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PROTEINS: Structure, Function, and Genetics 35:89–100 (1999)
Molecular Dynamics Simulations of the Ras:Raf
and Rap:Raf Complexes
Jun Zeng,1,2 Herbert R. Treutlein,2 and Thomas Simonson1*
1Laboratoire de Biologie Structurale (CNRS), IGBMC Illkirch (C.U. de Strasbourg), France
2Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria, Australia
The protein Raf is an immediate
downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Rasbinding domain (RBD) of Raf has been modelled by
homology to the (E30D,K31E)-Rap1A:RBD complex,
and both have been subjected to multiple molecular
dynamics simulations in solution. While both complexes are stable, several rearrangements occur in
the Ras:RBD simulations: the RBD loop 100–109
moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and
Loop 4 of the Ras switch II region shifts upwards
toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with
available NMR and mutagenesis data on the Ras:
RBD complex in solution. The Ras switch II region
does not interact directly with the RBD, although
indirect interactions exist through the effector domain and bridging water molecules. No large-scale
RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the
Raf kinase domain (whose structure is unknown) is
not included in the model. Proteins 1999;35:89–100.
r 1999 Wiley-Liss, Inc.
Key words: signal transduction; homology modelling; protein structure
The protein Ras functions as a molecular switch, relaying signalling events from the cell surface to the nucleus,
regulating cell growth and differentiation.1,2 Ras cycles
between an active form in which guanosine triphosphate
(GTP) is bound and an inactive form where guanosine
diphosphate (GDP) is bound. Only the active form binds to
downstream targets of Ras, such as c-raf-1,3 A-raf,3 B-raf,3
PI-3 kinase,4,5 and RalGEF/RalGDS.6,7 Oncogenic mutants
of Ras exist predominantly in the active form, resulting in
constitutive Ras signalling and abnormal cell growth; such
mutants are found in more than 30% of human cancers.1
The protein Raf is an immediate downstream target of
Ras in the MAP kinase pathway. Activation of Raf by Ras
occurs partly or entirely through recruitment of Raf to the
cellular membrane by farnesylated Ras. Evidence also
exists for an additional allosteric mechanism, whereby Ras
binding induces conformational changes in Raf that promote activation.8 Such changes would presumably be
Ras-specific, i.e., homologous proteins such as Rap that do
not activate Raf significantly would not be able to induce
Over the last decade, extensive mutagenesis studies
have shown that critical binding and regulatory interactions between Ras and Raf occur through a 76-residue
‘‘Ras-binding domain’’ (RBD: residues 55–131) of Raf and
two specific regions of Ras.9–11 The latter regions, termed
the effector domain (residues 32–40) and the Switch II
region (residues 56–76), have different conformations in
the crystal structures of GTP- and GDP-bound Ras, and
presumably act as the molecular switches in the signal
transduction.9 A detailed analysis of Ras-RBD interactions
will, therefore, be of value to design antagonists of Raf,
with potential anti-cancer applications, and to understand
other Ras-dependent signalling pathways, such as those
involving PI-3 kinase and RalGDS.
To this end, we report molecular dynamics simulations
of the Ras:RBD complex in solution, which complement
existing structural studies. The solution structure of the
RBD of human Raf-1 has been determined by 3-D heteronuclear NMR spectroscopy,12 and the structure of Ras has
been determined both by crystallography13–16 and NMR.17
The exact structure of the Ras:RBD complex is not known.
However, the structure of a complex between RBD and
Rap1A, a protein extremely homologous to Ras, has been
solved by X-ray crystallography.18 More recently, the structure of the complex between the RBD and the more
‘‘Ras-like’’ double mutant (E30D,K31E)-Rap1A was
solved19: the latter protein is known as Raps. The sequence
identity between Raps and Ras is 50% (Fig. 1), and the
sequence identity for residues in the interface region is
95%. The binding constant of Raps:Raf is within a factor of
two of that of Ras:Raf. Therefore, the Ras:RBD structure is
expected to be very similar to those of Raps:RBD and
Rap:RBD. Indeed, many of the direct Rap-RBD interactions seen in the crystal structures could also be inferred
from NMR experiments on the Ras:RBD complex in solution.12
We report molecular dynamics simulations of both the
Ras:RBD and Raps:RBD complexes in solution. The former
Grant sponsor: Australian National Health and Medical Research
Council; Grant number: C.J. Martin Fellowship 967362; Grant sponsor: IDRIS Supercomputer Center of the Centre National de la
Recherche Scientifique.
*Correspondence to: T. Simonson, IGBMC, BP163, 67404 Illkirch,
France. E-mail:
Received 5 October 1998; Accepted 8 December 1998
Fig. 1. Sequence alignment between Ras and Raps. The sequences
were aligned using the CLUSTALX program.48 Fully conserved residues
are highlighted in grey. The effector domain and the switch II region
are indicated. Helices and ␤-strands are indicated below the sequences.
The sequences RASH and RAPA correspond to the proteins simulated
complex is initially model-built based on the latter. Both
complexes are shown in Figure 2. The simulations then
investigate the stability of the model and the differences in
structure and fluctuations between the two complexes,
providing insights into the role of various groups in
Ras-Raf recognition. In the Rap:RBD crystal structures,
the Rap effector domain (residues 32–40) forms a tight
network of electrostatic interactions with a 10-residue
␤-sheet (residues 59–68) and a 12-residue helix (residues
78–89) in the RBD. In the Raps structure, Glu31 and
Asp33 interact directly with 84Lys of the RBD.† However,
the role of the Ras Switch II region in the Ras-Raf
interaction, if any, is not evident from the Rap:RBD crystal
structures. This region has low sequence identity between
Ras and Rap, particularly in the loop L4 (residues 60–65;
Fig. 1). This loop is next to the GTP ␥-phosphate, and its
residue Q61 has been proposed as a general base for
catalyzing GTP hydrolysis, ‘‘switching’’ Ras from its active
state to its inactive state.20–22 Mutants at Ala59, Asn61,
and Asp63 of the loop L4 are found in many types of
oncogenic Ras proteins.1 Moreover, although the switch II
region does not interact directly with the RBD in the
Rap:RBD crystal structures, Ras mutants D57A and A59T
have significantly reduced Raf binding affinity, and are
incompetent for signalling in vivo.23 The possible existence
of Ras switch II interactions with the RBD is thus important for understanding the recognition of Raf by Ras, as
well as interactions of Ras with other downstream targets.
The present simulations of the Ras:RBD and Rap:RBD
complexes can be compared to the experimental structures
of Ras and the RBD alone, as well as to earlier simulations
of the RBD24 and Ras21,25 alone. Binding to Ras and/or Rap
may induce conformational changes in Raf, possibly leading to an allosteric activation of Raf.8 Such a mechanism
would presumably involve changes in the kinase domain of
Raf, whose structure is not known, and which is therefore
not included in the simulations. This limits our ability to
investigate such a mechanism. Nevertheless, the simula-
†In what follows, Ras and Raps residues are designated with their
name first and number last; RBD residues are designated with the
opposite order.
Fig. 2. Stereo view of the C␣ trace of the Raps:RBD and Ras:RBD
complexes. The RBD in both complexes is colored grey. The Raps:RBD
complex is shown in thick lines and the Ras:RBD complex in thin lines.
The effector domain, the loop L4 (Switch II region), and the helix H2
(Switch II region) in Ras/Raps are colored red, green, and blue, respectively; the rest of Raps/Ras is black.
tions were analyzed to detect possible conformational
changes induced in the RBD by Ras or Rap binding.
Recent experiments have demonstrated that a second
Ras-binding domain, the Cysteine Rich Region (CRR;
residues 139–184) of Raf could also bind to Ras, possibly to
the switch II region.26 The present model of the Ras:RBD
complex has also been used in a docking study of the
ternary Ras:RBD:CRR complex, as well as in free energy
simulations of the binding to Ras of a Raf point mutant,
presented elsewhere.24,28
at a position near residue 62 of Ras/Raps, containing the
entire RBD and half of Ras/Raps. The complexes were
solvated by 2,243 water molecules. The heavy atoms of
Ras/Raps in the outermost 3 Å (‘‘buffer’’ region) were
restrained by weak harmonic forces to their starting
positions. The partial charges of charged side chains in the
buffer region were reduced by scaling factors, to mimic
solvent screening, following Simonson et al.31 The scaling
factors are related to the electrostatic potential produced
by each charged side chain at the center of the model,
calculated from continuum electrostatics. Specifically, the
scaling factor is the ratio between the potentials calculated
with the spherical MD region surrounded either by vacuum
or by high-dielectric solvent. With this scheme, the potential near the center of the model accurately reproduces the
potential that would be observed in bulk solvent, even
though the actual MD model is surrounded by vacuum.
The residues scaled were Lys88, Asp92, Lys117, Lys 147,
Arg102, Lys104, Arg149, and Glu153 in the case of Ras,
and Arg2, Lys149, Lys151, Lys117, Arg102, and Lys104 in
Raps. A few residues outside the 29 Å boundary were
included in the model to preserve continuity of the polypeptide chain. Charges of ionized residues in this outermost
region were scaled by 80, the dielectric constant of bulk
water. These are Asp47, Glu49, and Arg97 in Ras, and
Asp47, Asp92, and Glu45 in Raps. In one Ras:RBD simulation (S2), the charges of residues 104–153 of Ras were
scaled to zero, providing a test of the simulations’ robustness with respect to the exact scaling protocol.
Water molecules were described by a modified TIP3P
water model,32,33 with internal geometry constrained by
the SHAKE algorithm.34 The CHARMM22 force field was
used for all other atoms.29 No electrostatic cutoff was
applied. Long-range interactions were treated efficiently
by using a multipole approximation for interactions be-
Starting Structures
The starting structure of the Raps:RBD complex was
taken from the (E30D,K31E)-Rap1A:RBD X-ray structure
in the PDB (entry 1gua). The initial structure of the
Ras:RBD complex was built from the Raps:RBD complex
by first superimposing the C␣ atoms of Ras onto those of
Raps via least-squares fit. Since the effector domain has
the same sequence in Ras and Rap (Fig. 1), its coordinates
were then taken explicitly from the Raps:RBD crystal
structure. GTP and magnesium were also positioned as in
the Raps:RBD complex. The resulting structure was then
optimized by 400 steps of conjugate gradient minimization
using the CHARMM22 force field,29 during which the
backbone atoms shifted by just 0.3 Å. Finally, 70 ps of
molecular dynamics simulation were carried out with the
protein atoms harmonically restrained, to further relax
bad contacts.
Molecular Dynamics Setup
Molecular dynamics (MD) simulations were carried out
at a constant temperature of 25°C with stochastic boundary conditions30 and a 1.5 femtosecond time-step. The
model included a spherical region of 29 Å radius, centered
tween groups of atoms more than 12 A apart.35 Atoms in
the buffer region experienced friction and random forces
mimicking the coupling to a thermal bath. Simulations
were performed using the CHARMM36 program.
B-Factor Calculation
To compare the crystallographic B-factors with the MD
results, the contribution of overall rotation and translation
of the protein, absent in the simulations, must be added to
the MD data. We proceed as follows. The rigid body motion
of the protein can be represented by two (unknown)
symmetric tensors T and L37; the mean square amplitudes
of translational motion in the direction of a unit vector l
and of libration about l are given by
兺兺T ll
ij j j
兺 兺 L ll.
ij i j
respectively, where the li, lj are the cartesian components
of l. The contribution of overall protein rotation and
translation to the B-factor of a protein atom is38
BRT ⫽ 1⁄3 Trace (GL ⫹ T)
⫽ 1⁄3 [(r 22 ⫹ r 23 )L11 ⫹ (r 12 ⫹ r 32 )L22 ⫹ (r 12 ⫹ r 22 )L33 ]
⫺ 2r2r3 L23 ⫺ 2r1r3 L13 ⫺ 2r1r2 L12 ⫹ t 2.
where G is an array involving the coordinates of the atom
(r1 · r2 · r3 ), and t 2 represents the isotropic mean-square
amplitude of translational vibration,
t 2 ⫽ 1⁄3 (T11 ⫹ T22 ⫹ T33 ).
Fig. 3. a: Rms deviation (Å) of the Raps Switch II region from its
starting structure (solid line) and final structure (dashed line) during the
512 ps Raps:RBD simulation. b: Occupancy vs. time of conformations
sampled by the Raps switch II region (defined by clustering based on C␣
rms deviations; see text).
To determine the unknown tensors T and L, the difference ⌬B between the atomic B-factors from the X-ray data
and the MD simulation are first computed. These differences should provide reasonable estimates of the BRT;
therefore, the tensor elements are adjusted so that the BRT
in Eq. 3 optimally reproduce the ⌬B. Finally, the resulting
BRT are added to the MD B-factors. The same procedure
was used in a simulation study of Ribonuclease S.39
The above procedure is circular, in the sense that the
X-ray and MD data are used jointly to determine the BRT,
then the BRT are added to the MD B-factors, and the
results are compared back to the Xray B-factors. This is
unavoidable, since experimental values of the BRT are not
available for this system. To obtain such experimental
values, the X-ray refinement of the structure would have
to be performed using a sophisticated model of the disorder
in which overall rotation-translation is treated explicitly,
as has been done for a few proteins.37,40,41 An alternative
procedure, used in many early MD studies, consists in
neglecting the BRT component of the experimental Bfactors altogether. However, crystallographic studies have
shown37,40,41 (and the present study confirms; see below)
that the BRT are far from negligible, and so the present
fitting procedure is preferable. The procedure is valid,
because the determination of T and L involves only twelve
adjustable coefficients (the tensors are symmetric), which
are fit to several hundred atomic B-factors (here, backbone
atoms of buried residues in the complexes were used in the
fit). The B-factors are then calculated and compared for
approximately 1,000 atoms, including side chains.
Simulation of the Raps:RBD Complex
The average Raps:RBD simulation structure is very
similar to the crystal complex. The rms deviation from the
X-ray structure increases during the first half of the 510-ps
simulation (Fig. 3a), then stabilizes at 0.9 Å (nonhydrogen
atoms; 0.7 Å for backbone atoms). The potential energy of
TABLE I. Atomic Fluctuations and Deviations From Starting Structures (Å)
Rmsd. backbonec
Rmsd. side chains
Rmsd. heavy atoms
Rmsf. heavy atomsd
B-factor. heavy atoms
Rmsd. backbone
Rmsd. side chains
Rmsd. heavy atoms
Rmsf. heavy atoms
B factor. heavy atoms
MD ⫹ TLb
over the Raps:RBD and Ras:RBD simulation trajectories.
overall rotation-translation correction (see Materials and Methods).
cRms deviation from starting structure.
dRms fluctuation around average structure.
eFrom NMR spectroscopy on the RBD in solution.12
the system converges over this 250-ps time segment (data
not shown). The rms deviation from the final structure
stabilizes during the last 150 ps. Details are listed in
Table I.
The main conformations sampled in the simulation were
determined by clustering the trajectory based on rms
deviations of the C␣s. The switch II region in Raps
(residues 56–76) displays the greatest conformational
variability. Five main conformations are sampled in this
region, separated by small energy barriers, as shown by
the frequent transitions between them (Fig. 3b). In the
predominant conformation, the ␣ carbon of Raps-Ala66 is
shifted upwards 2.2 Å, slightly distorting the Raps ␣2
In the RBD, significant changes (and large fluctuations)
occur in two loop regions (Fig. 2). The loop L3 (residues
100–109) moves 2.0 Å down towards the Raps switch II
region, probably due to long-range interactions between its
charged amino acids (106Lys, 108Lys, and 109Lys) and the
switch II region. The loop L2 (residues 73–76) maintains a
backbone position similar to the crystal structure, but
different sidechain rotamers of residues 73Arg, 74Asn, and
76Met were sampled in the simulation, giving a large rms
fluctuation (1.6 Å) for these residues. In the crystal
structure, crystal contacts occur in these two loop regions,
resulting in smaller observed B-factors. In the Raps:RBD
interface region, six salt bridges seen in the crystal complex (84Lys/Asp33, 84Lys/Glu31, 59Arg/Glu37, 67Arg/
Glu37, and 89Arg/Asp38) are preserved during the entire
The simulation samples a solvent structure between the
Raps Loop 4 in the switch II region and the GTP ␥-phosphate that differs from the Raps:RBD crystal structure. In
the latter, a water molecule next to the ␥-phosphate forms
a channel connecting the GTP to bulk solvent. In Ras, a
water molecule in this position is thought to attack the
␥-phosphate in GTP hydrolysis. The channel, bounded by
the Thr35-C␥ and the Ala59-C␤, is just large enough to
allow a water molecule to enter, and inorganic phosphate
to leave the GTP pocket.18 This water is also found in the
simulation, with a B-factor of 17.5 Å2 (including the
protein translation/libration contribution: see below, Methods), close to the crystallographic value of 19.0 Å2. However, the simulation places a second water molecule between the ␥-phosphate and O␥1 of Thr61. The latter water
molecule is more mobile than the first, with a B-factor of
28.5 Å2. The presence of the second water molecule could
be due to the fully solvated simulation conditions. Another
structural difference is the direct formation of a hydrogen
bond between the Tyr32-OH and an oxygen atom of the
␥-phosphate, which were separated by a water molecule in
the crystal structure. The existence of such a hydrogen
bond in unbound Ras (free in solution) is suggested by
NMR data.42
The average rms fluctuation of nonhydrogen atoms
around the mean structure is calculated to be 0.94 Å (Table
I). This gives an average B-factor of 23.1 Å2, comparable to
the crystallographic value of 18.6 Å2.19 However, the
experimental B-factors include overall rotation and translation of the protein, which is not present in the simulations. By the method outlined above (see Materials Methods), this contribution is estimated to be 8.7 Å2 for Raps,
5.6 Å2 for the RBD, and 7.1 Å2 for all heavy atoms. The
amplitudes of overall rotation and translation are thus
estimated to be 0.44 Å and 0.30 Å, respectively, close to the
values observed in other proteins of similar size (e.g., 0.64
Å and 0.24 Å in ribonuclease A crystals).37,40,41 Adding
these to the intramolecular fluctuations from the simulation, the overall B-factors are calculated to be 21.5 Å2 for
Raps, 39.6 Å2 for the RBD, and 30.2 Å2 for all heavy atoms.
While the Raps value is close to the crystallographic
result, the RBD value is much larger. It is in fair agree-
Fig. 4. Comparison between atomic rms fluctuations (Å) in the
Raps:RBD simulation and experiment.19 Simulation results with (dots;
MD/TL) and without (x: MD) rotation-translation contribution. Results
averaged over entire residues. a: Raps: b: RBD.
ment with the flexibility of the uncomplexed RBD in
solution, suggested by the rms deviation from the mean of
the 30 structures in the experimental NMR ensemble12
(1.1 Å, corresponding to an effective B-factor of 32.0 Å2 ).
This is consistent with the geometry of the simulation
model, in which the inner part of Raps is involved in the
interface with the RBD and the outer part is in the
restrained buffer region (where the restraint force constants are actually determined by the experimental
B-factors). Most of the RBD surface, on the other hand, is
exposed to solvent. Thus, the Raps flexibility is similar to
that in the crystal environment, while that of the RBD is
solution-like. Similarly, the variations of the calculated
Raps B-factors along the polypeptide chain (Fig. 4) are in
good agreement with the crystallographic values, except
Fig. 5. a: Rms deviation (Å) of the Ras Switch II region from its starting
structure (solid line) and final structure (dashed line) during the 635 ps
Ras:RBD simulation S1. b: Occupancy vs. time of conformations sampled
by the Ras switch II region (defined by clustering based on C␣ rms
deviations; see text).
for residues Glu62 and Gln63 (whose larger calculated
values arise from the sampling of multiple sidechain
orientations in the simulation), while the calculated RBD
values are larger. Residues Ala57 and Asp131 at the two
Raps chain termini have large rms fluctuations of approximately 1.4 Å and 1.6 Å, respectively, typical of protein
chain termini in solution.25
The RBD structure in the complex, averaged over the
MD simulation, was compared to the average NMR structure of the RBD in solution. Both structures were slightly
minimized first, to remove stereochemical distortion associated with the averaging. While the RBD in the complex
folds in a similar manner to the RBD alone in solution,
some local conformational changes occur. In the complex,
the ‘‘Ras-binding helix’’ extends from residue 84 to 89: in
solution, two distinct conformations are seen for residues
87–90: one where the helix extends to residue 89 as in the
Fig. 6. Snapshots of the interface between Ras and the RBD during
the Ras:RBD simulation S1. Orientation as in Figure 2. The backbone as
well as charged sidechains in the interface are shown. Residues 59Arg,
67Arg, 73Arg, and 89Arg are dark blue: residues Asp33, Glu37, and
Asp38 are red. Snapshots corresponding to the first 100 ps of simulation
have their backbone colored black; similarly for the 73Arg side chain. The
downwards drift of the RBD backbone around 73Arg can be seen.
complex, and one where residues 87–90 form a ␤ turn.12,24
The backbone rms deviation between the MD complex and
the NMR structure, averaged over non-loop RBD residues,
is 1.2 Å. Deviations in the surface loops are larger, ranging
from 2.9 to 3.6 Å. These loops (residues 63–66, 73–77,
90–95, 100–110, and 122–125) are very flexible and only
moderately conserved throughout evolution, based on alignment of six RBD sequences (human, pig, rat, C. elegans, D.
melanogaster, X. laevis: not shown).
most of the analysis of the Ras:RBD complex below. In
addition, the RBD loop 72–76 undergoes a 1.3–1.7 Å shift
going from conformation 2 to conformations 3–6, with a
salt bridge forming between RBD-73Arg and Ras-Asp33.
In the Raps:RBD simulation, in contrast, the separation
between the RBD-73Arg and Raps-Asp33 always remained greater than 5.0 Å, with a water molecule bridging
the 73Arg-NH and the Asp33-OD.
Principal component analysis43 of S1 (not shown) indicates that a significant portion of the RBD motion corresponds to an overall rotation towards the switch II region
of Ras, correlated with an upwards motion of the switch II
␣2 helix towards the RBD. The motion of the switch II
region and the RBD are thus coupled, possibly through
long-range electrostatic interactions.
The stability of the interface structure during the three
simulations is illustrated in Figure 6 by a set of snapshots
from the trajectories. Hydrogen bonds between Ras and
the RBD are listed with their occupancies in Table II.
Three salt bridges, RBD-59Arg/Ras-Glu37, 67Arg/Glu37,
and 89Arg/Asp38, and two backbone hydrogen bonds,
Glu37-O/69Val-N and Ser39-N/67Arg-O, in the core of the
interface remain stable during all three simulations. Mutations of charged amino acids in or around the interface will
form or disrupt Ras-Raf salt bridges, and consequently
affect the Ras-Raf binding affinity. The relationship between the Ras:RBD structure and experimental mutagenesis results is summarized in Table III.
Some variations are seen between the three simulations
(Table II). In Ras, Asp31 and Glu33 flank the effector
domain and are exposed to bulk solvent. Their charged
sidechains form stable salt bridges to RBD-84Lys throughout S1 and S3: these salt bridges are partially broken
during S2. While Glu33 directly contacts RBD-73Arg
during S1 and S3, it is separated from it by a water
Simulations of the Ras:RBD Complex
The Ras:RBD structure was built and solvated as described above (see Materials and Methods), and three
simulations were carried out, lasting 635, 600, and 600 ps,
respectively (referred to below as S1, S2, and S3). These
provide a dynamic model of the Ras:RBD complex, whose
experimental structure is not known.
Structure and dynamics of the Ras:RBD complex
Table I reports deviations from the starting model
structure (which is similar to the Raps:RBD complex; see
Materials and Methods). In simulation S1, the average
deviation is 0.88 Å for backbone atoms and 1.22 Å for all
heavy atoms, larger than the values from the Raps:RBD
simulation, as expected. Figure 5a shows the rms deviation as a function of time. From the clustering protocol
used above, six main conformations were sampled during
S1 (Fig. 5b). The six conformations are mainly distinguished by the C␣ positions in the Ras switch II region.
The first 225 ps sampled two well-defined conformations,
each for approximately 100 ps. These are unstable, and the
structure then shifts about 1.3 Å (C␣ deviations) into a set
of four conformations that differ by only 0.5–0.8 Å from
each other and are separated by low energy barriers. The
stable 225–635 ps trajectory segment of S1 was used for
TABLE II. Occupancy of Selected Hydrogen Bonds
During Simulations
Hydrogen bond partners
Hydrogen bond partners
Simulation run
Simulation run
water molecule.
molecule during S2, similar to the Raps:RBD simulation
and the crystal structure. Tyr32-OH directly bonds to the
GTP ␥-phosphate during S1 and S2; this hydrogen bond
was only stable for half of S3. The conformation of the Ras
Loop 4 varies significantly between simulations. While the
hydrogen bonds Gly60-O/Tyr96-OH and Asn61-NE/Gly13-O
are present in S1, they are less stable in S2 and completely
broken in S3. Stable hydrogen bonds between the guanidinium group of Arg68 and Tyr71-OH are only observed in S2.
The best-defined hydrogen bond in the Ras switch II region
is Ser17-OG/Asp57-OD. This hydrogen bond strongly links
the Switch II region and the helix ␣1 in all three simulations. The Asp57Ala mutation, which decreases Ras-Raf
binding, may break this linkage, and consequently cause a
conformational change in Ras (see Table III).
The solvation around the Ras effector domain and
switch II region is stable and similar in the three simulations (Fig. 7a). A chain of water molecules connects Asp57
of the Ras switch II region, Asp37 and Asp38 of the effector
domain, and Arg89 of the RBD. In the GTP pocket, 2–3
water molecules are observed between loop L4 and the
GTP ␥-phosphate (Fig. 7b). While two water molecules
consistently bind to the GTP ␥-phosphate as in the Raps:
RBD simulation, a third water molecule only enters the
GTP pocket after 420 ps of simulation S2.
The atomic rms fluctuations of the Ras:RBD interface
are in good agreement with both the observed and simulated B-factors of the Raps:RBD complex (Figs. 4 and 8).
The RBD does not interact directly with the switch II
region in either the Ras:RBD or the Raps:RBD complexes.
The effect of switch II mutations on Ras-RBD binding
TABLE III. Relative Binding Affinity of Ras, Raf Mutants†
Mutation Affinity
Breaks salt bridge
Breaks salt bridge
Breaks salt bridge
Breaks salt bridge
Lost 89R-S39
hydrogen bonds
Lost hydrogen bond
Long-range effect/conformation change
Conformation change
Breaks salt bridge
Lost hydrogen bond
Breaks salt bridge
Lost hydrogen bond
Forms salt bridge
Breaks salt bridge
Breaks salt bridge
Breaks salt bridge
Breaks salt bridge
Forms salt bridge
Breaks salt bridge
Forms salt bridge
Forms salt bridge
Breaks salt bridge and
hydrogen bonds
D38/S39 Breaks salt bridge and
hydrogen bonds
D38/S39 Increased desolvation
cost/lost hydrogen
46 a
11,46 a
46 a
11 a
11 a
46 a
24,28 a
and ⫺ indicate increased or decreased binding affinity, respectively.
affinity measured for the Ras:RBD complex.
bMutation to any uncharged residue.
cM. Fridman and H. Maruta, unpublished data.
would thus be expected to be minor. Nevertheless, two
mutations at the switch II N-terminus of Ras, i.e., Asp57Ala
and Ala59Tyr, have been shown to decrease the binding
affinity significantly (Table III). These residues are buried
beneath the effector domain of Ras, and interact with the
RBD indirectly through a water channel shown in Figure
7a. The channel was also found in the Rap:RBD crystal
structures (see above), with a water molecule forming a
bridge between RBD-89Arg89 and Ras-Asp38,19 having a
B-factor of 17.9 Å2. The average B-factor of the channel
water molecules is calculated to be 20.1 Å2 from the simulations (including protein translation/libration, as appropriate for comparison to the X-ray value).
The RBD structure in the complex, averaged over the MD
simulation, was compared to the average NMR structure of
the RBD in solution. As in the Rap:RBD complex above, the
‘‘Ras-binding helix’’ extends from residue 84 to 89 in the
Ras:RBD MD complex, whereas this region occupies a mixture
of two conformations in solution. The backbone rms deviation
between the MD complex and the NMR structure, averaged
over non-loop RBD residues, is 1.5 Å. Deviations in the surface
loops are larger, ranging from 3.2 to 4.4 Å.
Fig. 7. Stereo view of the solvation structure around the effector
domain of Ras, the Ras switch II region, and the GTP pocket, from the
Ras:RBD simulation S1. a: Solvation structure around the N-terminus of
the Ras switch II region. The lower two ␤-strands belong to Ras, the upper
two to the RBD. b: Solvation structure in the GTP pocket. The magnesium
ion is shown as a sphere.
Comparison to the Raps:RBD complex
bonds directly to Ras-Asp33, with two N␩-O␦ distances of
2.9 Å and 2.6 Å. The newly formed 73Arg/Asp33 salt bridge
appears to drive the backbone rearrangement of the
RBD-72-77 loop. In Ras, the Loop 4 of Switch II (residues
62–65) shifts away from its starting position; the ␣2 helix
moves by approximately 2.0 Å and shortens by two residues, from 64–75 to 66–75.
The solvation structure in and near the GTP pocket
(shown in Fig. 7b) is similar to the Raps:RBD simulation,
with a water channel bounded by Thr35 and Ala59, and
two water molecules bound to the GTP ␥-phosphate. The
B-factors of these two waters (including protein translation/
The overall orientation of the RBD is slightly different
from that in the Raps:RBD complex, as shown in Figure 2.
However, all the Rap-RBD salt bridges are preserved in
Ras:RBD. Two loop regions of the RBD (residues 100–109
and residues 72–77) have different conformations from the
Raps:RBD simulation (and the crystal structure), as well
as large fluctuations (Fig. 8). Both regions move toward
Ras during the simulations; e.g., the C␣ atom of Asn74
moves 4.0 Å during S1. In the starting structure, RBD73Arg was more than 5.0 Å away from Ras-Asp33; after
225 ps of S1, the RBD-73Arg guanidinium group hydrogen
Fig. 8. Comparison between atomic rms fluctuations (Å) in the
Ras:RBD simulation S1 and experimental data for Raps:RBD.19 Simulation results with (dots) and without (x) rotation-translation contribution.
Results averaged over entire residues. a: Ras; b: RBD.
libration) are calculated to be 10.4 Å2 and 22.1 Å2, respectively, slightly smaller than the Raps:RBD X-ray and
simulation results.
Energy component analysis
Interaction energies were calculated between different
residues of the RBD and charged side chains in the effector
domain and the switch II region of Ras. Atoms within 6 Å
of an Arg-C␨, or 4 Å of an Asp-C␥ or Glu-C␦, were taken to
define a first interaction shell. (No Lys occurs in the
regions considered.)
All six charged residues in the effector domain have an
RBD salt bridge partner in their first interaction shell.
Corresponding interaction energies are between ⫺111 and
⫺83 kcal/mol, dominated by electrostatics. Mutation of
any single salt bridge partner will destroy one such
interaction, and the unbound state will be overwhelmingly
stabilized by solvation of the remaining, unbalanced partner. This is confirmed by experimental mutagenesis data
(Table III). Joint mutation of both salt bridge partners, on the
other hand, is likely to have a much smaller, possibly negligible effect on binding.44 Solvent is present in the first interaction shells of the effector domain, with coordination numbers
of 3–7 and interaction energies of ⫺35.0 to ⫺66.0 kcal/mol.
This gives on average ⫺10.0 kcal/mol interaction energy per
water molecule, typical of short-ranged charge-water interactions. No RBD amino acids are found in the first interaction
shells of the Ras Switch II region. Asp57 is buried inside Ras,
interacting with three water molecules (Fig. 7a). Charged
residues in the segment 60–73 are entirely exposed to solvent,
experiencing interaction energies of ⫺10.0 kcal/mol per water
The interaction energy differences between Ras and
Raps interacting with the RBD were also calculated.
Several large differences are seen for residues conserved
between Ras and Raps. First, although the sequence of the
effector domain (residues 30–42) is identical in Ras and
Raps, Ras-Asp33 forms a salt bridge to RBD-73Arg not
found in Raps. Thus, Ras-Glu31 and Asp33 contribute
interaction energies more negative by ⫺28 and ⫺52 kcal/
mol, respectively. The contribution of Ras-Arg68 is increased by 72 kcal/mol in the Ras:RBD complex, as a result
of a structural difference: Arg68 hydrogen bonds to RasTyr71 in the Ras:RBD complex (Table II), but forms a salt
bridge to Raps-Asp92 in the Raps:RBD complex.
Several large differences arise because of sequence
differences between Ras and Raps. For example, His27 in
Ras has an unfavorable interaction with the RBD residues
89Arg and 87Lys, and contributes ⫹153 kcal/mol to the
RBD:Ras interaction energy. Some other differences are
seen that are expected to be largely compensated by
changes in the unbound state. For example, Ras-Glu63
contributes ⫺236 kcal/mol to the interaction energy, compared to ⫺34 kcal/mol for its homologue Gln63 in Raps. At
the same time, Glu63 is expected to interact strongly with
solvent in the unbound state, destabilizing the bound
form. The extent of this destabilization is expected to be of
the same order of magnitude as, but somewhat smaller
than, the Glu63/Glu63 interaction energy difference (e.g.,
the solvation free energy difference between Gln and Glu is
on the order of 80 kcal/mol, favoring Glu45 ).
Overall, all the significant changes in the interaction
energy between Ras-RBD and Raps-RBD are due to
charged amino acids, underlining the importance of longrange electrostatics for Ras-RBD recognition.
The present simulations probe the structure and dynamics of the Raps:RBD complex in solution, and provide a
dynamical model of the Ras:RBD complex, whose structure has not been determined experimentally. The model
combines a high quality force field with an accurate
treatment of electrostatics, where long-range interactions
are treated with a multipole approximation and bulk
solvent is represented by scaling factors obtained from
Poisson-Boltzmann calculations on the fully solvated complexes. Repeated simulations and comparison to both the
Rap:RBD crystal structures and NMR data on the Ras:
RBD complex suggest that the present Ras:RBD model is
stable and representative of the actual complex in solution.
Structural differences between the Ras:RBD and Raps:
RBD complexes occur mainly in three regions. In the
Ras:RBD case, the RBD loop 100–109 moves closer to Ras;
73Arg in the RBD loop 72–77 moves towards Ras to form a
salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II
region shifts up towards the RBD. Loop 4 has a low
sequence identity between Ras and Raps; in particular, the
charged Glu63 is present in Ras but not Raps. This
additional charge may help drive the conformational
change, as well as attracting RBD-73Arg. These structural
differences are reflected by the interaction energy differences between Ras and Raps: Glu31 and Asp33 of Ras
interact much more strongly with the RBD than in the
Raps:RBD case, and Glu63 of the Ras switch II (Gln in
Raps) stabilizes the Ras:RBD complex by an additional
⫺142 kcal/mol. This residue may also ‘‘pull’’ the 100–109
loop downwards through a long-range electrostatic interaction, since the latter loop contains several positive charged
residues, i.e., 106Lys, 108Lys, and 109Lys.
The sequence of the effector domain in the interface with
the RBD is identical in Ras and Raps. The conformation of
this region is very similar throughout the Raps:RBD and
Ras:RBD simulations, and very stable, with an average
B-factor and rms atomic fluctuation of 19.7 Å2 and 1.0 Å,
respectively (from the Ras:RBD simulation S1). The RasRBD interactions seen in the simulations are consistent
with those observed by NMR spectroscopy in solution. In
the NMR experiment, the RBD residues with significantly
perturbed amide chemical shifts were in the N-terminal
region (i.e., residues 56–61, 65–69, 78–91), which directly
contacts the effector domain of Ras in the simulations. The
detailed correspondence between chemical shift perturbations and Ras-RBD interactions is almost perfect (see
ref.28 ). The direct contact between 73Arg of the RBD and
Asp33 of Ras predicted from our simulations can also be
inferred from a large broadening of the experimental
resonance of a 73Arg side chain proton, even though this
residue was not originally classified as an interacting
one.12 Experimental measurements of the Ras-RBD binding affinity change associated with point mutations of
73Arg are desirable to further validate this interaction.
The solution structure and dynamics of the Ras:RBD
complex obtained from the molecular dynamics simulations are also consistent with the available mutagenesis
data (Table III). The simulations confirm the Ras:RBD
interface structure suggested by the Rap:RBD crystal
structures. Salt bridges connect the effector domain of Ras
and the RBD, and binding is correspondingly sensitive to
mutations in this region. The Ras switch II region does not
interact directly with the RBD, and most of its side chains
are exposed to solvent, so that mutations in this region
should not affect Ras-RBD binding strongly. The Asp57Ala
mutation at the Ras N-terminus does have a significant
effect on Ras-Raf binding, even though Asp57 does not
directly interact with the RBD. The simulations suggest
several possible mechanisms for this effect. First, Asp57 is
connected with the Ras effector domain (Asp38, Tyr40)
through bridging water molecules, and its mutation may
therefore disrupt the conformation of the effector domain.
Second, the energy decomposition described above indicated that 89Arg of the RBD and Asp57 have a large
interaction energy (⫺22 kcal/mol); loss of this direct interaction may contribute to the binding free energy change.
Third, the hydrogen bond between Asp57 and Ser17 links
the Ras switch II region to the Ras ␣1 helix. The Asp57Ala
mutation is expected to break this linkage, possibly leading to a conformational change of the effector domain.
Some differences in the solvation of the active site were
observed, compared to the crystal structures. In addition
to the water molecule observed between the Loop L4 of
Switch II and the GTP ␥-phosphate in the crystal structures of both Ras and the Rap:RBD complex, the present
simulations (of both Raps:RBD and Ras:RBD) place a
second water molecule, and possibly a third, inside the
GTP pocket. These water molecules are expected to play a
role in GTP hydrolysis and, consequently, in Ras-Raf
Comparing the RBD in solution to the RBD in the
complexes, significant local conformational differences are
seen, mainly in surface loops far from the interface. These
loops are very flexible and only moderately conserved
throughout evolution. It remains to be seen whether these
motions are involved in the signalling mechanism. Comparing the Rap:RBD and Ras:RBD complexes, no large-scale
shift of the RBD relative to Ras or Rap is seen (Fig. 2) that
would provide evidence for a putative allosteric activation
of Raf by Ras. This could be a consequence of the limited
size of the model (29 Å radius) and simulation time, and
possibly of the absence of the Raf kinase domain.
J.Z. gratefully acknowledges a C.J. Martin fellowship
(967362) awarded by the Australian National Health and
Medical Research Council. The CHARMM program was
kindly provided by Professor Martin Karplus.
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