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

Abstract

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

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Dynamic properties of the Ras switch I region and its
importance for binding to effectors
Michael Spoerner*, Christian Herrmann†, Ingrid R. Vetter†, Hans Robert Kalbitzer*, and Alfred Wittinghofer†‡
*Universita ¨t Regensburg, Institut fu ¨r Biophysik und Physikalische Biochemie, Universita ¨tsstrasse 31, 93053 Regensburg, Germany; and†Max-Planck-Institut
fu ¨r Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
Edited by Henry R. Bourne, University of California, San Francisco, CA, and approved February 23, 2001 (received for review September 14, 2000)
WehaveinvestigatedthedynamicpropertiesoftheswitchIregion
of the GTP-binding protein Ras by using mutants of Thr-35, an
invariant residue necessary for the switch function. Here we show
that these mutants, previously used as partial loss-of-function
mutations in cell-based assays, have a reduced affinity to Ras
effector proteins without Thr-35 being involved in any interaction.
The structure of Ras(T35S)?GppNHp was determined by x-ray crys-
tallography. Whereas the overall structure is very similar to wild-
type, residues from switch I are completely invisible, indicating
that the effector loop region is highly mobile.31P-NMR data had
indicated an equilibrium between two rapidly interconverting
conformations, one of which (state 2) corresponds to the structure
found in the complex with the effectors.31P-NMR spectra of Ras
mutants (T35S) and (T35A) in the GppNHp form show that the
equilibrium is shifted such that they occur predominantly in the
nonbinding conformation (state 1). On addition of Ras effectors,
Ras(T35S) but not Ras(T35A) shift to positions corresponding to the
binding conformation. The structural data were correlated with
kinetic experiments that show two-step binding reaction of wild-
type and (T35S)Ras with effectors requires the existence of a
rate-limiting isomerization step, which is not observed with T35A.
The results indicate that minor changes in the switch region, such
as removing the side chain methyl group of Thr-35, drastically
affect dynamic behavior and, in turn, interaction with effectors.
The dynamics of the switch I region appear to be responsible for
the conservation of this threonine residue in GTP-binding proteins.
A
been identified and shown to regulate a diverse array of signal
transduction reactions and?or transport processes. These small
GTP-binding proteins, but also the larger G? proteins or the
proteinsynthesiselongationfactors,containconservedsignature
motifs in the primary sequence. Accordingly, the structures of
many of these proteins show a common structural core called the
G domain, an ??? fold consisting of six ? strands and five ?
helices(1).Thecommonstructuralcoreismodifiedbyinsertions
and?or additional structural elements. The structures of several
proteins have been solved in both the GDP- and GTP-bound
form, which showed that the conformational change is mostly
confined to the loop L2-?2 (by using the Ras nomenclature) and
the ?3??2 regions, which have accordingly been called switches
I and II (2). It turned out that the active site of the GDP-bound
conformations shows large variations, whereas the triphosphate
structures are very similar (3). It was also deduced from these
studies that the conformational change is triggered when two
hydrogen bonds to the ?-phosphate from switches I and II,
involving invariant Thr and Gly residues, respectively, are re-
leased after GTP hydrolysis (3). NMR structural studies have
shown that the switch regions show an inherent mobility much
higher than the rest of the G domain (4–6). From temperature-
dependence measurements and line-shape analysis of31P-NMR
spectra of Ras?GppNHp, it had been deduced earlier that in
solution, the effector region interconverts between two main
conformations that are characterized by different chemical shift
values for the resonances of the ?- and ?-phosphate groups (7).
n estimated 60–100 different GTP-binding proteins of the
Ras superfamily belonging to different subfamilies have
Furthermore, EPR studies of the Mg2?-binding site of Ras
suggested that the coordination of the Thr-35 to the ?-phosphate
might be transient in solution, indicating a high flexibility of the
side chain and?or the loop containing the Thr-35 (8, 9).
The switch I region contains only one invariant residue, Thr-35
in Ras (10), which is involved via its side chain hydroxyl in the
coordination of the crucial metal ion and, via its main chain NH, in
contacting the ?-phosphate. Thr-35 is most likely invariant in all
Ras-related proteins (not counting pseudogene products) and is
never substituted by a Ser, in contrast to the P-loop motif
GxxxxGKS?T, where the interaction with the metal ion is per-
formed by either Ser or Thr. Following the observation that the
T35S mutation—frequently used in biological studies as a partial
loss-of-function mutation—has a reduced affinity to RalGDS (see
below) without Thr making a significant contribution to the inter-
action (11, 12), we have investigated the role of Thr-35 for the
propertiesofH-Ras(subsequentlyreferredtoasRas)andfoundits
methyl group to be responsible for the dynamic behavior of the
switchregion.Thisobservation,inturn,ismostlikelythereasonfor
the complete invariance of this threonine residue.
Methods
Protein Purification. Wild-type and mutants of H-Ras (1–189) were
expressed in Escherichia coli and purified as described (13). Nu-
cleotide exchange to GppNHp as well as N-methylanthraniloyl-
GppNHpwasdoneasdescribedbyJohnetal.(14).Freenucleotide
and phosphates were removed by gel filtration. The final purity of
the protein was ?95%, as judged by SDS?PAGE. The concentra-
tionoftheproteinsolutionwasdeterminedbyBradfordassay,with
BSA as standard (15). The amount and nature of protein-bound
nucleotide were analyzed by C18 reverse-phase HPLC and quan-
tified with a calibrated detector (Beckman Coulter) and integrator
(Shimadzu). The Ras-binding domains of human c-Raf-1 and
RalGDSwereexpressedinE.coliandpurifiedasdescribed(16,17).
NMR Spectroscopy.31PNMRspectrawererecordedwithaBruker
DRX-500 NMR spectrometer operating at 202 MHz. Measure-
ments were performed in a 10-mm probe by using 8-mm Shigemi
sample tubes at various temperatures. Ras protein solutions (1
mM) in 40 mM Hepes, pH 7.4?10 mM MgCl2?150 mM NaCl?2
mM dithioerythritol contained 10% D2O to get a lock signal.
2,2-Dimethyl-2-silapentane-5-sulfonate (0.1 mM) was added to
calibrate the spectra by indirect referencing. A ?-value of
0.4048073561 reported by Maurer and Kalbitzer (18) was used,
which corresponds to 85% external phosphoric acid contained in
a spherical bulb.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: RBD, Ras-binding domain.
Datadeposition:Theatomiccoordinatesreportedinthispaperhavebeendepositedinthe
Protein Data Bank, www.rcsb.org (PDB ID code 1iaq).
‡To whom reprint requests should be addressed. E-mail: alfred.wittinghofer@mpi-
dortmund.mpg.de.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
4944–4949 ?
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Stopped-Flow Measurements. Stopped-flow experiments using N-
methylanthraniloyl-GppNHp bound to Ras were used to mea-
sure the interaction of different Ras-binding domains (RBDs)
with Ras in an Applied Photophysics (Surrey, U.K.) SX16MV
apparatus, as described (19, 20). The reactions were carried out
in 40 mM Hepes, pH 7.4?10 mM MgCl2?150 mM NaCl?2 mM
dithioerythritol at 10°C by using an excitation wavelength of
360 nm and a cutoff filter (408 nm) in front of the detector.
Exponential and hyperbolic fits to the data, respectively, were
done by using the program GRAFIT (Erithakus Software,
Horley, U.K.).
Binding Affinity of Mg2?to Ras. The affinity of Mg2?to Ras
nucleotide complexes was estimated by measuring the depen-
denceofRas?mGppNHpdissociationrateconstantsontheMg2?
concentration at 25°C. Different amounts of Mg2?were added
to 0.5 ?M Ras?mGppNHp in 40 mM Hepes, pH 7.5?150 mM
NaCl?2 mM dithioerythritol. Low Mg2?concentrations were
adjusted with EDTA by using an apparent dissociation constant
of 1.7 ?M for the Mg2??EDTA complex (21). Fluorescence
decrease (excitation?emission at 360?450 nm) because of dis-
sociation of N-methylanthraniloyl nucleotide was started by
addition of 200 ?M of nonlabeled GppNHp, and time traces
were fitted exponentially.
X-Ray Crystallography. Crystals of the T35S mutant of Ras in
complex with GppNHp and magnesium were grown with a
reservoir of 24% polyethylene glycol 1500?50 mM CaCl2?100
mM Tris, pH 7.5, by using 20 mg ml?1protein stock solution. The
drop was composed of 5 ?l protein stock solution, 4 ?l reservoir,
and1?lSpermine-tetraHCl.Crystalsgrewtoafinalsizeof0.4?
0.1 ? 0.05 mm in the space group P212121with three molecules
in the asymmetric unit. The crystals were very sensitive to
changes in the mother liquor, therefore no ideal cryoprotectant
was found, and the crystals were measured at room temperature.
Data were collected on an FR591 (Nonius Delft, The Nether-
lands) rotating anode equipped with a MAR345 (Mar Research,
Hamburg) image plate detector and processed with XDS (22).
The crystals diffracted to better than 2.5 Å, but because of the
small size of the crystals, the data were usable only to 2.9 Å
(Table 3). The structure was solved by using the program AMORE
from the CCP4 suite (23) with Ras?GppNHp (Protein Data Bank
ID code 5p21) as a search model and yielded unambiguous
solutionsforthethreemoleculesintheasymmetricunit(Rfactor
38.4%andcorrelation62.9%,nexthighestpeak44.8and48.3%).
The model was built with O (24) and refined with CNS (25) with
noncrystallographic symmetry restraints to a final Rfreeof 28.2%
(Rcrys24.2%) with good model geometries (Table 3). The final
model comprises residues 1–29,38–60,70–166 for molecule A,
1–29,39–61,68–166 for molecule B, and 1–31,38–63,71–166 for
molecule C.
Results and Discussion
Affinity of Wild-Type Ras, Ras(T35S), and Ras(T35A) to Different
Effectors. As is generally accepted, Ras and other Ras-like
proteins use different effectors to relay signals into different
cellular responses (26). The T35S mutation has originally been
identified in a screen for mutations that selectively disrupt
specific signaling pathways of the Ras protein (27). It has been
proposed, by using either the two-hybrid method or pull-down
assays, that the mutation affects only the interaction with
RalGDS and is thus used as a tool to prove or disprove the
participation of RalGDS in a particular pathway (28–35). How-
ever, quantitative measurements by the GDI method (16, 17) of
the interaction of Ras(T35S) with effectors show that the
mutation also drastically effects the interaction with other
effectors. Table 1 shows that the affinity to Raf-RBD is de-
creased 60-fold, at least 29-fold to RalGDS-RBD, and effects
binding to AF6-RBD and Byr2-RBD (36) as well. Because the
absolute affinity of wild-type Ras to Raf-RBD is much higher
than to the other effectors, this would be a possible explanation
for the finding that in vivo T35S appears to selectively activate
the Raf pathway. Replacing Thr-35 in Ras by an alanine residue
leadstoafurtherreductionoftheaffinitytoRaf-RBDbyafactor
of three (16). For measuring binding of Ras(T35A) to the RBDs
ofRalGDSandAF6,theaffinitiesweretoolowtobedetermined
by the GDI method.
That the affinity between Ras and the effectors is substantially
weakened by the T35S mutation is surprising at first glance,
considering that in the three-dimensional structures of the
Raps?Raf-RBD complex, which mimics the Ras?Raf-RBD com-
plex, no direct interaction between T35 and the effector can be
detected(37).Furthermore,theinteractionofaRasmutantwith
RalGDS-RBD showed only a weak water-mediated contact of
Thr-35 (11) and none for wild-type Ras (12).
Conformational Transitions in Ras Proteins. We have shown earlier
that the switch I region of Ras in the GppNHp-bound state
adopts at least two conformations, which can be detected by
different chemical shifts in the31P-NMR spectrum. From the
two conformational states, conformation 2 is slightly preferred.
From temperature-dependence measurements and line-shape
analysis,theinterconversionbetweenthetwoconformationswas
found to be strongly temperature dependent, with a rate of 130
s?1at 5°C and 1,900 s?1at 25°C (7). Fig. 1 shows the31P-NMR
spectra of wild-type Ras and selected mutants with substitutions
intheeffectorloop.Whereaswild-typeRasshowstworesonance
lineseachforthe?-and?-phosphate,asdescribedearlier(7,38),
T35S shows only single peaks for the ?-phosphate (?11.10 ppm)
and ?-phosphate (?2.57 ppm) (Table 2). The T35A mutation,
also originally described as a partial loss-of-function mutation
(27), again shows only single resonance lines for the ?- and
?-phosphate. This result indicates that in the time average, the
effector loops of Ras(T35S) and Ras(T35A) adopt a similar
conformation as in state 1 of wild-type Ras, and state 2 is not
populated significantly. Because effector binding induces state 2
(7, 38, 39), it most likely corresponds to a rather well-defined
conformation (Fig. 2), as found in the complexes with effectors
(40), where the OH of Thr-35 coordinates the Mg2?ion (dis-
tance 2.2 Å) and its methyl packs against the effector loop, and
where Tyr-32 is close to the phosphates (closest approach to ?-P
5.2 Å). State 1, however, may represent either a fixed confor-
mation or an equilibrium of conformational substates in fast
exchange on the NMR time scale. Both models would result in
only one resonance each for the ?- and ?-phosphates. Such a
spectrum would thus also be expected for an effector loop that
Table 1. Interaction of Ras(wt) and Ras(T35S) with different
effectors
H-Ras-variant Effector
KD??M
Relative affinity
KD
(mutant?wt)
rel
Ras(wt)?mGppNHp Raf-RBD
RalGDS-RBD
AF6-RBD
Byr2-RBD
Raf-RBD
RalGDS-RBD
AF6-RBD
Byr2-RBD
0.02
1.0
3.0
0.3
1.2 Ras(T35S)?mGppNHp60
?100
?100
?29
?25
123.7
The apparent dissociation constants KD were determined by the GDI
method at 37°C, according to Herrmann et al. (17).
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has become more flexible (see below), because of the loss of the
methyl group of Thr-35, an effect that would be even more
pronounced for Ras(T35A), which has lost its hydroxyl group as
well.
The notion that ‘‘state 1’’ is not a well-defined arrangement of
atoms fixed in space is supported by results from three glycine
mutants of Ras, Ras(V29G), Ras(I36G), and the double mutant
Ras(V29G?I36G). The mutated residues are sitting at positions
where they are supposed to function as ‘‘hinges’’ for the effector
loop, as their mutation to glycine was shown to increase the
flexibility of the loop (41). Indeed, the31P-NMR spectra of those
mutants show single peaks for the phosphates at positions
corresponding to the proposed flexible state 1 (Fig. 1; the
spectrum of the double mutant is shown). In principle, the
internal mobility of switches I and II could be studied directly in
isotope-enriched Ras protein by relaxation time measurements.
However, the relevant residues are not visible in wild-type
Ras?Mg2??GppNHp (5, 42), probably because they are exchange
broadened beyond detection. It would be interesting to study the
behavior of the T35 mutants by heteronuclear NMR spectros-
copy. From our data, one would predict that for the mutants, the
residues of the effector loop should now be visible in the
multidimensional spectra because they occur only in state 1.
Relaxation time measurements now feasible with these mutants
should indicate that there is a fast internal mobility with a
low-order parameter S (2) describing the conformational sub-
states of the effector loop.
Crystal Structure of Ras(T35S)?Mg2??GppNHp. To get more informa-
tion about the conformation of the effector loop, we solved the
structure of Ras(T35S) by x-ray crystallography. The mutant
crystallizes in a space group different from wild-type Ras
(orthorhombic in contrast to the trigonal wild-type crystals),
Fig.1.
ofMg2?).SpectraofdifferentRasproteins(1mM)asindicatedwererecorded
at5°C,asdescribedinMethods.Thechemicalshiftsofthe?-and?-phosphates
corresponding to states 1 and 2 are summarized in Table 2.
31P-NMRspectraofwild-typeormutantRas?GppNHp(inthepresence
Table 2.31P chemical shifts and conformational states of Ras and Ras mutants in the presence and absence of
different RBDs
? phosphate
? phosphate
? phosphate
K*
?1?ppm
?2?ppm
?1?ppm
?2?ppm
?1,2?ppm
Ras(wt)?GppNHp
? Raf-RBD*
? RalGDS-RBD†
Ras(T35S)?GppNHp
? Raf-RBD
? RalGDS-RBD
Ras(T35A)?GppNHp
? Raf-RBD
? RalGDS-RBD
?11.20
?11.70
?11.7
?11.6
?2.56
?3.31
?3.6
?3.4
?0.25
?0.3
?0.4
?0.27
?0.29
?0.25
?0.33
?0.35
?0.32
1.09
?11.10
?2.57
?0.05
?11.60
?11.53
?3.41
?3.26
?11.09
?10.52
?11.09
?2.49
?2.69
?2.49
?0.05
Data were recorded at 5°C and pH 7.4. The equilibrium constant K* between states 1 and 2 is calculated from integrals of the ?
resonances defined by K* ? k1?k?1? [(2)]?[(1)].
*Data from Geyer et al. (7).
†Data from Geyer et al. (38).
Fig.2.
structureofRaps?GppNHpincomplexwithRaf-RBD(37).Rasisshownwithvan
der Waals surface, highlighting the switch I region (red worm), with the
indicated side chains and the guanine nucleotide in ball-and-stick represen-
tation. Mg2?is shown as a yellow sphere. The image was prepared by using
GRASP (49).
StructureofthenucleotideandeffectorregionofRastakenfromthe
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whichindicatesaratherdrasticchangeinthestructure?dynamics
(Table 3). The overall fold is very similar, as expected (rms
1–29,39–60,71–166 for molA, 0.73 Å; molB, 0.47 Å; and molC,
0.5 Å) and contains three molecules in the asymmetric unit. The
nucleotide is located in a similar position and makes basically the
same contacts as in the wild-type protein (Fig. 3A). Although
Ser-35 is not visible (see below), the position of the magnesium
ion can be identified, which is somewhat (0.3 Å) further away
from the ?- and ?-phosphate oxygens as compared with wild-
type Ras?GppNHp, where the hydroxyl group of Thr-35 contacts
OH of Ser-17 (3.0 Å), the ?-phosphate oxygens (3.2 Å), the
magnesium ion (2.3 Å), and two water molecules. However,
there are significant differences in the switch regions. In all three
T35S molecules, the switch I and II regions are disordered (Fig.
3A). In detail, the invisible regions comprise residues 30–37 and
61–69 (molA), 30–38 and 62–67 (molB), and 31–37 and 64–70
in molC, respectively, indicating that the effector loop is ex-
tremely mobile in the crystals (Fig. 3 A and B). The remainder
of the structure is essentially unaltered. It can be shown that the
differences are not because of (missing) crystal contacts by
comparison with the structure of Ras?GppCH2p [Protein Data
Bank ID code 6q21 (43)], which was crystallized in space group
P21 with four molecules per asymmetric unit. In this space
group, two molecules have no crystal contacts at all in the
effector loop region, and still the effector loop shows density.
The temperature of data collection, the crystallization condi-
tions,andpHvaluesweresimilarandthuscannotaccountforthe
observed differences in flexibility. In the T35S structure, one
molecule (B) has no crystal contacts in the effector loop region
(the effector loop was modeled according to the structure in
5p21 because no density was visible; see Fig. 3B), the other
moleculecouldpossiblyhavecontactsbetweeneitherPro-34and
Arg-135 (A) or Tyr-32, and the symmetry-related Arg-41 and
Asp-54 as well as Pro-34 with Glu-3 and Glu-31 with Ser-39 (C).
Here, the putative effector loop taken from 5p21 would clash
with the symmetry-related molecules so that the effector loop
must be somewhere else (and flexible, because no density is
visible).
Conformational State of the Effector Loop of Ras Bound to Effectors.
We have shown earlier that the two conformations represented
by the31P-NMR spectrum can be correlated to the biological
function of Ras, as only one conformation (state 2) is found in
the complex of Ras with either Raf-RBD (7), RalGDS-RBD
(38), or AF6-RBD (39). The dynamic equilibrium of the switch
region is thus of prime importance for the function of Ras in its
interactions with effectors, probably also with regulators (7). In
the spectrum shown in Fig. 1, Ras(T35S) can be seen exclusively
in the first nonbonding conformation. Fig. 4 shows that increas-
ing amounts of Raf-RBD or RalGDS-RBD induce a complete
shift of the ? and ? resonances in the31P-NMR spectrum to state
2. The chemical shift values are summarized in Table 2. Thus,
although the mutant has a lower affinity and higher flexibility,
it most likely adopts a conformation similar to that seen in the
wild-type complex. The binding affinity of RalGDS-RBD, not
Table 3. Crystallographic data
Space group: P212121
Unit cell: a ? 63.89 Å, b ? 79.06 Å, c ? 94.04 Å, ? ? ? ? ? ? 90.0
Resolution: 50.0–2.9 Å
Unique reflections: 10,369; observed reflections, 60,892
Completeness: 95.2% to 2.9 Å (last shell 3.0–2.9 Å: 91.6%)
I??: 7.13 (last shell 3.0–2.9 Å: 1.8)
Rmrgd-F*: 20.4% (last shell 3.0–2.9 Å: 38.7%)
Model: Protein atoms3,595
Nucleotide atoms96
Magnesium atoms3
Refinement: Resolution50.0–2.9 Å
Reflections 9,016
Rcrys†
24.2%
Rfree
28.2%
rms deviations from expected geometry:
Bond lengths, Å0.008
Bond angles, deg1.2
Overall B value, Å2
58.0
B value Mg atoms, Å2
30.85
*Quality of amplitudes (F) in the scaled data set; for definition, see ref. 50.
†Rcrys? (hkl?Fobs? ? ?Fcalc??(hkl?Fobs?, where Fobsdenotes the observed structure
factor amplitude, and Fcalcdenotes the structure factor amplitude calculated
from the model. Ten percent of reflections were used to calculate Rfree.
Fig. 3.
Superimposition of the structures (in worm plot) of wild-type Ras (Protein
Data Bank ID code 5p21) in blue and molA of Ras(T35S) in red. Neither switch
I nor II of the mutant is visible. (B) Part of the final 2Fo? Fcmap at 1.2 ? with
the Ras(T35S) model (molecule C in the asymmetric unit) as a stick molecule
and with the three phosphates of GppNHp in purple and Mg2?as a ball in
magenta, as indicated. The effector region from Tyr-32 to Glu-37, including
Ser-35, is not visible. The corresponding region from the wild-type
Ras?GppNHp structure (45) is shown in green, with corresponding residues as
indicated.
X-ray structural analysis of Ras(T355S) in the GppNHp-state. (A)
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measurable by the GDI or stopped-flow method, can be quali-
tatively determined with NMR titration, because the relation of
unbound and complexed Ras protein can be taken from the
integrals of the separated ? lines. Using these, we get a disso-
ciation constant between Ras(T35S)?GppNHp and RalGDS-
RBD of about 360 ?M, very much higher than the wild-type
constant of 1 ?M (Table 1).
In contrast to the above, the T35A mutant shows only a
broadening of the resonance lines and a slight chemical shift on
addition of the Raf-RBD (Table 2) but no shift to positions
characteristic for state 2. Addition of RalGDS-RBD produces
neither a shift of the ? phosphate nor a pronounced line
broadening, even at millimolar concentrations. When no special
effectshavetobeconsideredsuchaslinebroadeningbychemical
exchange, the transverse relaxation rate 1?T2and hence the line
width should increase after binding of the effector because of an
increase in the rotational correlation time of the complex. In a
first approximation, the increase of 1?T2 is expected to be
proportional to the increase of the molecular mass. Using the
line broadening of the relatively narrow ?-phosphate line ob-
served as function of the RalGDS-RBD concentration, we can
estimate a dissociation constant of about 10 mM for the inter-
action with Ras(T35A).
Binding of Ras(V29G?I36G) to Raf-RBD again causes a shift
toward state 2 (data not shown). However, much higher con-
centrations of RBD are needed compared with wild-type protein
to shift the equilibrium. A dissociation constant of about 1 mM
can be estimated. These results support the idea that the
observed ‘‘state 1’’ in Ras(T35S) indeed corresponds to a highly
flexible effector loop.
Kinetics of the Ras–Effector Interaction. Kinetic stopped-flow ex-
periments by using the fluorescent analogue mGppNHp bound
to Ras showed that the description of the binding of effectors to
Ras requires (at least) a two-step model. The data could be fitted
satisfactorily by assuming that an initial low-affinity encounter
complex isomerizes to the final high-affinity complex (19, 39),
and that the Ras effector complex is highly dynamic, showing
both fast association and dissociation (19, 44). The binding
reaction has been described by Eq. 1.
[1]
For wild-type Ras, plotting the observed pseudofirst-order
association rate constants against the concentration of Raf-RBD
(Fig.5)andfittingthedataaccordingtoEq.1givesadissociation
constant K1for the first step of 12 ?M and 415 sec?1for the
rate-limiting isomerization reaction at 10°C. For Ras(T35S), the
equilibrium constant K1 of the initial complex formation is
higher by a factor of 5.9, and the isomerization rate constant
drops down to 211 s?1. The overall affinity KD? K1? K2drops
42-fold to 2 ?M, very close to what has been found by the
equilibrium method (Table 1). The T35S mutant thus interacts
with Raf-RBD qualitatively similar to the wild-type protein,
although both the kinetic and equilibrium parameters of the
binding are affected by the mutation. Ras(T35A) behaves quite
differently: not only does it show a fluorescence increase on Raf
binding (not shown), as opposed to a decrease observed for
wild-type and T35S, but it also shows no saturation of the
observed rate constants (Fig. 5, Table 4). This seems to indicate
that alanine in position 35, which cannot coordinate to the Mg2?
ion as shown for the threonine residue by the three-dimensional
structure (2, 45) and expected for serine, forms a different
effector-binding conformation, and the association does not
involve a rate-limiting conformational change. The ratio of
Fig. 5.
complexed with mGppNHp was mixed with increasing concentrations of
Raf-RBD in a stopped-flow apparatus, and the resulting pseudo-first-order
reactions were measured (not shown). The observed pseudo-first-order rate
constantsareplottedagainsttheconcentrationsofeffector,asindicated,and
the points are fitted to a two-step binding equation shown in the text. The
resulting parameters are shown in Table 4.
Kinetics of binding of Raf-RBD to Ras and Ras-mutants. 0.5 ?M Ras
Fig. 4.
Ras(T35S)(AandC)andRas(T35A)(BandD)intheGppNHpformandcRaf-RBD
(AandB)andRalGDS-RBD(CandD).Increasingamountsoftheeffectorswere
added to 1–2 mM Ras protein, and spectra were recorded as described in Fig.
1 and Methods. The ratio of RBDs to Ras proteins is indicated. The chemical
shift values are summarized in Table 2.
31P-NMR spectroscopic analysis of complex formation between
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www.pnas.org?cgi?doi?10.1073?pnas.081441398Spoerner et al.