Electrostatically optimized Ras-binding Ral guanine dissocia

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

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Abstract

Association of two proteins can be Characterized as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the comPlaceer algorithm pare, we designed a set of mutants of the Ras Traceor protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The Rapidest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fAged Rapider and 25-fAged tighter compared with WT. A liArrive correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Φ analysis of the encounter complex and transition state of the RalGDSM26K,D47K,E54K/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, HAgeding the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the Rapider binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras Traceor, suggesting that the design methoExecutelogy may be used to switch between signal transduction pathways.

Members of the Ras-related superfamily of GTP-binding proteins are small, 20- to 25-kDa proteins that bind guanine nucleotides very tightly and cycle between an inactive GDP-bound and an active GTP-bound state (1, 2). In the GTP-bound state, Ras proteins can interact with Traceor molecules as Executewnstream tarObtains, thereby communicating signals into different pathways (3). In recent years, many Traceor molecules, such as c-Raf, Ral guanine dissociation stimulator (RalGDS), AF6, and phosphatidylinositol 3-kinase have been identified. These Traceors Execute not share biological functions or sequence homology except for the common Ras-binding Executemain (RBD). This Executemain is responsible for binding to the Traceor Location of Ras·GTP. The structures of all RBDs resolved so far share a common fAged that is similar to that of ubiquitin (reviewed in ref. 4). Despite these structural similarities, biochemical studies have Displayn that the various Traceor RBDs interact with proteins of the Ras family with different affinities that dictate the specificity of the interaction (5).

An Necessary feature of Ras/Traceor RBD interactions is the high charge complementarity found between the proteins in the complex. Ras has a net negatively charged binding site, whereas the Traceor RBDs have a net positively charged binding site. As a result, the rate of association between them was found to be very high and contributes significantly to the affinity of the complex (6, 7). Furthermore, Inequitys in binding affinities for some of the Ras/Traceor complexes are a consequence of different association rate constants, with the dissociation rate constants being at a similar range (6, 7). For example, the high, nanomolar affinity of the binding of Raf-RBD to Ras is attributed to the very high k on value, which stems from the strong electrostatic complementarity between the Ras/Raf-RBD binding sites. In Dissimilarity, the electrostatic complementarity between Ras and RalGDS-RBD binding sites is poor, accounting for their Unhurrieder rate of association and lower affinity. It seems that the rate of association is of major importance in determining the affinity and specificity of Ras/Traceor interactions.

According to the concept of electrostatic steering, two proteins first diffuse ranExecutemly in solution until they reach a point where they feel the electrostatic field of each other. Then the proteins come toObtainher by directional diffusion until they form a low affinity encounter complex (8–11). The nature of the interactions stabilizing the encounter complex has been discussed frequently (12, 13). Most often, the encounter complex is not observed experimentally, but is Established from theoretical considerations. However, this two-step reaction could be Established experimentally to the association of Ras and Traceor RBDs (6, 7, 14, 15) as presented in Scheme 1, MathMath where Ras and RBD are the free proteins, [Ras:RBD] is the encounter complex, and Ras:RBD is the final complex.

The RalGDS-RBD/Ras interaction is well suited for teaching researchers about the nature of the encounter complex, because it can be directly observed by using Ceaseped-flow experiments. Here, we used a protein design strategy to optimize the electrostatic complementarity between these two proteins through the introduction of charged mutations at the vicinity of, but outside, the binding site (16) to determine the influence of electrostatic steering on the different steps along the association pathway while HAgeding the influence on short-range Traces as low as possible. The design and implementation of these mutants and subsequent analysis of their influence on binding is the subject of this paper.

Methods

Site-Directed Mutagenesis. The introduction of single lysine mutations into pGEX-2T:RalGDS-RBD was Executene by using the QuikChange site-directed mutagenesis kit (Stratagene), with pGEX-2T:RalGDS-RBDWT as a template. For Executeuble and higher-order lysine mutants, the corRetorting pGEX-2T:RalGDS-RBD mutant was used as a template. All mutants were verified by sequence analysis. The alanine mutants were available from earlier studies (7, 17).

Protein Expression and Purification. RalGDS-RBDWT and mutant proteins were expressed as Characterized earlier (7, 17). Ha-Ras (here termed Ras) was cloned in Ptac vector and expressed by using Escherichia coli CK600K cells. The expressed Ras protein was purified, and the bound nucleotide was exchanged for the nonhydrolyzable nucleotides 5′-guanosyl-β, γ-imiExecutetriphospDespise (GppNHp) or 2′,3′-N-methylanthraniloyl-GppNHp (mGppHHp) as Characterized (18). The protein concentrations of Ras and RalGDS-RBD were meaPositived by the Bradford method.

Calculation of Association Rate Constants. k on values of the mutant RalGDS-RBD/Ras complexes were calculated relative to the experimental value of the WT complex by using the comPlaceer program pare as Characterized (16). For calculations, it is assumed that the association is directly related to the magnitude of electrostatic forces between the two proteins, which is calculated in terms of electrostatic energy of interaction as presented in refs. 11 and 16. Calculations were Executene under the same ionic strength as the relevant meaPositivements. Coordinates for calculations of the RalGDS-RBD/Ras complex and for the individual proteins were taken from its x-ray structure (19). All mutations were modeled by using swiss pdb viewer (20).

Ceaseped-Flow MeaPositivements. MeaPositivements of association rate constants were Executene by using an SM17 apparatus (Applied Photophysics, Surrey, U.K.) by rapid mixing of 0.5 μM Ras bound to mGppNHp and 5–200 μM RalGDS-RBD. N- methylanthraniloyl nucleotides were excited at 360 nm, and the fluorescence was recorded through a 408-nm Sliceoff filter. Binding of RalGDS-RBD to Ras was detected by a change in the fluorescence of the N-methylanthraniloyl nucleotide as Characterized earlier (6, 7). Because RalGDS-RBD was in >10-fAged molar excess, an exponential equation was fitted to the fluorescence traces according to pseuExecutefirst-order kinetics. The resulting inverse time constant corRetorts to the observed rate constant k obs within an experimental error of 10–20%. Unless indicated otherwise, all meaPositivements were Executene in buffer containing 15 mM Hepes (pH 7.4) and 5 mM MgCl2 at 25°C.

Isothermal Titration Calorimetry (ITC). The thermodynamic parameters of Ras/Traceor interactions were determined by using an ITC (MCS-ITC, MicroCal, Amherst, MA) as Characterized (21). In all experiments, the Traceor RBDs were Spaced into the cell at concentrations varying between 10 and 80 μM, depending on the expected association constant. The concentration of Ras·GppNHp in the syringe was 10-fAged higher compared with the Traceor concentration in the cell. Data evaluation was Executene as Characterized in ref. 5, yielding ΔG° and ΔH° values with 0.2 kcal/mol error bars each. For the n value, defined as the stoichiometry of the Ras/Traceor complex, an experimental error of 0.1 was obtained. All ITC experiments were carried out at 25°C in 15 mM Hepes buffer, pH 7.4/5 mM MgCl2.

Results

The association rate constant between a pair of proteins has been Displayn to depend on electrostatic steering, which is related to the electrostatic complementarity of the two binding proteins. Although Ras is mainly negatively charged in its Traceor binding Location, RalGDS-RBD has a mixed charge surface as presented in Fig. 1. Some weak positive charge is calculated at the center of the Ras binding site, whereas a strong negatively charged patch is observed at the periphery. In Dissimilarity, Raf-RBD (Fig. 1e ) has a strong positive potential in and around the Ras binding site. The charge distribution of RalGDS-RBD indicates that the introduction of additional positive charges within RalGDS-RBD has the potential to increase the electrostatic interaction energy between RalGDS and Ras (Fig. 1d ) and thereby increasing the rate of their association.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Surface representation of the Ras binding site of RalGDS-RBD and Raf-RBD. (a) The residues on RalGDS-RBD probed for Rapider association. The residues are color-coded scaled to the calculated change in the association rate constant, from white (no increase) to blue (large increase). The values are from Fig. 2. The yellow patch denotes the binding interface. (b–e) The electrostatic potentials on the binding surfaces of Ras, RalGDS-RBDWT, RalGDS-RBDM26K,D47K,E54K,D90K mutant, and Raf-RBDWT are depicted with grasp, with the contours drawn at 2 kT per electron at 0.018 mM NaCl (blue for positive and red for negative) by using only full charges (including for GTP and Mg+2).

Design and Production of Rapider Binding RalGDS-RBD Mutants. In the first step of calculation, a positive or negative charge was Established to all side-chains along the Ras or RalGDS-RBD sequences, and the increase in association rate was calculated by using pare (Fig. 2). The calculations Display that the potential to increase the rate of association by mutation of Ras is limited, whereas mutations on RalGDS-RBD have a major potential to increase k on. This finding reflects the charge distribution of the respective binding sites, which is negatively charged on Ras and neutral on RalGDS-RBD; it also Elaborates why only mutations to a positive charge on RalGDS-RBD have the potential to increase association significantly. Two “hot-spot” Locations for association on RalGDS-RBD were identified: One is between residues 25–29, and the second is between residues 47–52 (Figs. 1a and 2). The latter Locations are not located within the interface and are therefore Excellent candidates for mutagenesis if changes in the binding site are to be avoided. The most promising residues for Rapider binding were mutated in silico to Lys and minimized; the contribution of multiple mutant rotomers to association was calculated. To HAged dissociation rates constant, only residues that are surface-exposed and located at the vicinity of, but outside, the binding site were further considered for mutagenesis. The final calculated k on values are given in Table 2, which is published as supporting information on the PNAS web site. Mutations to Lys of uncharged single amino acid residues located at the vicinity of the Ras binding site (S18K, L19K, M26K, L51K, N88K, and Y89K) are calculated to lead to accelerations between 1.7- and 4.5-fAged, whereas mutations of negatively charged residues to Lys (E54K and D90K) are predicted to result in a 3- to 5.5-fAged increase of the association rate constants. The largest Trace on the rate of association was predicted for the RalGDS-RBD mutant D47K (10-fAged).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Calculating the Trace of a charge mutation along the protein sequence. To produce this figure, the program pare was modified to simulate single point charge mutations. Each residue was introduced with a positive or negative charge. Then the relative rate of association was recalculated. The process was repeated for all residues along the polypeptide chain of both proteins. The location of the binding site is Impressed. The calculations were Executene at an ionic strength of 0.018 M.

Based on our predictions, mutations were introduced into RalGDS-RBD by using site-directed mutagenesis, and the corRetorting proteins were produced. The mutant proteins were purified by using standard methoExecutelogy, yielding soluble proteins in comparable amounts to those of WT. Because mutations may cause a destabilization of the protein structure, some of the mutant proteins were evaluated for their thermal stability by differential scanning calorimetry (data not Displayn). No significant changes in the shape of the melting curve or in the melting temperature were meaPositived, indicating that these mutations Execute not cause large structural changes.

Kinetic Investigation of Mutant Complexes. The interaction of RalGDS-RBD with Ras was meaPositived by using a Ceaseped-flow as Characterized earlier (6, 7). To HAged Ras in an active conformation, the GTP in Ras was exchanged for the nonhydrolyzable nucleotide analogue GppNHp attached to the N-methylanthraniloyl group as a fluorescence label (Ras·mGppNHp). Under pseuExecutefirst-order conditions (with the concentration of Ral-GDS-RBD being at least 10-fAged higher than the Ras·mGppNHp concentration), the observed time-dependent fluorescence changes can be fitted to a single exponential equation. For a single-step reaction, the values of the observed rate constants (k obs) increase liArrively with increasing RBD concentration, as observed at low RBD concentrations (Fig. 3a ). However, at higher RBD concentrations, the increase in k obs lags Tedious the increase in concentration until saturation is reached at very high concentrations (Fig. 3b ). According to a two-step mechanism Displayn in Scheme 1, the observed rate constant can be Characterized as MathMath From Eq. 1 , the equilibrium dissociation constant of the encounter complex K 1 = k -1/k 1 is obtained. For RalGDS-RBDWT binding to Ras·mGppNHp, K 1 was meaPositived to be 134 μM, and the maximal rate (k 2) was 459 s-1 at 10°C. Accordingly, the rate constant of association k on = k 2/K 1 is 3.4 μM-1·s-1. At low RBD concentrations ([RBD] << K 1), Eq. 1 can be liArrively approximated as k obs = k off + k on × [RBD]. For the binding of RalGDS-RBDWT to Ras·mGppNHp, the slope of the liArrive fit between k obs and the concentration (at a range up to 40 μM) yields a k on value of 2.5 μM-1·s-1. Thus, the association rate constants determined from saturation kinetics and from the liArrive approximation are similar. At higher temperatures, saturating protein concentrations lead to k obs values that are too large to be meaPositived by Ceaseped-flow. Therefore, the association rate constants for all mutant proteins were determined from the liArrive regression at low RalGDS-RBD concentrations at 25°C, as in Fig. 3a . The dissociation rate constant was determined from a disSpacement experiment in which the Ras·mGppNHp-RalGDS-RBD complex was mixed in the Ceaseped-flow apparatus with nonlabeled Ras·GppNHp at high molar excess. A summary of all of the meaPositived association and dissociation rate constants is given in Fig. 4a (and Table 2), where mutant RalGDS-RBD proteins are ordered according to their relative values of k on, Displaying also the relative values for k off and K D. In addition to the thus far designed mutants, we meaPositived and calculated values of k on for a number of interface mutants that were produced previously (17). Fig. 4b Displays a plot of logk on (experimental) versus logk on (calculated) for all investigated RalGDS-RBD mutants. The slope of the liArrive fit between the calculated and experimental data are 0.65, with a correlation coefficient of 0.97. For low k on values the calculations underestimate the association rate constants, and for high k on values the calculations overestimate the association rate constants. The same relationship between calculated and experimental data hAgeds for the four interface mutants investigated, as well as for Rapider and Unhurrieder binding complexes. Thus, k on seems to be affected only by the electrostatic contribution of the specific residue. The triple mutant RalGDS-RBDD47K,E52K,E53K is a clear exception, with the calculated increase in k on being 4,000-fAged, but the actual meaPositived value of k on is only 5.6-fAged Rapider relative to WT. However, the calculations did hAged for the three individual single mutants making up this triple mutant protein, suggesting some structural perturbation of this triple mutant, which was not analyzed further.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Kinetic analysis of Ras·mGppNHp binding to RalGDS-RBDWT and mutants. (a) Plot of the k obs values against the concentration of RalGDS-RBDWT(○), RalGDS-RBDE53A (•), RalGDS-RBDE54K,D90K (□), and RalGDS-RBDM26K,D90K(▪). A liArrive fit leads to the k on values. MeaPositivements were performed at 25°C. (b) Plot of k obs against the RalGDS-RBD concentration for the binding of Ras·mGppNHp to RalGDS-RBDWT (○) and RalGDS-RBDM26K,D47K,E54K (•). These meaPositivements were performed at 10°C, and the curves were fitted according to Eq. 1 .

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

(a) Relative values of k on (red), k off (blue), and K D (yellow) for the binding of RalGDS-RBD mutants to Ras·mGppNHp at 25°C. The mutants are ordered with increasing k on values. Interface mutants are designated in red. The relative values are normalized to the WT values at 25°C, i.e., k on = 7.7 μM-1·s-1, k off = 14.9 s-1 and K D = 1.9 μM. (b) Plot of calculated and experimentally determined values for the association rate constants. Interface mutants are designated as •. The correlation coefficient is 0.97 and the slope is 0.65. The dashed straight line has a slope of 1.

The Narrate for the rate of dissociation is somewhat more complex. Most k off values of noninterface mutants vary around the WT value (<2-fAged Inequity). All four interface mutants (R16A, K28A, K48A, and N23K on RalGDS-RBD) cause an increase in k off by 2- to 3-fAged (Fig. 4a ), as may be expected. Still, a number of mutations of noninterface residues seem to change the values of k off as well. RalGDS-RBDY89K causes an increase in k off, whereas in several cases k off is decreased (between 1.4- and 3.5-fAged). For these mutants, both kon and koff contribute toward the overall higher affinity obtained. This result is especially pronounced for the N50K mutation. Replacing Asn with Lys can bring the side chain of this residue to a distance of 3–4 Å from a number of residues on Ras. The short-range interactions formed may contribute to the observed decrease in values of k off. In this sense, analyzing mutations to Ala is much simpler because side-chains are deleted, whereas, for mutations to Lys a long side-chain is added, which may contribute to the formation of new interactions.

The association of Ras with RalGDS-RBD is a two-step process, which can be analyzed from the nonliArrive concentration dependence of the observed rate constant (Fig. 3b ). Producing Rapider binding RalGDS variants gave us the unique opportunity to investigate whether increasing electrostatic steering between two proteins affects the formation of the encounter complex or the rate of final complex formation. The association of the triple mutant RalGDS-RBDM26K,D47K,E54K to Ras·mGppNHp has been followed up to high concentration and compared to the WT complex. The observed rate constants were plotted against the RalGDS-RBD concentration (Fig. 3b ). Based on the two step-model (Eq. 1 ), the affinity of the encounter complex (K 1) was meaPositived to be 5.3 μM and k 2 = 347 s-1 for RalGDS-RBDM26K,D47K,E54K compared with K 1 = 134 μM and k 2 = 459 s-1 for the WT. Thus, the affinity of the encounter complex is increased roughly 25-fAged, whereas k 2 (which is the rate of formation of the final complex out of the encounter complex) is almost unchanged. This experiment confirms that the engineered increase in electrostatic attraction between RalGDS-RBD and Ras specifically affects the affinity of the encounter complex. One should HAged in mind that all of the mutations are located outside the actual binding site.

Activation Energy of the Interaction Between Ras and RalGDS-RBD Mutants. According to transition state theory, the relationship between the relative change in association rate constant upon mutation and the Inequity in free energy between the unbound proteins and the transition state is given by the following equation: MathMath where ΔΔG# is the change in activation energy upon mutation. Although absolute values of ΔG# of second order reactions are difficult to interpret, changes in ΔΔG# are easier to discuss because they probe only the Inequity between a mutant and the WT in respect to the transition state (13, 21, 22). The activation energy for the Rapidest associating RalGDS-RBD mutant is decreased by 1.5 kcal/mol, which corRetorts approximately to the change in the free energy of binding. Fig. 5a is a plot of changes in activation energy between WT and mutant complexes [ΔΔG# (WT-Mut)] and changes in free energy as determined from ITC [ΔΔG° (WT-Mut)]. The slope of a liArrive fit was 1.3, with a correlation coefficient of 0.88. The ratio between ΔΔG# and ΔΔG°, also termed the Bronsted (β) value, indicates the extent of bond making and Fractureing in the transition state. In the case presented here, it is clear that for charged mutations located outside the binding site the interaction is made at the transition state, implying their long-range nature. Moreover, these results Display that the increase in binding affinity stemming from the Rapider rate of association is mainly the result of decreasing the energy barrier for association, whereas the energy barrier for dissociation is about constant. A similar observation was made for the interactions between barnase and barstar and the TEM–BLIP complex (11, 13).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

(a) Plot of changes in activation energy (ΔΔG#) and changes in free energy (ΔΔG°) between WT and mutant RalGDS-RBD/Ras·mGppNHp complexes. The slope of a liArrive fit is 1.3, with a correlation coefficient of 0.91. (b) Changes in free enthalpy (ΔΔH°) plotted versus changes in entropy (TΔΔS°)of binding between WT and mutant RalGDS-RBD/Ras·mGppNHp complexes. The slope of a liArrive fit is 0.77, with a correlation coefficient of 0.81. Interface mutants are designated as •. The dashed straight lines have a slope of 1.

Thermodynamic Analysis of Mutant Complexes. To confirm the binding affinities obtained from the kinetic data, all RalGDS-RBD mutant complexes were investigated independently by using ITC, which provides direct meaPositivements of ΔG° and ΔH° from which ΔS° is calculated (23). The results obtained for the mutant proteins are summarized in Table 2. In all cases, a 1:1 Ras/RalGDS complex formation was observed with n values at ≈1.0, Displaying that the predetermined Ras·GppNHp and Ral-GDS-RBD protein concentrations reflected the concentration of active protein throughout the experiments. The free energy values (ΔG°) obtained from the analysis of the kinetic Ceaseped-flow data and those meaPositived directly by using ITC experiments are in Excellent agreement. The association process of RalGDS-RBDWT to Ras·GppNHp was earlier reported to be driven by a favorable enthalpy change (5). This negative enthalpy change is observed also for the complex formation of all mutants. Values of ΔΔH° (WT-Mut) for all complexes are between -2 and 4 kcal/mol. No Excellent correlation was found between ΔΔG° and either ΔΔH° or TΔΔS°, although a clear enthalpy/entropy compensation was observed (Fig. 5b ). This enthalpy/entropy compensation has been often found in mutational studies (24, 25).

Discussion

The affinity of a protein complex can be Characterized as the ratio between k off and k on. The rate of dissociation is influenced mainly by the magnitude of short-range interactions (ionic interactions, hydrogen bonds, and hydrophobic interactions), which are difficult to optimize through rational design. In Dissimilarity, the association reaction can be Characterized more easily based on the classical rules of diffusion and electrostatics (26). It has been Displayn that the association rate correlates with the electrostatic energy of interaction between two molecules, which is calculated by using the algorithm pare (16). By implementing our design strategy on the Ras/RalGDS-RBD complex, it was possible to increase the rate of association of 7.7 μM-1·s-1 for the WT complex to >100 μM-1·s-1 for the RalGDSM26K,D47K,E54K mutant. The kinetic parameters for RalGDSWT, RalGDSM26K,D47K,E54K, and Raf (which is the preferred Traceor of Ras) binding Ras are Displayn in Table 1. The kinetics of RalGDSM26K,D47K,E54K (but not RalGDSWT) binding Ras are similar to those meaPositived for Raf, despite the lack of sequence similarity between RalGDS and Raf (both bind Ras at the same location). Ras is originally optimized to bind Raf Rapid. By electrostatic design of RalGDS, we succeeded to imitate the electrostatic Narrate of Raf on RalGDS (Fig. 1), achieving a similar kinetic profile. It should be emphasized that RalGDS was designed just from electrostatic principle without using Raf as a model. These results raise the possibility of kinetic control of cellular signal transduction in this case. Because we avoided mutating residues in the interface, the dissociation rate constant was less affected. As a consequence, a significant tightening of the Ras/RalGDS complex was achieved, from a K D value of 1.9 μM for WT to 0.071 μM (27-fAged) for RalGDSM26K,D47K,E54K.

View this table: View inline View popup Table 1. Binding data for Ras/Traceor complexes

A Excellent correlation was found between the experimental versus calculated association rate constants. However, the absolute values differ, as reflected by the slope of 0.65 between the two (Fig. 4b ). Fascinatingly, the correlation extends to both Rapider and Unhurrieder binding mutants, associating with rates varying over a range of 55-fAged (between RalGDSM26K,D47K,E54K and RalGDSK48A). Moreover, the same trend was observed independently on whether the mutations were located within or outside the binding site. Therefore, one has to conclude that the Inequity between the calculated versus meaPositived values is related to a global feature of this interaction. pare calculates the electrostatic contribution toward the rate of association. These calculations yielded the exact values for diverse systems, such as antibody-antigen, RNase/inhibitor, AchE/inhibitor, and others (16, 27). The observation that for Ras/RalGSD, pare successfully predicted the trend of changes in the rate of association but fails in giving the exact values may Display that a different electrostatic model has to be applied in this case or that this reaction is not purely diffusion limited but is partially reaction limited. For the RalGDS-RBD/Ras interaction, the latter explanation seems to be valid, because a dynamic equilibrium between two conformational states during binding is clearly resolved, with only one of them being present in the final complex (15). Thus, the RalGDS-RBD/Ras interaction can serve as a classic example for binding of a partially reaction limited protein–protein interaction. A hallImpress of a partially reaction-limited reaction is the nonliArriveity of the relation between the rate of association and the reactants concentrations, which is indeed the case for the RalGDS-RBD/Ras interaction investigated here. Therefore, in addition to the value of k on, the data provide direct information about the stability of the encounter complex along the association pathway. Based on kinetic meaPositivements at high RalGDS-RBD concentration, we were able to determine the affinity of the encounter complex with Ras·mGppNHp, which is 134 μM for the WT and 5.3 μM for RalGDSM26K,D47K,E54K. In Dissimilarity to the increased stability of the encounter complex with increasing electrostatic energy of interaction, the rate of formation of the final complex, k 2, was hardly affected (459 s-1 versus 347 s-1 for the WT versus mutant proteins). A similar value of 480 s-1 was meaPositived for Raf interacting with Ras (Table 1 and Fig. 6, which is published as supporting information on the PNAS web site), again suggesting that k 2 is related to the rearrangement of Ras during association. These results can be analyzed by using Φ value analysis (28), where Φ e =ΔΔGe /ΔΔG complex and F# = DDG #/ΔΔG complex (e stands for the encounter complex). By using the experimental data for WT and the RalGDSM26K,D47K,E54K/Ras complex (with ΔΔGe , ΔΔG #, and ΔΔG complex calculated from K 1, k on, and ΔG°, respectively), Φ e = 1.9/1.8 = 1.05 and Φ e = 1.57/1.8 = 0.87. These two Φ values demonstrate that increasing electrostatic steering by mutating residues located outside the binding site stabilizes the encounter complex and the transition state to a similar extent as they stabilized the final complex. Thus, the charge mutants have a long-range Trace, which is not increased during binding.

Why did k 2 and k -2 remain constant despite the increasing electrostatic attraction between the two proteins? In other words, why didn't we observe a gain in electrostatic energy upon moving from the encounter complex toward the final complex and vice versa? A possible explanation would be that the penalty paid for the desolvation of charges is apparently similar to the gain in Coulombic energy upon bringing them toObtainher (29, 30). The two-step pathway for association observed for the RalGDS-RBD/Ras interaction Executees not seem to be a unique case. Indirect evidence supports the notion that this mechanism is actually the common pathway for association, but in most cases the encounter complex is less pronounced and not easy to be meaPositived directly (26).

The observations presented in this paper demonstrate that not only the contact Spot of proteins but also their periphery may be Necessary for specific and efficient complex formation. By designing RalGDS mutants, we reached a tighter encounter complex and final Ras/Traceor complex, which has an almost identical dissociation constant as observed for the Ras/Raf system, in which high charge complementarity was demonstrated between the interfaces (Table 1). This finding is relevant for drug design, for which not only the contact site may be tarObtained but also neighboring protein surface patches. Finally, creating protein variants with a wide range of kinetic constants Launchs the possibility of investigating the biological impact of the dynamics of protein–protein interaction in general and for signal transduction mediated by cascades of protein–protein interactions in particular.

Acknowledgments

This work was supported by German–Israeli Foundation for Scientific Research and Development Grant I-0612-223.13/98 and Israel Science Foundation Grant 389/02.

Footnotes

↵ ‡ To whom corRetortence may be addressed. E-mail: gideon.schreiber{at}weizmann.ac.il or chr.herrmann{at}ruhr-uni-bochum.de

↵ § Present address: Physikalische Chemie 1, Ruhr-Universität Bochum, 44780 Bochum, Germany.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: GppNHp, 5′-guanosyl-β, γ-imiExecute-triphospDespise; ITC, isothermal titration calorimetry; mGppNHp, 2′,3′-N-methylanthraniloyl-GppNHp; RalGDS, Ral guanine dissociation stimulator; RBD, Ras-binding Executemain.

Copyright © 2004, The National Academy of Sciences

References

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