slow conformational dynamics of the guanine nucleotide-binding protein ras complexed with the gtp...

Slow conformational dynamics of the guaninenucleotide-binding protein Ras complexed with the GTPanalogue GTPcSMichael Spoerner1, Andrea Nuehs1, Christian Herrmann2, Guido Steiner1 andHans Robert Kalbitzer1

1 Universitat Regensburg, Institut fur Biophysik und physikalische Biochemie, Germany

2 Ruhr Universitat 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 typical31P 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 fur Biophysik und

physikalische Biochemie,

Universitatsstraße 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)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 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|1a1b of 61 kJÆmol)1 and 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

KA 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 GTPcScomplex.

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).

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)•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-phosphateis involved, as shown previously for the GTP ana-

logues GppNHp and GppCH2p [2]. The pK3 values 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

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

GppCH2p, indicating a more complex pH perturbation

of the electronic system in these analogues.

Fig. 1. Titration curves of free and Mg2+

bound GTPcS. (A,C) 31P chemical shift val-

ues of the a-, b- and c-phosphate groups

were determined on a 2.5 mL of a 1 mM

GTPcS solution in 100 mM Tris, 95% H2O

and 5% D2O 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 Mg2+ complexes were per-

formed in the presence of 3 mM MgCl2. The

dependence of chemical shifts on the pH

values was fitted to Eqn (7). The 31P reso-

nances were assigned by selective 1H- and31P-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 mM MgCl2 in 95% H2O ⁄ 5% D2O. In a first approximation d2, d3, and d4 correspond to the chemical shifts of two-,

three-, and fourfold negatively charged nucleotide. pK2 and pK3 are the corresponding pKa values of the three phosphates of the nucleotide.

d2 values are given in parentheses the titration up to pH 1.5 does not allow the precise estimation of this value. For d3 and d4 the estimated

error is ± 0.05 p.p.m.

Nucleotide

Phosphate

group d2 ⁄ p.p.m. pK2 d3 ⁄ p.p.m. pK3 d4 ⁄ 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

Mg2+•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

GppCH2p 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•GppCH2p 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


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 K1a1b 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. 31P NMR spectra of wild-type Ras complexed with

Mg2+•GTPcS at various temperatures. The samples contained

1 mM Ras(wt)•Mg2+•GTPcS in 40 mM Hepes ⁄ NaOH pH 7.4,

10 mM MgCl2, 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM

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 corres-

A B

Fig. 3. Conformational equilibria of wild-type Ras and Ras mutants complexed with Mg2+•GTPcS. (A) The sample contained 1 mM

Ras(wt)•Mg2+•GTPcS (lower), 1.2 mM Ras(T35S)•Mg2+•GTPcS (middle), and 1 mM Ras(T35A)•Mg2+•GTPcS (upper) in 40 mM Hepes ⁄ NaOH

pH 7.4, 10 mM MgCl2, 150 mM NaCl, 2 mM 1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfonate in 5% D2O, 95% H2O,

respectively. Data were recorded at 278 K. The assignment was determined by a 31P–31P NOESY experiment on Ras(wt)•Mg2+•GTPcS. 31P

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) 31P NMR saturation transfer experiment on Ras(wt)•Mg2+•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)•Mg2+•GTPcS at different temperatures. The sample contained 1.2 mM

Ras•Mg2+•GTPcS in 40 mM Hepes ⁄ NaOH pH 7.4, 10 mM MgCl2, 2 mM 1,4-dithioerythritol and 0.1 mM 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 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 ⁄ T2 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).



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 the31P 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

T2 relaxation 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. 31P 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 K12 between state 1 and 2 is calculated from inte-

grals of the c-thiophosphate resonances defined by K12 ¼ k12 ⁄ k21 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

K12 K1a1b

d1

(p.p.m.)

d2

(p.p.m.)

d1

(p.p.m.)

d2

(p.p.m.)

d1

(p.p.m.)

d2

(p.p.m.)

Ras(wt)•Mg2+•GTPcS )11.30 )16.67 37.01 > 10 ND b

Ras(T35S)•Mg2+•GTPcS )10.70 )17.96 a

)17.22 a

32.73 a

37.89 a

36.87 0.06 0.5

Ras(T35A)•Mg2+•GTPcS )10.80 )17.92 a 32.79 a < 0.05 0.5

)17.19 a 37.91 a

Ras(wt)•Mg2+•GTPcS )11.19 )16.55 36.85 > 10 ND b

+ Raf-RBD

Ras(T35S)•Mg2+•GTPcS )11.22 )16.52 36.54 > 10 ND b

+ Raf-RBD

Ras(T35A)•Mg2+•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.



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 k12 and k21 (k1a1b and

k1b1a) 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 and k21 are given. The

states are defined as in Table 1. DG12 or DG1a1b is the difference in free enthalpy between state 2 (1b) and 1 (1a). T2 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

j1a1b DH

j1a1b TDS

j1a1b DG1a1b

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

Ras(T35S)•Mg2+•GTPcS 278 70 137 41 ± 2 61 ± 1 18 ± 1 1.56 ± 0.15a

288 170 330

298 430 810

k12 k21 DGj12 DH

j12 TDS

j12 DG12

Ras(wt)•Mg2+•GppNHp a 278 80 42 42 ± 5 70 ± 3 28 ± 2 ) 1.48 ± 0.15

288 250 135

298 700 387

Ras(wt)•Mg2+•GppCH2p a 278 80 39 41 ± 5 63 ± 3 29 ± 2 ) 1.65 ± 0.15

288 260 131

298 740 391

Relaxation times T2 (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)•Mg2+•GTPcS 278 4.0 4.0 4.2 4.9 5.3 5.3

Ras(wt)•Mg2+•GppNHp a 278 5.8 3.9 4.8 4.8 4.1 7.1

Ras(wt) •Mg2+•GppCH2p 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.



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 Ddin 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 mM Ras•Mg2+•GTPcS (lower), 1.2 mM Ras(T35S)•Mg2+•GTPcS (middle) or 1.0 mM Ras(T35A)•Mg2+•GTPcS (upper) in

40 mM Hepes ⁄ NaOH pH 7.4, 10 mM MgCl2, 150 mM NaCl, 2 mM

1,4-dithioerythritol and 0.1 mM 2,2-dimethyl-2-silapentane-5-sulfo-

nate in 5% D2O, 95% H2O, 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. 31P 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 KA between Raf-RBD and Ras com-

plexed with different GTP analogues was determined using ITC. Measurements were performed at 298 K in 40 mM Hepes ⁄ NaOH pH 7.4,

10 mM MgCl2, 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 Gcomplex ) Gfree ¼ -RTlnKA.

Raf-RBD complexed

with

KA

(lM)1)

DG�(kJÆmol)1)

DH�(kJÆmol)1)

TDS�(kJÆmol)1)

Ras(wt)•Mg2+•GppNHp 2.50 ± 0.4 )36.5 ± 0.6 )13.4 ± 1.5 23 ± 2.1

Ras(wt)•Mg2+•GppCH2p 2.50 ± 0.4 )36.5 ± 0.6 )18.4 ± 2.0 18 ± 2.6

Ras(wt)•Mg2+•GTPcS 2.44 ± 0.6 )36.4 ± 0.9 )7.5 ± 1.5 29 ± 2.4

Ras(T35S)•Mg2+•GppNHp 0.12 ± 0.04 )29.0 ± 0.06 )9.7 ± 1.0 19 ± 1.1

Ras(T35S)•Mg2+•GppCH2p 0.09 ± 0.04 )28.2 ± 0.06 )15.3 ± 1.5 13 ± 1.6

Ras(T35S)•Mg2+•GTPcS 0.18 ± 0.04 )30.0 ± 0.06 )13.6 ± 1.5 16 ± 1.6



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 conformational

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

A B

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 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-resonancesof 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



with Gly60 and Lys16 is still possible, in the two other

cases either the metal ion coordination is weakened

when the sulfur is oriented towards the metal ion

(Fig. 6B) or the hydrogen-bond interaction with Gly60

is weakened (Fig. 6C). It seems plausible that a wild-

type-like arrangement is the energetically most

favoured; meaning that state 1a should be assigned to

this stereoisomer. For the resonances assigned to

state 1b it is more difficult to derive a structural hypo-

thesis. However, the chemical shifts of the c-phosphateresonance 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 with GppNHp and

GppCH2p are 42 and 41 kJÆmol)1, respectively [1,2].

For the complex between wild-type protein and

GTPcS the activation energy cannot be determined by

NMR because only state 2 is visible. However, it is

reasonable to assume that the activation energy is sim-

ilar. For the transition between states 1a and 1b we

determined a DG| value of 41 kJÆmol)1. Thus the acti-

vation energies are identical within the limits of error.

This may be due to chance or may suggest that a sim-

ilar transition state is involved in the transition

between states 1 and 2 and between states 1a and 1b.

Dynamic equilibria in Ras complexed with GTP and

different GTP analogues have been described previ-

ously [4,19,20] in 15N-enriched Ras. It was shown that

a number of amide resonances are not visible in 2D

heteronuclear NMR spectra, most 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 reso-

nances can be detected. Some of these resonances are

broader in the GTPcS complex than in the GTP com-

plex [4].

It is clear that any protein exists in multiple con-

formational states (now often called excited states)

with different populations. Ito et al. [4] called this phe-

nomenon regional polysterism. Different probes are

also differentially sensitive to different conformational

states. The two main conformational states 1 and 2

coexist with almost equal populations in the GppNHp

complex, and exchange between theses two states,

which probably involves structural changes in loop L1,

switch 1 and switch 2, can qualitatively explain the

excessive line broadening of the resonances of residues

located in these regions. Additional local conforma-

tional 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 stereo-

isomers around the thiophosphate group may contrib-

ute 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 con-

stant is identical within the limits of error for all three

GTP analogues (Table 4). At first this seems surprising

because NMR spectroscopy shows that the bound ana-

logue clearly influences the equilibrium between the

two conformational states. However, NMR spectrosco-

py indicates that, after binding of the RBD, Ras most

probably exists in its correct structural state. Because

the data analysis used assume a two-state model and

the free enthalpy difference between state 2 in the free

and complexed form can be assumed to be very small,

only the conformational equilibrium with state 1 could

influence the total enthalpy change measured by ITC.

However, in the GppNHp or GppCH2p complexes of

the wild-type protein, the difference in DG12 between

the two states is � 2 kJÆmol)1 (Table 3) and thus much

smaller than DG� involved in effector binding, which is

of the order of 36 kJÆmol)1 (Table 4). In addition, only

the relatively small fraction of the protein occurring in

state 1 in GTPcS would contribute to a nucleotide-spe-

cific variation in DG� and thus would be scaled down

proportionally to K12)1.

In the T35S-mutant the population of free Ras is

shifted to state 1, but the binding of Raf-RBD restores

the correct conformation (similar to state 2). The wild-

type and mutant proteins differ mainly in the methyl

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

largest increase seen for GppCH2p. DG� increases by

7.5, 8.3, and 6.5 kJÆmol)1 for GppNHp, GppCH2p and

GTPcS, respectively. The small differences may reflect



the energy difference between states 1 and 2 in this

mutant, which cannot be derived from our NMR data.

Relatively small, but in some cases significant, differ-

ences in DH� and TDS� 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 is suggested to be oriented in

the correct position already.

The nucleotide analogue bound to Ras influences

the equilibrium between states 1 and 2. Replacing the

oxygen bridging the b- and c-phosphate group in

GTPcS with an imido or methylene group shifts the

population of state 2 to state 1. Although it was

shown that P–O–P bonds have very open bond angels

[21], which should lead to delocalization of the electron

density into the neighbouring atoms, the bridging oxy-

gen may still be a weak hydrogen bond acceptor. That

is an interaction involving a hydrogen bond donator

and ⁄or a group with positive partial charge could be a

reason for stabilization of state 2 in the GTPcS com-

plex that is abolished by replacement of the bridging

oxygen. Taking a closer look at the crystal structure of

Ras in the GTP-bound state [22], only the main-chain

NH of Gly13 and ⁄or the amino group of Lys16 and

the bridging Pb–O–Pc oxygen seem to be able to con-

tact each other by forming a hydrogen bond. The

interacting groups are also close enough when

GppNHp is bound, as derived from X-ray structure

[23], although in this case a strong hydrogen bond is

not to be expected.

Conclusions

Ras bound to triphosphate nucleosides exists in (at

least) two conformational states which can be identi-

fied using 31P NMR spectroscopy. One of these states

(state 2) represents the high-affinity binding state for

effectors; the second state (state 1) represents a differ-

ent state of the protein with much reduced affinity to

effectors. The equilibrium between the states can be

shifted 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 fac-

tor responsible for stabilization of state 2 in the GTP

complex. Thus, Ras(wt)•Mg2+•GTPcS exists predom-

inantly in state 2. Other factors stabilizing state 2 are

clearly the interactions of the amide and side-chain

hydroxyl groups of Thr35 with the c-phosphate group

and metal ion, respectively. Ras variants existing in

state 1 show two substates states 1a and 1b. The trans-

ition 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) were

expressed in Escherichia coli strain CK600K with ptac vector

plasmids and purified as described previously [18]. Nucleo-

tide exchange to GppNHp, GppCH2p or GTPcS was done

using alkaline phosphatase treatment in the presence of a

twofold excess of the GTP analogue as described at John

et al. [24]. Free nucleotides and phosphates were removed

by gel filtration. The final purity of the protein was > 95%

as judged from the SDS ⁄PAGE. The Ras-binding domain

of human cRaf-1 (Raf-RBD, amino acids 51–131) was

expressed in E. coli and purified as described previously [25].

Sample preparation

Typically 1 mm Ras•Mg2+•GTPcS was dissolved in 40 mm

Hepes ⁄NaOH pH 7.4, 10 mm MgCl2, 150 mm NaCl, 2 mm

1,4-dithioerythritol and 0.1 mm 2,2-dimethyl-2-silapentane-

5-sulfonate in 5% D2O, 95% H2O. For binding studies a

solution of 5 or 7 mm Raf-RBD contained in the same buf-

fer was added in appropriate amounts to the samples.

NMR spectroscopy

31P NMR spectra were recorded with an Avance-500 NMR

spectrometer (Bruker Biospin, Karlsruhe, Germany) oper-

ating at a 31P frequency of 202 MHz. Measurements were

performed in a 10 mm probe using 8 mm Shigemi (Tokyo,

Japan) sample tubes at various temperatures. Seventy-degree

pulses were used together with a total repetition time of 7 s.

Protons were decoupled during data acquisition by a GARP

sequence [26] with strength of the B1-field of 980 Hz. For

referencing a X-value of 0.4048073561 reported by Maurer

and Kalbitzer [27] was used, which corresponds to 85%

external phosphoric acid contained in a spherical bulb. The

assignment of the phosphate resonances was established

by a 31P–31P NOESY experiment on 1.2 mm solution of

Ras(wt)•Mg2+•GTPcS at 283 K with a mixing time of 1.5 s

and a total repetition time of 14 s. Saturation experiments

were performed at 278 K using a B1-field of 18 Hz for a per-

iod of 1 s for presaturation.



Sample temperature was checked by using the line separ-

ation (methylene-hydroxyl) of external ethylene glycol [28].

Thus, the absolute accuracy of the temperatures given in

here is better than ±0.5 K.

Calculation of the exchange rates and the

thermodynamic parameters

Exchange rates were calculated by a line shape analysis

using full-density matrix formalism [29] based on a pro-

gram described by Geyer et al. [1] and modified later. Vis-

cosity-dependent changes of the rotational correlation time

srot were corrected as described by Spoerner et al. [2].

Care was taken that the spin systems were largely in ther-

modynamic equilibrium before each scan in the experiments

by using a repetition time of 7 s together with 70 � pulses.

Before fitting the data the noise level was decreased by mul-

tiplying the FID with an exponential filter leading to an

additional line broadening of 5 Hz. The difference in the

free activation energy DG|, the activation enthalpy DH|, and

the activation entropy DS|, were obtained by fitting the

temperature dependence of the exchange rates sex to the

Eyring equation with

1=sex ¼ k1 þ k�1 ð1Þ

For the fit of the data the two-bond phosphorus–phosphorus

coupling constants were taken from proton decoupled spec-

tra of GTPcS measured at 278 K in the same buffer used for

the experiments with Ras. For free GTPcS the absolute

value of 2Jab and2Jbc are 19.7 Hz and 29.1 Hz, respectively.

pH dependence of chemical shifts

The pH dependence of chemical shifts was measured in

samples containing 2.5 mL solution containing 1 mm nuc-

leotide, 0.1 mm 2,2-dimethyl-2-silapentane-5-sulfonate in

5% D2O, 95% H2O. To estimate the optimal magnesium

concentration it was varied in the range from 0 to 40 mm.

Whereas at pH 2 no plateau in the chemical shifts could be

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 is

often described by a modified Henderson–Hasselbalch

equation using microscopic equilibrium constants Kji and

chemical shift contributions dji for the description of the

equilibrium between the protonated and the deprotonated

form of a functional group. Even in small molecules such

as GTP the description can become very complex because

different forms with the same total charge exist in the

equilibrium. For a molecule with N sites 2N different states

and thus 2N ) 1 independent equilibrium constants and

chemical shifts are required for a full description. The prob-

lem can be simplified by defining macroscopic equilibrium

constants Ki and chemical shift contributions di to N + 1

dissociation steps. As long as fast exchange conditions pre-

vail the di can be calculated from the microscopic dij andthe concentrations pij of the M components of a dissoci-

ation step i by

di ¼1

PM

j¼1

pji

XM

j¼i

pjidji ð2Þ

and is independent of the ligand L (H+) concentration,

because the relative concentrations in the different states

(ji) with the same number of bound ligands are constant

(defined by the respective microscopic equilibrium con-

stants). The concentration of the molecule pi in a given

macroscopic dissociation state i is then

pi ¼XM

j¼i

pji ð3Þ

With the macroscopic equilibrium constants Ki

Ki ¼piL

pi�1ð4Þ

one obtains

pi

p0¼ P

i

j¼1

Kj

Lð5Þ

The average chemical shift is then

d ¼d0 þ

PN

i¼1

di Pi

j¼1

Kj

L

1þPN

i¼1

Pi

j¼1

Kj

L

ð6Þ

With the definition of the pH the chemical shift is then

given as a function of pH by

d ¼d0 þ

PN

i¼1

di10ipH�

Pi

j¼1

pKj

1þPN

i¼1

10ipH�

Pi

j¼1

pKj

ð7Þ

A more detailed derivation of Eqn (7) is found in the

PhD thesis by Freund [30].

Isothermal Titration Calorimetry (ITC)

Measurements were performed with a MicroCal MCS ITC

apparatus at 298 K. Ras(wt) and Ras(T35S) bound to

Mg2+•GppNHp, Mg2+•GppCH2p or Mg2+•GTPcS and



also Raf-RBD were dissolved in identical buffer (40 mm

Hepes ⁄NaOH pH 7.4, 10 mm MgCl2, 150 mm NaCl, 2 mm

1,4-dithioerythritol). A 60 or 80 lm solution of Ras protein

in the cell was titrated with 0.6, 1.0 or 1.5 mm solution of

Raf-RBD. Data were analysed using origin for itc 2.9

(MicroCal Software Incorporation, Northampton, MA,

USA) assuming a 1 : 1 complex formation [31].

Acknowledgements

This work was supported by the Volkswagenstiftung

and the DFG.

References

1 Geyer M, Schweins T, Herrmann C, Prisner T, Witting-

hofer A & Kalbitzer HR (1996) Conformational transi-

tions in p21 (Ras) and in its complexes with the effector

protein Raf-RBD and the GTPase. Biochemistry 35,

10308–10320.

2 Spoerner M, Nuehs A, Ganser P, Herrmann C, Witting-

hofer A & Kalbitzer HR (2005) Conformational states

of Ras complexed with the GTP-analogs GppNHp or

GppCH2p: implications for the interaction with effector

proteins. Biochemistry 44, 2225–2236.

3 Kraulis PJ, Domaille PJ, Campbell-Burk S-L, Van Aken

T & Laue ED (1994) Solution structure and dynamics

of ras p21.GDP determined by heteronuclear three- and

four-dimensional NMR spectroscopy. Biochemistry 33,

3515–3531.

4 Ito Y, Yamasaki K, Iwahara J, Terada T, Kamiya A,

Shirouzu M, Muto Y, Kawai G, Yokoyama S, Laue

ED et al. (1997) Regional polysterism in the GTP-

bound form of the human c-Ha-Ras protein.

Biochemistry 36, 9109–9119.

5 Stumber M, Geyer M, Graf R, Kalbitzer HR, Scheffzek

K & Haeberlen U (2002) Observation of slow dynamic

exchange processes in Ras protein crystals by 31P

solid state NMR spectroscopy. J Mol Biol 323,

899–907.

6 Iuga A, Spoerner M, Kalbitzer HR & Brunner E (2004)

Solid-state 31P NMR spectroscopy of microcrystals of

the Ras protein and its effector loop mutants: compari-

son between crystalline and solution state. J Mol Biol

342, 1033–1044.

7 Nassar N, Horn G, Herrmann C, Block C, Janknecht R

& Wittinghofer A (1996) Ras ⁄Rap effector specificity

determined by charge reversal. Nat Struct Biol 3, 723–

729.

8 Vetter IR, Linnemann T, Wohlgemuth S, Geyer M,

Kalbitzer HR, Herrmann C & Wittinghofer A

(1999) Structural and biochemical analysis of

Ras-effector signaling via RalGDS. FEBS Lett 451,

175–180.

9 Spoerner M, Herrmann C, Vetter I, Kalbitzer HR &

Wittinghofer A (2001) Dynamic properties of the Ras

switch I region and its importance for binding to effec-

tors. Proc Natl Acad Sci USA 98, 4944–4949.

10 Bellew BF, Halkides CJ, Gerfen GJ, Griffin RG &

Singel DJ (1996) High frequency (139.5 GHz) electron

paramagnetic resonance characterization of Mn(II)–

H2(17)O interactions in GDP and GTP forms of p21

ras. Biochemistry 35, 12186–12193.

11 Ye M, Shima F, Muraoka S, Liao J, Okamoto H,

Yamamoto M, Tamura A, Yagi N, Ueki T & Kataoka

T (2005) Crystal structure of M-Ras reveals a GTP-

bound ‘off’ state conformation of Ras family small

GTPases. J Biol Chem 280, 31267–31275.

12 Ford B, Skowronek K, Boykevisch S, Bar-Sagi D &

Nassar N (2005) Structure of the G60A mutant of Ras;

implications for the dominant negative effect. J Biol

Chem 280, 25697–25705.

13 Spoerner M, Wittinghofer A & Kalbitzer HR (2004)

Perturbation of the conformational equilibria of Ras by

selective mutations as studied by 31P NMR spectro-

scopy. FEBS Lett 578, 305–310.

14 Geyer M, Assheuer R, Klebe C, Kuhlmann J, Becker J,

Wittinghofer A & Kalbitzer HR (1999) Conformational

states of the nuclear GTP-binding protein Ran and its

complexes with the exchange factor RCC1 and the

effector protein RanBP1. Biochemistry 8, 11250–11260.

15 Sigel H (1987) Isomeric equilibria in complexes of

adenosine 5¢-triphosphate with divalent metal ions.

Eur J Biochem 165, 65–72.

16 Rosch P, Kalbitzer HR & Goody RS (1980) 31P NMR

spectra of thiophosphate analogues of guanine nucleo-

tides; pH dependence of chemical shifts and coupling

constants. FEBS Lett 121, 211–214.

17 Allin C, Ahmadian MR, Wittinghofer A & Gerwert K

(2001) Monitoring the GAP catalyzed H-Ras GTPase

reaction at atomic resolution in real time. Proc Natl

Acad Sci USA 98, 7754–7759.

18 Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS

& Wittinghofer A (1986) Expression of p21 proteins in

Escherichia coli and stereochemistry of the nucleotide-

binding site. EMBO J 5, 1351–1358.

19 Miller AF, Papastavros MZ & Redfield AG (1992)

NMR studies of the conformational change in human

N-p21ras produced by replacement of bound GDP with

the GTP analog GTPcS. Biochemistry 31, 10208–10216.

20 Miller AF, Halkides CJ & Redfield AG (1993) An

NMR comparison of the changes produced by different

guanosine 5¢-triphosphate analogs in wild-type and on-

cogenic p21ras. Biochemistry 32, 7367–7376.

21 Cheng H, Sukal S, Deng H, Leyh TS & Callender R

(2001) Vibrational structure of GDP and GTP bound to

RAS: an isotope-edited FTIR study. Biochemistry 40,

4035–4043.



22 Scheidig AJ, Burmester C & Goody RS (1999) The pre-

hydrolysis state of p21 (ras) in complex with GTP: new

insights into the role of water molecules in the GTP

hydrolysis reaction of ras-like proteins. Struct Fold Des

7, 1311–1324.

23 Pai E, Krengel U, Petsko GA, Goody RS, Kabsch W &

Wittinghofer A (1990) Refined crystal structure of the

triphosphate conformation of H-ras p21 at 1.35 A reso-

lution: implications for the mechanism of GTP hydroly-

sis. EMBO J 9, 2351–2359.

24 John J, Sohmen R, Feuerstein J, Linke R, Wittinghofer

A & Goody R (1990) Kinetics of interaction of nucleo-

tides with nucleotide-free H-ras p21. Biochemistry 29,

6058–6065.

25 Herrmann C, Martin GA & Wittinghofer A (1995)

Quantitative analysis of the complex between p21ras

and the Ras-binding domain of the human Raf-1 pro-

tein kinase. J Biol Chem 270, 2901–2905.

26 Shaka AJ, Baker PB & Freeman R (1985) Computer-

optimized decoupling scheme for wideband applications

and low-level operation. J Magn Reson 64, 547–552.

27 Maurer T & Kalbitzer HR (1996) Indirect Referencing

of 31P and 19F NMR spectra. J Magn Reson B113,

177–178.

28 Raiford DS, Fisk CG & Becker ED (1979) Calibration

of methanol and ethylene glycol nuclear magnetic reso-

nance thermometers. Anal Chem 51, 2050–2051.

29 Rao BD (1989) Nuclear magnetic resonance line-shape

analysis and determination of exchange rates. Methods

Enzymol 176, 279–311.

30 Freund J (1994) Biophysikalische und proteolytische

Charakterisierung des HIV-Proteins Nef und Zuord-

nung der kernmagnetischen Resonanzen eines Fusions-

proteins aus Tendamistat und HIV-1-Tat. Dissertation,

University of Tubingen, Germany.

31 Wiseman T, Willistone S, Brandts JF & Lin LN (1989)

Rapid measurement of binding constants and heats of

binding using a new titration calorimeter. Anal Biochem

179, 131–137.

32 Frey PA & Sammons RD (1985) Bond order and charge

localization in nucleoside phosphorethioates. Science 22,

541–545.



slow conformational dynamics of the guanine nucleotide-binding protein ras complexed with the gtp...

Documents