The Importance of Two Conserved Arginine Residues for Catalysis by the Ras GTPase-activating Protein, Neurofibromin*

Beth A. Sermon, Peter N. LoweDagger , Molly Strom, and John F. Eccleston§

From the Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom and Dagger  Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 epsilon 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.
k<SUB><UP>obs</UP></SUB>=k<SUB>1</SUB>[<UP>NF</UP>1-334]+k<SUB><UP>−</UP>1</SUB> (Eq. 1)
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.
k<SUB><UP>obs</UP></SUB>=<FR><NU>k<SUB><UP>max</UP></SUB><FENCE>P−<RAD><RCD>P<SUP>2</SUP>−4[<UP>Ras</UP>]<SUB><UP>0</UP></SUB>[<UP>NF</UP>1-334]<SUB>0</SUB></RCD></RAD></FENCE></NU><DE>2[<UP>Ras</UP>]<SUB><UP>0</UP></SUB></DE></FR> (Eq. 2)
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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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·mantGTPgamma 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·mantGTPgamma S was not accelerated by p120-GAP.

To clarify this discrepancy, we have repeated the experiment using N-Ras·mantGTPgamma S. A solution of 2.5 µM N-Ras·mantGTPgamma 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·mantGTPgamma 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.
<UP>Ras</UP> · <UP>mantGTP</UP>+<UP>NF</UP>1-334 <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB><UP>−</UP>1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>Ras</UP> · <UP>mantGTP</UP> · <UP>NF1-334</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>2</SUB></UL></LIM>  (Scheme 1)
<UP>Ras</UP> · <UP>mantGDP</UP> · <UP>P</UP><SUB><UP>i</UP></SUB> · <UP>NF</UP>1-334
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

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.

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.

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

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma  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 Galpha subunit of heterotrimeric G proteins (24-26), and there appear to be roles for similar arginines in RhoGAPs (22, 23).

    ACKNOWLEDGEMENTS

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.

    Note Added in Proof

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 alpha  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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 44-181-959-3666; Fax: 44-181-906-4419; E-mail: j-eccles{at}nimr.mrc.ac.uk.

1 The abbreviations used are: GAP, GTPase-activating protein; NF1, neurofibromin; NF1-334, catalytic domain of NF1; mant, 2'(3')O-N-methylanthraniloyl.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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