From the Division of Physical Biochemistry, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United
Kingdom and
Glaxo Wellcome Medicines Research Centre,
Gunnels Wood Road, Stevenage,
Hertfordshire SG1 2NY, United Kingdom
Ras proteins are guanine-nucleotide binding
proteins that have a low intrinsic GTPase activity that is enhanced
105-fold by the GTPase-activating proteins (GAPs)
p120-GAP and neurofibromin. Comparison of the primary sequences of
RasGAPs shows two invariant arginine residues (Arg1276 and
Arg1391 of neurofibromin). In this study, site-directed
mutagenesis was used to change each of these residues in the catalytic
domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras·GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate
contributions of each of these residues to catalysis and binding
affinity to Ras were measured. Both the R1276A and the R1391A mutant
NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured at saturating concentrations. In
contrast, there was only a minor effect of either mutation on NF1-334
affinity for wild-type Ha-Ras. These data are consistent with both
arginines being required for efficient catalysis. Neither arginine is
absolutely essential, because the mutant NF1-334 proteins increase the
intrinsic Ras·GTPase by at least 100-fold. The roles of
Arg1276 and Arg1391 in neurofibromin are
consistent with proposals based on the recently published x-ray
structure of p120-GAP complexed with Ras.
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INTRODUCTION |
Members of the Ras superfamily of guanine-nucleotide binding
proteins, comprising the Ras, Rho, Rab, and Arf families, share common
structural and biochemical properties (1-3). Each protein exists in a
GTP-bound conformation that is converted into the GDP-bound
conformation through hydrolysis of the bound GTP. The intrinsic GTPase
rate of these proteins is relatively slow, around 10
3-10
4 s
1. This intrinsic
GTPase is activated by ~105-fold by interaction with
GTPase-activating proteins
(GAPs).1 GAPs have been
characterized for members of all branches of the Ras superfamily.
Since the first identification of p120-GAP as a RasGAP, several other
RasGAPs have been cloned, such as neurofibromin (NF1), IRA,
and GAP1. The catalytic (Ras-binding) domain of these proteins has been
identified, and it contains within its most conserved region 13 invariant residues. The x-ray structures of the GTPase-activating domain of p120-GAP both alone and in complex with Ras have recently been published (4, 5). It can be seen from these structures that the
role of most of these conserved residues is likely to be structural.
From the structural data, it was proposed that Arg789 and
Arg903 (equivalent to Arg1276 and
Arg1391 of NF1) (Fig.
1a) might play an essential
role in catalysis by directly contributing a residue to the GTPase
active site. Although there is support for this hypothesis based on
mutagenesis studies of these conserved arginines in p120-GAP and NF1,
there have been conflicting data reported (6-12). We believed that
confusion may have resulted because the fundamental parameters
describing catalysis, the maximal rate (kcat)
and the affinity (Kd), have not always been measured
in these mutagenesis experiments. To verify the proposed mechanism, we
considered that it was important to undertake a more detailed
examination of the biochemical properties of these arginine residues.
Therefore, we mutated either Arg1276 or Arg1391
to alanine in the catalytic domain of NF1, i.e. NF1-334
(13), and measured the effects of these mutations on both catalysis and
on binding affinity with wild-type and Leu61Ha-Ras.

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Fig. 1.
Comparison of sequences of RasGAPs and
RhoGAPs surrounding conserved basic residues. Panel a shows
sequences from NF1, surrounding its conserved Arg1276 and
Arg1391 residues, and the homologous regions from other
RasGAPs. The sequences are NF1 (P21359), p120-GAP (human, P20936),
dmGAP1 (M86655), Ira1p (M24378), Sar1 (Schizosaccharomyces
pombe, P33277), and GAP1IP4BP (X89399). Panel
b shows sequences from RhoGAP, surrounding its conserved
Arg282 and Lys319 residues, and the homologous
regions from other GAPs active on the Rho family. The proteins are
RhoGAP (Z23024), bcr (A28765), n-chimerin (P15882), p190 (P27986), and
p85alpha (P27986). The arrow indicates the position of the
catalytic arginine residue, and the filled circle indicates
the position of the basic residue involved in positioning this
arginine. Where at least 60% of the sequences show identical residues,
they are shown in boldface. Acid residues (Asp or Glu),
basic residues (Lys, Arg, or His) and hydrophobic residues (Ile, Leu,
Val, Phe, Trp, Tyr, or Met) were grouped together, and residues of the
same type are boxed. The derived consensus sequences use
X to refer to hydrophobic residues and x to refer
to unspecified residues.
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MATERIALS AND METHODS |
Proteins and Nucleotide Complexes--
Harvey Ras (wild-type
truncated at residue 166 and full-length Leu61Ha-Ras) were
prepared as described previously (14, 15). Wild-type and mutant NF1-334
proteins were expressed in Escherichia coli as glutathione
S-transferase fusion proteins and cleaved from the
glutathione S-transferase and purified as described
previously (13). Protein concentrations of NF1-334 were determined
using
280 value of 32520 M
1cm
1 calculated from the
extinction coefficients of Tyr and Trp residues (16)
Ras·mant-nucleotide complexes were prepared, and their concentrations
were determined, as described previously (17).
Site-directed Mutagenesis of NF1-334--
The R1276A and R1391A
mutations in glutathione S-transferase-NF1-334 were made
using the two-step PCR method described by Landt et
al. (18). In the first PCR, oligonucleotides BS1
(5'-dCTGTTGCCTGCGAAGAGAGTC-3' for the R1276A mutation) and BS2
(5'-dGTTCCTCGCATTTATCAATCC-3' for the R1391A mutation), were used in
combination with a "flanking" oligonucleotide located either 5' to
the unique XhoI site for R1276A (RS113:
5'-dTTTGACAGATCTACGCTAGCAGAAACAGTATTGGCT-3', where the last 24 nucleotides correspond to amino acids 1195-1202 of NF1) or 3' to the
unique NdeI site for R1391A (RS131:
5'-dCGTGCTGCATCAAAGTT-3', which is complementary to the DNA sequence
corresponding to amino acids 1450-1455) to amplify 265- and 200-base
pair DNA fragments, respectively. These DNA fragments were gel purified
and used in a second PCR with the other flanking oligonucleotide (RS114
(5'-dTTTGTGGTCGACTTAGAACTCCTCTGGGGGCCCTAGGTATGCAAG-3') for R1276A
and RS113 for R1391A). The PCR products of the second reaction were
isolated as a XhoI/NdeI DNA fragment that was
ligated into pGEX-NF1-334 DNA digested with XhoI and
NdeI. The presence of each mutation was determined by DNA
sequencing, and the entire DNA between XhoI and
NdeI restriction sites was sequenced to confirm that no
other mutations had occurred.
Fluorescence Measurements--
All measurements were made
in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM MgCl2, and 0.1 mM dithiothreitol
at 30 °C unless otherwise stated. Time courses of fluorescence
changes over periods of less than 10 s were made in a Hi-Tech MX61
stopped-flow fluorometer operated in the single push mode. Excitation
was at 366 nm, and emitted light was monitored through a Wratten 47B
filter. For monitoring fluorescence changes over longer time periods,
measurements were made on a SLM 8000S fluorometer with excitation at
366 nm, and emission was monitored at 440 nm. High performance liquid chromatography of mant nucleotides was as described previously (17).
Data Analysis--
Data on the effect of concentration of NF1 on
the observed rate constant (kobs) for the fast
phase of the fluorescence change consequent to mixing Ras·mantGTP and
NF1-334 was fitted to Equation 1. This describes the kinetics of
initial formation of Ras·mantGTP·NF1-334 complex (Scheme 1, below).
The assumption is made that the fluorescence changes reflect formation
of the Ras·mantGTP·NF1-334 complex.
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(Eq. 1)
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Data on the effect of concentration of NF1 on the observed rate
constant (kobs) for the slow phase of the
fluorescence change consequent to mixing Ras·mantGTP and NF1 was
fitted to Equation 2 using non-linear regression.
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(Eq. 2)
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where P = [Ras]0 + [NF1-334]0 + K1,
[Ras]0 = total concentration of Ras,
[NF1-334]0 = total concentration of NF1-334, and kmax is the limiting value of the slow
phase.
This equation describes the process shown in Scheme 1 below, in which
the initial binding step is a rapid equilibrium. This assumption is
valid for the mutant proteins, in which k1 and
k
1 are very fast compared with
k2. It is not valid in the case of wild-type
NF1-334, where k
1 < k2. However, kinetic simulation experiments show
that this treatment gives K1 within a factor of
2 of the true value. The assumption is also made that the fluorescence decrease reflects formation of
Ras·mantGDP·Pi·NF1-334.
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RESULTS |
Characterization of Mutant NF1--
Wild-type, R1276A, and R1391A
glutathione S-transferase-NF1-334 proteins were all
expressed at the same levels and with the same solubility. The
wild-type and mutant NF1-334 proteins were >95% homogeneous by
SDS-polyacrylamide gel electrophoresis, and electrospray mass
spectrometry gave the predicted molecular masses. The tryptophan
fluorescence emission spectrum and near-UV and far-UV circular
dichroism spectra of the three proteins were indistinguishable, suggesting that the mutations do not cause any gross structural perturbation.
The Interaction of Ha-Ras·mantGTP with Wild-type and Mutant
NF1-334--
Mant nucleotides were used in these studies because they
act as close analogues of guanine nucleotides and are fluorescent. The
intrinsic rate constants for the hydrolysis of Ha-Ras·mantGTP was
determined in experiments similar to those used for N-Ras·mantGTP to
be 1.6 × 10
4s
1 (17). The same result
was obtained whether chemical cleavage or fluorescence changes were
measured.
The interaction of Ha-Ras·mantGTP with wild-type, R1276A, and R1391A
NF1-334 was investigated using single-turnover conditions (i.e. excess NF1-334) and monitoring mant fluorescence in a
stopped-flow instrument. With wild-type NF1-334, on mixing with
Ha-Ras·mantGTP, a biphasic fluorescence signal was obtained: first, a
rapid increase in fluorescence followed by a slower decrease in
fluorescence. The time course of this biphasic reaction is shown over
80 ms (Fig. 2a) and 800 ms
(Fig. 2d). The data were fitted to a double exponential. In
this example, at 2 µM wild-type NF1-334, the observed first-order rate constants are 22 s
1 and 12 s
1 respectively. The dependence of these rate constants
on the concentration of NF1-334 was investigated and the results shown
in Fig. 3, a and
d.

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Fig. 2.
Fluorescence records of the interaction of
Ha-Ras·mantGTP with wild-type NF1-334. One syringe of the
stopped-flow instrument contained Ha-Ras·mantGTP, and the other
contained NF1. The concentrations after mixing were 0.2 µM Ha-Ras·mantGTP and 2 µM wild-type
NF1-334 (a and d), 2 µM R1276A
NF1-334 (b), or 2 µM R1391A NF1-334
(c). For the slow time scale reactions of the two mutant
proteins, measurements were made in a steady-state fluorometer to
reduce photobleaching, and hence the rapid phase of fluorescence
increase was not seen. To a solution containing 0.2 µM
Ha-Ras·mantGTP was added 4 µM R1276A NF1-334
(e) or 4 µM R1391A NF1-334 (f)
(final concentrations). The solid lines are the best fits of
the data to a double exponential in a and d and
to single exponentials in the other panels. The first-order rate
constants are 22 (a), 12 (d), 26 (b),
0.02 (e), 70 (c), and 0.02 (f)
s 1.
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Fig. 3.
Dependence of the rate constants involved in
the interaction of Ha-Ras·mantGTP with wild-type and mutant NF1-334
on the concentration of NF1-334. Experiments similar to those
shown in Fig. 2 were performed at final concentrations of NF1 between 1 and 12 µM. Observed first-order rate constants of both
processes were plotted against NF1-334 concentration. The rate
constants for the fast phase of wild-type NF1-334 (a),
R1276A NF1-334 (b), and R1391A NF1-334 (c) show a
linear dependence of NF1-334 concentrations with slopes of 7.9 × 106, 5.4 × 106, and 3.8 × 106 M 1 s 1 and
intercepts of 25, 16, and 50 s 1, respectively. The rate
constants for the slow phase of wild-type NF1-334 (d),
R1276A NF1-334 (e), and R1391A NF1-334 (f) showed
a hyperbolic dependence on NF1 concentration with Kd
values of 1.2, 1.2, and 2.6 µM and limiting rate
constants of 23, 0.02, and 0.04 s 1 respectively. Errors
in these values are discussed in the text.
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The above experiments were repeated using R1276A NF1-334 (Fig. 2,
b and e) and R1391A NF1-334 (Fig. 2, c
and f) proteins. With both mutant proteins, an initial rapid
increase in fluorescence occurred (Fig. 2, b and
c) followed by a decrease of fluorescence at rates about 3 orders of magnitude slower than the wild-type protein (Fig. 2,
e and f).
The effect of NF1-334 concentration on both the fast phase and slow
phase of the reactions was studied for both mutant NF1-334 proteins.
Because the fast and slow processes for both mutant proteins differ by
more than 2 orders of magnitude, the data over the appropriate time
range were fitted in each case to a single exponential. The effect of
NF1-334 concentration on the observed rate constants of the initial
rapid increase in fluorescence and of the subsequent decrease in
fluorescence for R1276A (Fig. 3, b and e) and
R1391A (Fig. 3, c and f) are shown.
In a separate experiment, 0.2 µM Ha-Ras·mantGTP was
mixed with 10 µM R1276A NF1-334 at 30 °C. Samples were
taken at intervals and analyzed for mantGTP and mantGDP by high
performance liquid chromatography. The fluorescence intensity was also
monitored with time. The fluorescence decrease was fitted to a single
exponential with a rate constant of 0.025 s
1. The
formation of mantGDP could also be fitted to a single exponential with
the identical rate constant, indicating that the fluorescence decrease
monitored mantGTP hydrolysis (data not shown).
Interpretation of the Kinetic Data Obtained upon the Interaction of
Ha-Ras·mantGTP with Wild-type, R1276A, and R1391A NF1-334--
The
binding of N-Ras·GTP with a GAP (in this case p120-GAP) has
previously been proposed (17, 19) to occur by a two-step mechanism in
which the initial binding step is followed by a conformational change
that precedes and limits the cleavage step. This was based on the
observation that although mantGMPPNP is not hydrolyzed by Ras,
incubation of N-Ras·mantGMPPNP was accompanied by an exponential decrease in fluorescence that occurred with a similar rate constant to
the hydrolysis of N-Ras·mantGTP (19) and that this rate constant of
the N-Ras·mantGMPPNP fluorescence decrease was accelerated by
p120-GAP (17). However, Rensland et al. (20) showed that a
similar fluorescence decrease with Ha-Ras·mantGTP
S occurred that
was 10-20-fold slower than for Ras·mantGTP and probably associated with hydrolysis, although this was not measured directly. Furthermore, the fluorescence change with Ha-Ras·mantGTP
S was not accelerated by p120-GAP.
To clarify this discrepancy, we have repeated the experiment using
N-Ras·mantGTP
S. A solution of 2.5 µM
N-Ras·mantGTP
S was incubated at 37 °C. The fluorescence
intensity was monitored with time and samples were also taken at
intervals and analyzed for conversion of N-Ras·mantGTP
S to
N-Ras·mantGDP. A 20% exponential decrease in fluorescence
occurred with a rate constant of 2.2 × 10
5s
1, which was within the experimental
error of the rate constant for the formation of N-Ras·mantGDP
(2.7 × 10
5s
1), inconsistent with the
conformational change being rate-limiting. Furthermore, the experiment
of Moore et al. (17) (see Fig. 4 of Ref. 17) could not be
repeated when NF1-334 replaced p120-GAP. With R1276A NF1-334 mutant
proteins (see above), we have compared the chemical cleavage rate with
the fluorescence change at a single NF1-334 concentration and have
shown that they occur at the same rate. Therefore, we have assumed that
the fluorescence intensity changes occur at the same rate as the
cleavage process, as was previously done in Eccleston et
al. (13) and Ahmadian et al. (21), and that
cleavage is not controlled by a preceding conformational change.
In this two-step model (Scheme 1) the initial fluorescence increase is
due to binding and the subsequent fluorescence decrease is due to
cleavage, and this process can be described by Equation 1.
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(Scheme 1)
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A mechanism involving a two-step binding process before cleavage
has recently been proposed by Ahmadian et al. (21). However, for a comparison of the effects of mutations R1276A and R1391A on their
interaction with Ha-Ras·mantGTP, Scheme 1 is at present sufficient.
Fig. 3, a-c, shows that under these conditions, there is a
linear relationship between kobs of the initial
binding step and NF1-334 concentration for wild-type and mutant
proteins. From Equation 1, the slope of the line gives
k1 and the intercept gives k
1 + k2. Values for
k1 are 7.9 × 106
M
1s
1 for wild-type protein,
5.4 × 106
M
1s
1 for R1276A, and 3.8 × 106 M
1 s
1 for
R1391A. Values of k
1 + k2 (from the intercept) are 25, 16, and 50 s
1 for wild-type, R1276A, and R1391A respectively. For
both mutant proteins, k2 is very small compared
with these values, so they are essentially values for
k
1. By use of the relationship Kd = k
1/k1, values of
Kd for the two mutant proteins can be calculated to
be 3.0 and 13.0 µM for R1276A and R1391A,
respectively.
For the wild-type protein, the intercept is at 25 s
1,
which represents k2 + k
1. k2 is obtained from
the limiting rate constant of the subsequent slow phase of decreasing
fluorescence (Fig. 3d) as discussed below and was found to
be 23 s
1. It is therefore difficult to calculate
accurately a value of k
1 because it involves
the difference between two relatively large numbers. However, if an
upper limit of 5 s
1 is placed on the difference between
the intercept and k2, an upper limit for
Kd = k
1/k1 is 0.6 µM.
The relationship between concentration of wild-type (Fig.
3d), R1276A (Fig. 3e), and R1391A (Fig.
3f) NF1-334 concentration on the observed rate constant of
the slow phase is shown in each case to be hyperbolic. Fitting this
data to Equation 2, values of K1 were calculated
to be 1.2, 1.2, and 2.6 µM and values for k2 were 23, 0.02, and 0.04 s
1 for
wild-type, R1276A, and R1391A NF1-334, respectively. It is difficult to
obtain data for the mutant proteins with very slow time course
reactions (Fig. 3, e and f) as good as the data
for the faster reactions of the wild-type protein, and there could be
an error of a factor of 2 for the values of k2
for mutant proteins. Also, the value of K1 for
R1276A is an upper limit, as the observed rate constants are
essentially independent of [NF1-334] (Fig. 3e). The
hyperbolic fit is based on the rate constant at zero NF1-334 being
1.6 × 10
4s
1, which is the rate
constant for intrinsic mantGTP hydrolysis.
The calculated equilibrium and kinetic rate constants for the
interaction of Ha-Ras·mantGTP with wild-type, R1276A, and R1391A obtained by the above analysis are shown in Table
I.
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Table I
Equilibrium and kinetic rate constants of the interaction of
Ha-Ras · mantGTP with wild-type, R1276A, and R1391A NF1-334
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Interaction of Leu61Ha-Ras·mantGTP with Wild-type and
Mutant NF1-334--
Leu61Ha-Ras·mantGTP hydrolyses to
the mantGDP complex with a rate constant of 1 × 10
5s
1, and this is activated by only a
factor of 6 by wild-type NF1-334 (7) This fact allows the binding of
wild-type and mutant NF1-334 to Leu61Ha-Ras·mantGTP to be
studied in isolation from the GTP cleavage step. On mixing
Leu61Ha-Ras·mantGTP with excess wild-type (Fig.
4a), R1276A (Fig.
4b), or R1391A (Fig. 4c) NF1-334, a single
exponential increase in fluorescence occurred, and the observed rate
constants from these experiments were linearly dependent on NF1-334
concentration (Fig. 4, d-f). From these data, using
Equation 1, where k2 = 0, values of
k1 of 1.1 × 107, 5.2 × 106, and 3.0 × 106
M
1s
1 and
k
1 of <1, <1, and 40 s
1 were
obtained for wild-type, R1276A, and R1391A respectively. It should be
noted that because of the significantly reduced binding of R1391A
NF1-334 to Leu61Ha-Ras·mantGTP, signals were much
smaller, and thus the errors in the derivative plot (Fig.
4f) were greater.

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Fig. 4.
Stopped-flow fluorescence records of the
interaction of Leu61Ras·mantGTP with wild-type, R1276A
and R1391A NF1-334. One syringe contained
Leu61Ha-Ras·mantGTP and the other contained NF1-334. The
concentrations after mixing were as follows: a, 0.2 µM Leu61Ha-Ras·mantGTP and 2 µM wild-type NF1-334; b, 0.2 µM
Leu61Ha-Ras·mantGTP and 2 µM R1276A
NF1-334; and c, 1 µM
Leu61Ha-Ras·mantGTP and 5 µM R1391A
NF1-334. The solid lines are best fits of the data to a
single exponential with observed first-order rate constants of 23, 10, and 55 s 1 for a, b, and
c, respectively. The dependence of these rate constants on
NF1 concentration is shown for wild-type NF1-334 (d), R1276A
NF1-334 (e), and R1391A NF1-334 (f). The solid
lines are best fits of the data to a straight line with slopes of
1.1 × 107, 5.2 × 106, and 3.0 × 106 M 1s 1 for
d, e, and f, respectively, and
intercepts of <1, <1, and 40 s 1, respectively.
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Values of k
1 were independently determined by
displacement experiments in which the complex
Leu61Ha-Ras·mantGTP·NF1-334 was rapidly mixed with
excess Leu61Ha-Ras·GTP (Fig.
5). An exponential decrease in
fluorescence occurred, representing the dissociation of
Leu61Ha-Ras·mantGTP from NF1-334. Dissociation rate
constants (k
1) were 0.8, 0.9, and 39 s
1 for wild-type, R1276A, and R1391A NF1-334,
respectively.

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Fig. 5.
Stopped-flow fluorescence record of
displacement of Leu61Ha-Ras·mantGTP from its complex with
NF1-334 by excess Leu61Ras·GTP. One syringe
contained Leu61Ha-Ras·mantGTP and NF1 and the other
contained Leu61Ha-Ras·GTP. Concentrations after mixing
were as follows: a, 0.1 µM
Leu61Ha-Ras·mantGTP, 0.2 µM wild-type
NF1-334, 18 µM Leu61Ha-Ras·GTP;
b, 0.1 µM Leu61Ha-Ras·mantGTP,
0.2 µM R1276A NF1-334, 18 µM
Leu61Ha-Ras·GTP; c, 4 µM
Leu61Ha-Ras·mantGTP, 5 µM R1391A NF1-334,
46 µM Leu61Ha-Ras·GTP. The solid
lines are best fits of the data to single exponentials with
observed rate constants of 0.8 (a), 0.9 (b), and
39 (c) s 1.
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The values of k
1 obtained from the intercept
of the association reactions and from the displacement experiments are consistent with each other. The values for the Kd
were calculated from Kd = k
1/k1, where
k1 was obtained from the dependence of
kobs on [NF1-334], and
k
1 was obtained from the displacement
experiments. These values are 0.07, 0.17, and 13.0 µM for
wild-type, R1276A, and R1391A NF1-334, respectively. All of the values
for kinetic and equilibrium constants are given in Table
II.
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Table II
Equilibrium and kinetic constants for the interaction of Leu61
Ras · mantGTP with wild type, R1276A, and R1391A NF1-334
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DISCUSSION |
In this study, we have analyzed the effects of converting the
Arg1276 and Arg1391 residues of NF1-334 to
alanine residues.
The effect of these mutations on the interaction of NF1-334 with
Ha-Ras·nucleotide complexes has been shown by a combination of the
use of a fluorescent nucleotide analogue, mantGTP, and single turnover
conditions, in which the NF1-334 is in excess of Ha-Ras·mantGTP. An
advantage of this method is that values of the rate constants governing
the hydrolysis reaction can be measured over a time scale of
milliseconds to hours, so that values of the mantGTPase rate between
the Ha-Ras intrinsic rate constant of 1.6 × 10
4s
1 and the wild-type NF1 activated rate
constant of 23 s
1 can be measured. Single turnover
conditions also give information about the binding step that precedes
GTP hydrolysis.
The rate constant of the slow fluorescence change accompanying
hydrolysis showed a hyperbolic dependence on NF1-334 concentration, and
the limiting rate constant was the rate constant of mantGTPase cleavage. The rate constant for the intrinsic Ha-Ras·mantGTP
hydrolysis is 1.6 × 10
5s
1, and this
is accelerated to 23 s
1 (Fig. 3d) by wild-type
NF1-334 but only to 0.02 s
1 (Fig. 3e) and 0.04 s
1 (Fig. 3f) by R1276A and R1391A NF1-334,
respectively. These two arginine residues, therefore, play an important
role in the activation of the hydrolysis of GTP by NF1-334. However,
our data show that neither arginine is absolutely essential for
activation of catalysis, because the mutant protein can still increase
the Ras·mantGTPase by over 100-fold. Thus, it is likely that other
interactions of Ras with NF1-334, besides these with the conserved
arginine residues, contribute to catalysis by NF1. The Q61L and G12P
Ras mutants (13) bind RasGAP normally, and the L902I RasGAP mutant (7) binds Ras normally, yet these proteins all show around a 1000-fold reduction in GAP-stimulated GTPase. This supports the suggestion that
the catalysis of Ras GTPase by GAPs cannot be simply ascribed to a
single arginine residue. Rather, the role of the GAP interaction is
both to contribute a catalytically important arginine residue and also
to stabilize the precise geometry of the transition state for
catalysis. One possible mechanism would be that the conformation of Ras
is altered or a specific conformation is stabilized upon binding to
NF1-334. Indeed, it has been noted that the Switch II region of Ras,
which contains the catalytically important residue Gln61,
is well defined in the x-ray structure of the Ras·p120-GAP structure but is highly mobile in Ras alone (5). A similar observation has been
made by Rittinger et al. (22) for the interaction of Rho
with Rho-GAP.
In addition to measurements of the activation of Ras·mantGTP by the
wild-type and mutant NF1-334 proteins, the data can also be analyzed to
give information about the binding affinity of the initial interaction
of Ras·mantGTP with NF1-334. Two approaches were made to calculate
this affinity. First, an estimate was made for the value of
k
1 as described under "Results," and this value combined with k1 to give
Kd or an upper estimate of it. Values obtained for
wild-type, R1276A, and R1391A by this method were <0.6, 3.0, and 13.0 µM, respectively. Second, the initial binding step was
assumed to be a rapid equilibrium, and the value of the
Kd was obtained from the hyperbolic dependence of
the rate constant of the slow phase. The limitations of this approach
and the data are described under "Results." The values obtained by
this method for wild-type, R1276A, and R1391A NF1-334 are 1.2,
1.2,
and 2.6 µM, respectively. Values for wild-type and R1276A
NF1-334 are in reasonable agreement for the two methods, but those for
R1391A NF1-334 are not. The reason for this discrepancy may be that the
binding step is not a single step. Evidence that this is a more complex
process has been described (21). However, in comparison with the
dramatic effect of mutating Arg1276 and Arg1391
on the catalytic activity of NF1-334, there is only a small effect on
binding.
The analysis of the data of interaction of
Leu61Ha-Ras·mantGTP with wild-type and mutant NF1-334 is
more straightforward, as under the conditions used, this interaction
can be interpreted as a single second-order reaction. The second-order
rate constant for the binding of wild-type NF1-334 to
Leu61Ha-Ras·mantGTP is 1.1 × 107
M
1s
1, and this is reduced in
R1276A and R1391A NF1-334 to 5.2 × 106 and 3.0 × 106 M
1s
1,
respectively. This reduced rate of binding is similar to that of
wild-type Ha-Ras·mantGTP and could be caused by a change in the ionic
interactions of the two proteins. Although the dissociation rate
constants of wild-type and R1276A NF1-334 from
Leu61Ha-Ras·mantGTP are almost identical, it is increased
by a factor of >40 in the R1391A NF1-334 mutant. The effects of the
mutations on k1 and k
1
result in the Kd for the interactions between
wild-type, R1276A, and R1391A NF1-334 and
Leu61Ha-Ras·mantGTP being 0.07, 0.17, and 13.0 µM, respectively. R1276A, therefore, has little effect on
the binding between Leu61Ha-Ras and NF1-334, but R1391A has
a much greater effect.
The Kd for the interaction of
Leu61Ha-Ras·mantGTP to wild-type NF1-334 is 10-fold
tighter than for wild-type Ha-Ras·mantGTP. This differential binding
is maintained in the R1276A NF1-334 mutation but not in the R1391A
NF1-334 mutation, where the affinities are similar or weaker. This
suggests that the high affinity of Leu61Ha-Ras for
wild-type NF1-334 is related to an interaction with, or is dependent
on, Arg1391 of NF1-334. Examination of the structure of the
Ras·GDP·AlF3/p120-GAP complex (5) suggests that in
wild-type NF1 there might be a hydrophobic interaction between the side
chain of Leu61 of Ras and of the hydrophobic backbone of
the side chain of Arg1391 of NF1. This would be lost on
mutation of Arg1391 to R1391A, thus accounting for the
biochemical observation.
Although there are several reports on the effects of mutations of
Arg1276 and Arg1391 of NF1 and, in the
homologous residues of p120-GAP, Arg789 and
Arg903, the results and their interpretation are
conflicting (6-12). Very likely, part of the explanation for this is
that the effects of mutation on kcat and
Kd have not always been measured.
Although there is currently no x-ray structure of NF1, structures of
the Ras-binding domain of p120-GAP both on its own and in complex with
Ras·GDP·AlF3 have recently been published (4, 5).
Judging from the close sequence homologies between the catalytic domain
of p120-GAP and NF1, the overall structure of these two proteins in the
region of interaction with Ras is likely to be very similar, and hence
the role in catalysis of residues Arg1276 and
Arg1391 of NF1 is likely to be identical to that of the
homologous residues Arg789 and Arg903 of
p120-GAP. The x-ray structure of p120-GAP shows that both Arg789 and Arg903 of p120-GAP are in proximity
both to each other and to the proposed location of the
phosphate in
the complex with Ras-GTP. Because the guanidinium group of
Arg903 contacts the main chain in the region of
Arg789, the possibility was suggested that
Arg789 is used for catalysis, whereas Arg903
stabilizes the conformation of Arg789. The experimental
data presented here are fully consistent with this hypothesis.
The GAP domains of RhoGAP and RasGAP show no clear tertiary structural
similarity and hence are unlikely to be evolutionarily homologous.
Despite this, there are several parallels. Primarily, the x-ray
structures reveal that conserved arginine residues fulfill similar
structural (5, 22) and catalytic roles (data herein and Refs. 5 and
23). These residues are Arg1276 of NF1 (Arg789
of p120 GAP) and Arg282 (termed Arg85 in Ref.
22) of RhoGAP (Fig. 1). Interestingly, these residues are also in a
similar sequence environment (see Fig. 1). There appears to be a
further structural analogy relating to Arg1391 of NF1
(Arg903 of p120GAP). There is a similarity in the sequence
surrounding this residue and that near the conserved Lys319
of RhoGAP (Fig. 1) and also in their role in stabilizing the structure
of the catalytic arginine. Thus, the amino group of Lys319
in RhoGAP interacts with the main chain Arg282 (22), and in
RasGAP, the guanidino group of Arg903 (Arg1391
of NF1) interacts with the main chain carbonyl of Arg789
(Arg1276 of NF1) (5).
In conclusion, the two conserved arginines in NF1 are essential for
efficient catalysis. This is consistent with the hypothesis, supported
by structural and mechanistic studies, that the mechanism of RasGAPs
involves transition state stabilization by direct involvement of an
arginine residue contributed by GAP. It would seem likely that this
might represent a more general mechanism for catalysis in GTPases,
because an arginine residue has been demonstrated to be involved in
catalysis in the G
subunit of heterotrimeric G proteins
(24-26), and there appear to be roles for similar arginines in RhoGAPs
(22, 23).
We thank Richard Skinner for gifts of
expression systems and valuable advice, Stephen Martin for recording CD
spectra, Sohail Ahmed for pointing out some similarities between
RhoGAPs and RasGAPs, Liz Carpenter for assistance with the sequence
alignment, and Tracey Jenkins for the studies in N-Ras.
A structural similarity between RasGAPs
and RhoGAPs has now been identified (Rittinger, K., Taylor, W. R., Smerdon, S. J., and Gamblin, S. J. (1998)
Nature, in press). Both structures have a core made up of
seven
helices, which pack in a related but not identical manner.
Alignment of the structures puts Arg282 of RhoGAP and
Arg789 of RasGAP into related positions.