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

It has been shown using 31P 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-imidot

nucleotide-binding protein Ras complexed with the GTP analogue GTPcS

Michael Spoerner1, Andrea Nuehs1, Christian Herrmann2, Guido Steiner1and

Hans Robert Kalbitzer1

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 31P 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 (GppCH2p) [2] These two states interconvert with rate constants in the millisecond time scale They are characterized by typical

31P 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 K12 of 2.0, when the GTP analogue guanosine-5¢-(b,c-imido)tri-phosphate or guanosine-5¢-(b,c-methyleno)triguanosine-5¢-(b,c-imido)tri-phosphate 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 31P NMR spectra that indicate the prevalence of only one con-formational state with K12> 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|1a1bof 61 kJÆmol)1and an activation entropy

DS|1a1b of 65 JÆK)1Æmol)1 are derived Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity

KAfor 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

GppCH2p, 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).

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)•Mg2+•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 GppCH2p [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)•Mg2+•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 31P 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 31P 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 31P 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

GppCH2p) 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

Mg2+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 Mg2+ ions within a pH range of 2–13 The rate of exchange between Mg2+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 Mg2+concentration does not influence the observed chemical shifts (also see Experimental procedures)

Figure 1 shows the titration curves for GTPcS in the absence and presence of Mg2+ 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 GppCH2p [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 Mg2+•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 GppCH2p [2] The pK3values in GTPcS are much smaller than those reported for GppNHp and GppCH2p The value of pK2 does not depend much on the analogue when a relatively large error is taken into consideration pK2 and pK3 are usually associated with the first and the second

deprotonation step at the c-phosphate group of the

nucleotide for the transition from the threefold

negat-ively charged state to the fourfold negatnegat-ively 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 GppCH2p, 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 m M Tris, 95% H2O

and 5% D 2 O containing 0.1 m M

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 m M nucleotide in the absence or presence of 3 m M MgCl2in 95% H2O ⁄ 5% D 2 O In a first approximation d2, d3, and d4correspond to the chemical shifts of two-, three-, and fourfold negatively charged nucleotide pK2and pK3are the corresponding pKavalues 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

a

Data from Spoerner et al [2].

Conformational states of Ras complexed with

Mg2+•GTPcS

Figure 2 shows 31P 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 31P–31P NOESY

experiment on Ras(wt)•Mg2+•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 GppCH2p [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 GppCH2p 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 31P NMR In addition,

H2S 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 GTPcS with the mutant Ras(T35S) or Ras(T35A), additional 31P 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 GppCH2p 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 K1a1b¼ [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

1 m M Ras(wt)•Mg 2+ •GTPcS in 40 m M Hepes ⁄ NaOH pH 7.4,

10 m M MgCl 2 , 150 m M NaCl, 2 m M 1,4-dithioerythritol and 0.1 m M

2,2-dimethyl-2-silapentane-5-sulfonate in 5% D2O, 95% H2O,

respectively The absolute temperature was controlled by

immer-sing a capillary with ethylene glycol and measuring the

hydroxyl-methylene shift difference [28].

timescale < T1 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

Fig 3 Conformational equilibria of wild-type Ras and Ras mutants complexed with Mg 2+ •GTPcS (A) The sample contained 1 m M

Ras(wt)•Mg 2+ •GTPcS (lower), 1.2 m M Ras(T35S)•Mg 2+ •GTPcS (middle), and 1 m M Ras(T35A)•Mg 2+ •GTPcS (upper) in 40 m M Hepes ⁄ NaOH

pH 7.4, 10 m M MgCl 2 , 150 m M NaCl, 2 m M 1,4-dithioerythritol and 0.1 m M 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 a31P–31P 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 B1-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 31P NMR data of Ras(T35S)•Mg 2+ •GTPcS at different temperatures The sample contained 1.2 m M

Ras•Mg 2+ •GTPcS in 40 m M Hepes ⁄ NaOH pH 7.4, 10 m M MgCl2, 2 m M 1,4-dithioerythritol and 0.1 m M 2,2-dimethyl-2-silapentane-5-sulfonate

in 5% D2O, 95% H2O 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 broadenlead-ing 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)1for 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)1for state 1a and 1b of the bound c-thiophosphate group (values are given with an estimated error of ± 15 s)1).

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

31P NMR data from Ras(T35S)•Mg2+•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)•Mg2+•GppNHp or Ras(wt)•Mg2+•GppCH2p

Whereas DG| of the exchange in Ras(T35S)•Mg2+

•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 T2 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

T2relaxation times for the conformational states 1a and

1b of Ras(T35S)•Mg2+•GTPcS

Complex of Ras•Mg2+•GTPcS with the

Ras-binding domain of Raf-kinase

Addition of the Ras-binding domain of Raf-kinase

(Raf-RBD) to Ras(wt)•Mg2+•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 GTPcS is bound Correspondingly, in Ras(T35S)•Mg2+•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)•Mg2+•GTPcS corresponds to state 2 found

in the wild-type protein Complex formation between Raf-RBD and Ras(T35A)•Mg2+•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, GppCH2p 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 K12¼ 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

d1 (p.p.m.)

d2 (p.p.m.)

d1 (p.p.m.)

d2 (p.p.m.)

d1 (p.p.m.)

d2 (p.p.m.)

)17.22 a

32.73 a 37.89 a

+ Raf-RBD

+ Raf-RBD

a Chemical shifts in state 1a (lower) and 1b (upper) b Values could not be determined since signal cannot be detected.

association constant KA 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 GppCH2p may exist A

significant decrease in KA, 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 GppCH2p 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 k12 andk21are 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)_

DGj1a1b DH1a1bj TDS1a1bj DG 1a1b

k1a1b k1b1a (kJÆmol)1) (kJÆmol)1) (kJÆmol)1) (kJÆmol)1)

Relaxation times T 2 (ms) of the resonances of

(1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b) (1) or (1a) (2) or (1b)

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.

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 Mg2+–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 GppCH2p 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 Mg2+•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 Mg2+•GTPcS in complex with Raf-RBD Initially the samples

con-tained 1.0 m M Ras•Mg 2+ •GTPcS (lower), 1.2 m M Ras(T35S)•

Mg 2+ •GTPcS (middle) or 1.0 m M Ras(T35A)•Mg 2+ •GTPcS (upper) in

40 m M Hepes ⁄ NaOH pH 7.4, 10 m M MgCl 2 , 150 m M NaCl, 2 m M

1,4-dithioerythritol and 0.1 m M

2,2-dimethyl-2-silapentane-5-sulfo-nate in 5% D2O, 95% H2O, respectively A solution of 9.8 m M

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 MgCl2, 150 m M NaCl, 2 m M 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 (l M )1)

DG

(kJÆmol)1)

DH

(kJÆmol)1)

TDS (kJÆmol)1)

atom in the c-phosphate group are represented by

bro-ken lines

Influence of the nucleotide bound on the Ras

conformational states

31P 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 Mg2+–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•Mg2+•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 GppCH2p exist in a conform-ational equilibrium between two main conformconform-ational states 1 and 2, with a K12 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

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.

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 GppCH2p

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 sub-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 GppCH2p, 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 GppCH2p

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 Mg2+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 31P 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 31P 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•Mg2+•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