Table 2. 31 P chemical shifts and conformational states of Ras complexed with different GTP analogues. Data were recorded at various tem- peratures. Shifts were taken from spectra recorded at 278 K. The equilibrium constant K 12 between state 1 and 2 is calculated from inte- grals of the c-thiophosphate resonances defined by K 12 ¼ k 12 ⁄ k 21 ¼ [2]] ⁄ ([1a] + [1b]). State 2 is assigned to the conformation close to the effector binding state. The error is < 0.03 p.p.m. for the chemical shifts and < 0.1 for the equilibrium constants. ND, not detected. Ras-complex a-phosphate b-phosphate c-phosphate K 12 K 1a1b d 1 (p.p.m.) d 2 (p.p.m.) d 1 (p.p.m.) d 2 (p.p.m.) d 1 (p.p.m.) d 2 (p.p.m.) Ras(wt)•Mg 2+ •GTPcS )11.30 )16.67 37.01 > 10 ND b Ras(T35S)•Mg 2+ •GTPcS )10.70 )17.96 a )17.22 a 32.73 a 37.89 a 36.87 0.06 0.5 Ras(T35A)•Mg 2+ •GTPcS )10.80 )17.92 a 32.79 a < 0.05 0.5 )17.19 a 37.91 a Ras(wt)•Mg 2+ •GTPcS )11.19 )16.55 36.85 > 10 ND b + Raf-RBD Ras(T35S)•Mg 2+ •GTPcS )11.22 )16.52 36.54 > 10 ND b + Raf-RBD Ras(T35A)•Mg 2+ •GTPcS )10.50 )17.55 32.48 a < 0.05 0.5 + Raf-RBD 37.91 a a Chemical shifts in state 1a (lower) and 1b (upper). b Values could not be determined since signal cannot be detected. Conformational dynamics of Ras bound to GTPcS M. Spoerner et al. 1424 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS association constant K A between wild-type Ras and Raf-RBD is not influenced by the type of bound ana- logue (Table 4). However, in all cases, the contributions of enthalpy and entropy to DG° differ between nucleo- tide analogues. Although for the Thr35 mutant the error ranges for the three nucleotide analogues overlap, a dif- ference in affinities between Ras(T35S) bound to the analogue GTPcS, where the oxygen between b- and c- phosphate is still available, and GppCH 2 p may exist. A significant decrease in K A , by a factor of $ 20, is seen, independent of the analogue used when the wild-type protein is compared with Ras(T35S). The decrease in affinity is due to changes in DH° and DS°, which partly compensate. Discussion The environment of the nucleotide bound to the protein NMR spectroscopy very sensitively reports changes in the environment of a given atom by measuring a change in its resonance frequency. Whenever chemical shift changes are visible they indicate that there is a change in the environment of the observed nucleus. For phosphorus resonance spectroscopy on nucleo- tides, it is known that two factors mainly determine chemical shift changes, a conformational strain and electric field effects polarizing the oxygen atoms of the phosphate groups. In addition to these direct effects, long-range effects may occur that are caused by a structure-dependent change in the anisotropy of the magnetic susceptibility. Here, ring current effects may be the most dominant contribution. We have previously studied the complexes of Ras using the GTP analogues GppCH 2 p and GppNHp [2], which differ in the position of the b–c-bridging oxygen by replacing the naturally occurring oxygen either with an apolar group or a hydrogen-bond donator. We have now completed the picture using the slowly hydrolysing GTP analogue GTPcS, in which the b–c- bridging oxygen is not affected, but the physicochemi- cal properties of the c-phosphate group are modified. For a quantitative analysis of the chemical shift chan- ges induced by protein binding it was necessary to have reliable data for the system not perturbed by Table 3. Exchange rates and thermodynamic parameters in different Ras–nucleotide complexes. The rate constants k 12 and k 21 (k 1a1b and k 1b1a ) were calculated by a line-shape analysis based on the density matrix formalism as described in Experimental procedures. The free acti- vation energy DG | , the activation enthalpies DH | , and the activation entropies DS | , were calculated from the temperature dependence of the exchange rates on the basis of the Eyring equation. The values for the transition between state 1 and state 2 k 12 and k 21 are given. The states are defined as in Table 1. DG 12 or DG 1a1b is the difference in free enthalpy between state 2 (1b) and 1 (1a). T 2 times given are without exchange contribution and were obtained from the line shape analysis. The estimated error is ± 0.3 ms. Protein complex Temp. (K) Exchange rate constant (s )1 )_ DG j 1a1b DH j 1a1b TDS j 1a1b DG 1a1b k 1a1b k 1b1a (kJÆmol )1 ) (kJÆmol )1 ) (kJÆmol )1 ) (kJÆmol )1 ) Ras(T35S)•Mg 2+ •GTPcS 278 70 137 41 ± 2 61 ± 1 18 ± 1 1.56 ± 0.15 a 288 170 330 298 430 810 k 12 k 21 DG j 12 DH j 12 TDS j 12 DG 12 Ras(wt)•Mg 2+ •GppNHp a 278 80 42 42 ± 5 70 ± 3 28 ± 2 ) 1.48 ± 0.15 288 250 135 298 700 387 Ras(wt)•Mg 2+ •GppCH 2 p a 278 80 39 41 ± 5 63 ± 3 29 ± 2 ) 1.65 ± 0.15 288 260 131 298 740 391 Relaxation times T 2 (ms) of the resonances of Protein-complex a-phosphate b-phosphate c-phosphate (1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b) Ras(T35S)•Mg 2+ •GTPcS 278 4.0 4.0 4.2 4.9 5.3 5.3 Ras(wt)•Mg 2+ •GppNHp a 278 5.8 3.9 4.8 4.8 4.1 7.1 Ras(wt) •Mg 2+ •GppCH 2 p a 278 4.2 4.0 6.4 6.4 3.8 5.2 a Data from Spoerner et al. [2]. Note that the values given differ somewhat from those given by Geyer et al. [1] because absolute tempera- ture was controlled independently and the new assignment of the signals were considered. M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1425 protein binding that we provide here. Although data had been published previously for free GTPcS [16], they were measured under different experimental conditions and the referencing system (external stand- ard) in particular is not sufficiently reliable for precise comparisons. When one compares the chemical shift changes Dd in the free Mg 2+ –nucleotide complexes (Table 1) with those induced by protein binding (Table 2) one may obtain information on the change of the environment of the phosphate groups in the different complexes. In wild-type Ras in state 2, one finds Dd values of )0.26, 6.39 and 3.10 p.p.m., respectively for the a-, b- and c-phosphate of GTPcS. The corresponding shift chan- ges are )1.15, 7.51 and )2.41 p.p.m. for GppNHp and )2.44, 6.32 and )3.03 p.p.m. for GppCH 2 p. The a-phosphate groups in the three GTP analogues should be least influenced by the modifications. In accordance with this observation, in the absence of protein, their response to a change in pH (acidity) is very small, only an upfield shift of < 0.26 p.p.m. is observed when the c-phosphate group is protonated by a decrease in pH. After binding to the protein, for all three analogues an upfield shift between of 0.26 and 2.44 p.p.m. is observed, indicating that the environmental changes are qualitatively similar but differ in detail. Potential phosphate group interactions can be derived from the published X-ray structures, although one should be aware that they show differences in effector loop details that may reflect the occurrence of different conformational states in solution. Because NMR data indicate that the interaction of Ras with Raf-RBD stabilizes the effector loop in a well- defined, state 2-like conformation, the X-ray structure of the Ras-like mutant of Rap1A, called Raps [Rap(E30D,K31E)], complexed with Mg 2+ •GppNHp and Raf-RBD [7] can serve as a model. The most important interactions derived from the X-ray structure are depicted in Fig. 6. It is assumed to represent state 2 of the protein. Interactions assumed to be absent in state 2 and ⁄ or weakened (or abolished) by the replacement of an oxygen atom with a sulfur Fig. 5. 31p NMR spectra of wild-type Ras and Ras mutants bound to Mg 2+ •GTPcS in complex with Raf-RBD. Initially the samples con- tained 1.0 m M Ras•Mg 2+ •GTPcS (lower), 1.2 mM Ras(T35S)• Mg 2+ •GTPcS (middle) or 1.0 mM Ras(T35A)•Mg 2+ •GTPcS (upper) in 40 m M Hepes ⁄ NaOH pH 7.4, 10 mM MgCl 2 , 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfo- nate in 5% D 2 O, 95% H 2 O, respectively. A solution of 9.8 mM Raf-RBD dissolved in the same buffer was added in increasing amounts. The molar ratios of Raf-RBD ⁄ Ras are 1.5 for Ras(wt) and 2 in the mutant samples. Data were recorded at 278 K. 31 P reso- nances assigned to Ras–nucleotide complex in conformation of state 1a or state 1b are coloured red, the resonances assigned to state 2 are coloured green. Table 4. Affinities of Raf-RBD to Ras complexed with different GTP analogues. The association constant K A between Raf-RBD and Ras com- plexed with different GTP analogues was determined using ITC. Measurements were performed at 298 K in 40 m M Hepes ⁄ NaOH pH 7.4, 10 m M MgCl 2 , 150 mM NaCl, 2 mM 1,4-dithioerythritol. Data were analysed using ORIGIN FOR ITC 2.9 assuming a 1 : 1 complex formation [28] and DG° ¼ G complex ) G free ¼ -RTlnK A . Raf-RBD complexed with K A (lM )1 ) DG° (kJÆmol )1 ) DH° (kJÆmol )1 ) TDS° (kJÆmol )1 ) Ras(wt)•Mg 2+ •GppNHp 2.50 ± 0.4 )36.5 ± 0.6 )13.4 ± 1.5 23 ± 2.1 Ras(wt)•Mg 2+ •GppCH 2 p 2.50 ± 0.4 )36.5 ± 0.6 )18.4 ± 2.0 18 ± 2.6 Ras(wt)•Mg 2+ •GTPcS 2.44 ± 0.6 )36.4 ± 0.9 )7.5 ± 1.5 29 ± 2.4 Ras(T35S)•Mg 2+ •GppNHp 0.12 ± 0.04 )29.0 ± 0.06 )9.7 ± 1.0 19 ± 1.1 Ras(T35S)•Mg 2+ •GppCH 2 p 0.09 ± 0.04 )28.2 ± 0.06 )15.3 ± 1.5 13 ± 1.6 Ras(T35S)•Mg 2+ •GTPcS 0.18 ± 0.04 )30.0 ± 0.06 )13.6 ± 1.5 16 ± 1.6 Conformational dynamics of Ras bound to GTPcS M. Spoerner et al. 1426 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS atom in the c-phosphate group are represented by bro- ken lines. Influence of the nucleotide bound on the Ras conformational states 31 P NMR spectroscopy allows us to probe the con- formational states of nucleotide-binding proteins, such as Ras-related proteins, which lead to structural rear- rangement in the active centre. In principle, whenever chemical shift changes are visible they indicate that there is a change of the environment of the phospho- rus nuclei, although small changes in structure can lead to large differences in chemical shifts and vice versa. The main mechanisms leading to changes in chemical shifts are conformational strain and electric field effects polarizing the oxygen atoms of the phosphate groups. In addition to these direct effects, long-range effects may occur, caused by a structure- dependent change in the anisotropy of the magnetic susceptibility, with ring current effects making the most dominant contribution. Binding of the different GTP analogues to Ras leads to large changes in chemical shift, namely a strong upfield shift in the a-phosphate resonance and a strong downfield shift in the b-phosphate resonance compared with data from free Mg 2+ –nucleotide complexes (Table 2). In complexes with GTPcS, a relatively small upfield shift of 0.63 p.p.m. is observed for the a-phos- phate resonance and a strong downfield shift of 3.84 p.p.m. is observed for b-phosphate resonance. c-Phosphorus resonances do not show the typical shift changes common to all analogues. Thus, qualitatively the phosphorus of the a-phosphate group in the mag- nesium complexes of GTP and its analogues is less shielded when bound to the protein, whereas the strong downfield shift in the resonance most probably results from strong polarization of the phosphorus– oxygen bonds in the b-phosphate group. Such bond polarization in Ras•Mg 2+ •GppNHp has been dis- cussed by Allin et al. [17], as an explanation of strong infrared shifts seen in the P–O vibrational bands after complexation. It should be mentioned that the degree of shift differences in the chemical shift values cannot be related in a simple way to the degree of conforma- tional change causing this change. Whereas wild-type Ras complexes with the GTP analogues GppNHp or GppCH 2 p exist in a conform- ational equilibrium between two main conformational states 1 and 2, with a K 12 value of $ 2, the complex with the analogue GTPcS obviously exists in predom- inantly only one conformation. It shows the spectral characteristics of state 2 as the effector binding state. (a) The interaction with Ras-binding domains leads AB C Fig. 6. Schematic representation of the coordination sphere of the phosphate groups and the thiophosphate of GTPcS in wild-type and mutant Ras nucleotide complexes. G, guanosine. (A) Coordination that predominantly exists in wild-type protein containing Thr35. (B,C) Other possible complexes with Ras(T35S) or Ras(T35A). Note, that not all contacts between the nucleotide and the protein are included. Bonds that probably exist only in state 1 or are weakened or abolished in the thiophosphate group are represented by broken lines. The sul- fur atom was assumed to be negatively charged as shown previously for free ATPcS [32]. However, in the protein bound nucleotide the charge distribution is probably also influenced by the protein environment and could be thus different in different conformations. M. Spoerner et al. Conformational dynamics of Ras bound to GTPcS FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS 1427 only to small chemical shift changes. (b) Weakening or destruction of the naturally occurring hydrogen- bond interaction of the side-chain hydroxyl group of Thr35 with the metal ion, and of the main-chain amide with the c-phosphate by mutations to serine or alanine leads to large changes in chemical shift. (c) These chemical shift changes can usually be reversed in Ras(T35S) by Raf-binding because serine still con- tains a side-chain hydroxyl, however this is not the case in Ras(T35A). Geyer et al. [1] suggested that in the GTP-bound form, Ras(wt) also exists predomin- antly in one conformation. In terms of the conforma- tional equilibria of Ras, GTPcS seems to be the analogue which is more similar to physiological GTP than both other commonly used analogues GppNHp or GppCH 2 p. Structural states of Ras(T35S) and Ras(T35A) Mutation of Thr35 to serine or alanine leads to two new phosphorus lines of the c-thiophosphate group and the b-phosphate group, which both show charac- teristics of state 1. The two states are in a dynamic equilibrium as evident from their temperature depend- ence. They are therefore assumed to represent sub- states of state 1 and are called states 1a and 1b. The alanine mutation makes coordination of the side chain with the divalent ion typical for state 2 impossible and can therefore only exist in state 1. In the serine mutant, metal ion coordination is perturbed but still possible. It shows, in addition to lines assigned to sub- states 1a and 1b, a very weak line at the position of the c-phosphate resonance in wild-type Ras, suggesting that Ras(T35S) shows in equilibrium a sparse popula- tion of state 2. As in the case of the complexes of Ras(T35A) or Ras(T35S) with the two analogues GppNHp and GppCH 2 p, the resonance of the a-phos- phate is shifted downfield relatively to state 2, whereas the b-phosphate resonance is shifted upfield and is split into two. The c-phosphate resonance is also split into two well-separated lines, but one is shifted downfield and one upfield from the resonance positions obtained with the wild-type protein. As observed earlier for GppNHp and GppCH 2 p complexes of Ras, and now for GTPcS, not only is the hydroxyl group of Thr35 that interacts in the X-ray structures with the metal ion important for stabiliza- tion of state 2, but so too is its methyl group. This is evident because in Ras(T35S) an hydroxyl group remains available but state 2 is destabilized. Stabiliza- tion of state 2 by the side-chain methyl group of Thr35 does not seem to be due to a simple hydropho- bic interaction, but rather to sterical restraints, because it is located in a cavity formed by the side chains of Ile36 and the charged ⁄ polar side chains of Asp38, Asp57 and Thr58. In GTPcS bound to Ras three different stereoiso- mers of the thiophosphate group are possible (Fig. 6). In principle, they can occur in state 1 and state 2 of the protein, but the corresponding populations may differ greatly. However, they are not equivalent ener- getically because sulfur is coordinated more weakly to magnesium ions than oxygen and is a weaker acceptor of hydrogen bonds than oxygen. As a consequence, GTPcS binds more weakly to Ras than does GTP itself [18]. In state 2, the amide group of Thr35 is probably involved in a hydrogen bond with one of the nonbridging c-phosphate oxygen atoms and the diva- lent ion with the other oxygen; the third oxygen is probably involved in a hydrogen bond with the amide of Gly60 and the interaction with the positively charged side chain of Lys16. Energetically, a sterical position such as that shown in Fig. 6A is strongly favoured, in agreement with the experimental observa- tion of a single phosphorus resonance for the c-phos- phate (Fig. 6A). In the mutant proteins, state 1 is strongly preferred because the side-chain interaction of Thr35 with the Mg 2+ ion is perturbed (T35S) or impossible (T35A). It has been suggested previously [2] that weakening of metal ion coordination most prob- ably leads to a concerted breaking of the hydrogen bond between the amide group of amino acid 35 and the c-phosphate group. Indeed, M-Ras [11] and H-Ras(G60A) [12] in the GppNHp form show 31 P NMR spectra typical of state 1 and recently published X-ray structures show that the amide group of Thr35 is distant from the c-phosphate group. Ford et al. [12] proposed a third conformational state for human wild-type H-Ras because their spectrum contained three 31 P resonances corresponding to the a- and c-phosphate (note that a new resonance assignment published by Spoerner et al . [2] was not known to Ford et al. [12]). However, because the third state could not be observed in our experiments, and the chemical shifts are very close to those observed for H-Ras•Mg 2+ •GDP, they should most probably be assigned to the a- and b-resonances of Ras-bound GDP. When a hydrogen bond exists between the amide group of amino acid 35 and the c-phosphate in the mutant proteins, in the GTPcS-complex the free energy differences DG° and thus the equilibrium popu- lations of the three stereoisomers are changed (Fig. 6). In the stereoisomer that most probably dominates in wild-type Ras (Fig. 6A), coordination of the c-phos- phate group with the metal ion and the interaction Conformational dynamics of Ras bound to GTPcS M. Spoerner et al. 1428 FEBS Journal 274 (2007) 1419–1433 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... to the increased line width seen in some resonances in the GTPcS complex Affinity of Ras- binding domains of Raf-kinase to Ras complexed with different GTP analogues ITC measurements show that under our experimental conditions the affinities between Ras( wt) and the tightly binding Raf-RBD are not influenced much by the type of bound GTP analogue The association constant is identical within the limits of. .. qualitatively explain the Conformational dynamics of Ras bound to GTPcS excessive line broadening of the resonances of residues located in these regions Additional local conformational changes may strengthen this effect GTP and GTPcS exist predominantly in state 2 meaning that the line broadening associated with the transition will be smaller The equilibrium between different stereoisomers around the thiophosphate... by using different GTP analogues or by specific mutations of Ras A hydrogen bond of the amide group of Gly13 and ⁄ or the amino group of Lys16 with the b–c-phosphorus-bridging oxygen may be one factor responsible for stabilization of state 2 in the GTP complex Thus, Ras( wt)•Mg2+ GTPcS exists predominantly in state 2 Other factors stabilizing state 2 are clearly the interactions of the amide and side-chain... group of threonine which is missing in Ras( T35S) From the NMR point of view, Ras( wt) and the serine mutant seem to exist in the same conformation when bound to effectors This is not true for the complexes of Raf-RBD with Ras( T35A) where the interaction with the RBD cannot restore the correct conformation [9] For the T35S-mutant the dissociation constant increases by about one order of magnitude with the. .. probably because of exchange broadening In the complex between Ras( 1–171) and GppNHp the amide resonances of 22 nonproline residues are not visible, whereas in the complex with GTPcS or GTP $ 20 additional resonances can be detected Some of these resonances are broader in the GTPcS complex than in the GTP complex [4] It is clear that any protein exists in multiple conformational states (now often called... structural hypothesis However, the chemical shifts of the c-phosphate resonance in state 1b are close to those observed in metal-free GTPcS (Tables 1 and 2) suggesting that it represents the arrangement seen in Fig 6B, with coordination with the metal ion abolished Dynamics and energetics of the conformational transitions The DG| values for the transition between state 1 and 2 in complexes of wild-type protein. .. given macroscopic dissociation state i is then pi ¼ M X ð3Þ pji j¼i With the macroscopic equilibrium constants Ki 1=sex ¼ k1 þ kÀ1 ð1Þ For the fit of the data the two-bond phosphorus–phosphorus coupling constants were taken from proton decoupled spectra of GTPcS measured at 278 K in the same buffer used for the experiments with Ras For free GTPcS the absolute value of 2Jab and 2Jbc are 19.7 Hz and 29.1... obtained within this range of MgCl2 concentration, at 3 mm MgCl2 a plateau in the magnesium induced chemical shifts was observed for the GTPcS at pH 7 and pH 9 Therefore this concentration was used for the study of the nucleotide–metal complexes The pH of the solutions was adjusted by adding HCl or NaOH and was determined with a calibrated glass electrode The dependence of the chemical shift d on the pH... values are seen in the three nucleotide complexes Qualitatively, the differences may be rationalized with the help of the NMR results as follows In a dynamic equilibrium Ras in complex with GppNHp or GppCH2p has a mobile effector loop which is fixed upon RBD binding Therefore, the change in the configurational entropy (as part of the total entropy) is smaller than in the GTPcS complex, where the effector loop... 1b The transition velocity between these two states and thus the energy of the transition state is similar to that found for transition between states 1 and 2 of Ras bound to the analogues GppCH2p or GppHNp The activation barrier may reflect a transient breakage of the bond between the metal ion and the c-phosphate Experimental procedures Protein purification Wild-type and Thr35 mutants of human H -Ras( 1–189) . between the corresponding T 2 relaxation times for the conformational states 1a and 1b of Ras( T35S)•Mg 2+ GTPcS. Complex of Ras Mg 2+ GTPcS with the Ras- binding domain of Raf-kinase Addition of the. Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPcS Michael Spoerner 1 , Andrea Nuehs 1 ,. on Ras( wt)•Mg 2+ GTPcS (data not shown). Binding of GTPcS to the Ras protein leads to rather large chemical shift changes. In contrast to the observations made for the GTP analogues GppNHp and