Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity^*

Site-directed Mutagenesis

The introduction of single lysine mutations to DNA coding for Raf-RBD and Raf full-length was performed using the QuikChange^TM site directed mutagenesis kit (Stratagene) using pGEX-2T vector harboring the insert coding Raf-RBD 51–131 and pCDNA3 Raf full-length (wild type) as templates, respectively. The single lysine mutants were used as templates to generate multiple lysine mutants. All mutations were analyzed by sequencing.

Protein Expression and Purification

Raf-RBD and Ras wild type and mutants were synthesized in bacteria and purified as described earlier (13, 15, 34, 35). The nucleotide exchange with the non-hydrolyzable GTP analogue GppNHp and mantGppNHp, respectively, was performed according to Ref. 35. The loading of Ras with the fluorescent GDP analogue, mantGDP, was performed in the presence of 10 mm EDTA and a 5-fold excess of mantGDP and incubated at 4 °C overnight. For the last step of purification, all proteins were subjected to size exclusion chromatography (Superdex 75, GE Healthcare) equilibrated with the buffer containing 15 mm Hepes, pH 7.4, 5 mm MgCl₂, and 125 mm NaCl. The protein concentration was measured by the Bradford method (36) using bovine serum albumin as a standard.

Calculation of Association Rate Constants

The increase of the association rates of mutant Raf-RBD·Ras·GppNHp complexes were calculated as described earlier using the program HyPare, which automatically generates all of the mutant files (33). Coordinates for the calculation were taken from the crystal structure Raps-GppNHp, a Ras homologue complexed with Raf-RBD resolved at 1.8 Å resolution (15) (Protein Data Bank entry 1GUA). The surface-exposed residues were chosen, and the lysine mutations at the appropriate positions were modeled using SwissPdbViewer (37).

Stopped-flow Measurements

The measurements of association rate constants were performed with an SM17 apparatus (Applied Photophysics) by rapid mixing of 0.5 μm Ras bound to either mantGppNHp or mantGDP with Raf-RBD at various concentrations. Pseudo-first order conditions were established with Raf-RBD at a minimum concentration of 2.5 μm, allowing for single exponential curve fitting. Mant-nucleotides were excited at 360 nm, and the emitted fluorescence was monitored through the 408 nm cut-off filter. The binding of Raf-RBD was detected by the fluorescence change as described earlier (20–22).

Isothermal Titration Calorimetry

The thermodynamic parameters of the Ras·GppNHp interaction with Raf-RBD wild type and mutants were determined using an isothermal titration calorimeter (MCS-ITC, Microcal) as described by Wiseman et al. (38). Dependent on the affinity, Raf-RBD mutant concentrations between 10 and 80 μm were placed into the cell, and Ras was placed in the syringe at a 10-fold higher concentration. The data analysis was performed by the manufacturer's software as described earlier (39).

Guanine Nucleotide Dissociation Inhibitor (GDI) Assay

The GDI assay was performed as described earlier (40). Briefly, Ras loaded with either mantGppNHp or mantGDP was used for the GDI assay in a concentration of 0.05 μm in the presence of different concentrations of Raf-RBD wild type and mutants. The nucleotide dissociation was initiated by the addition of a 1000-fold excess of the non-fluorescent analogue GppNHp or GDP. The time-dependent fluorescence decrease was fitted using single exponential decay, and the observed rate constants of the nucleotide dissociation were plotted against the corresponding Raf-RBD concentration. The curves were fitted using the quadratic equation as described previously (35).

Phosphor NMR Measurements

³¹P NMR spectra have been recorded as described earlier (48). Ras proteins were dissolved in 40 mm Hepes, pH 7.4, 5 mm MgCl₂, 1 mm dithioerythritol, 150 mm NaCl. 5% D₂O was added to obtain a lock signal, and 0.1 mm 2,2-dimethyl-2-silapentane-5-sulfonate was added for referencing. ³¹P NMR spectra were recorded with a Bruker Avance 500 NMR spectrometer operating at ³¹P frequency of 202 MHz using a 70° pulse and a total repetition time of 7 s to avoid saturation effects. Measurements were performed in a 10-mm probe using 8-mm Shigemi sample tubes. Protons were decoupled during data acquisition by a GARP sequence (41) with a strength of the B₁-field of 830 Hz. For referencing, the Ξ-value of 0.4048073561 reported by Maurer and Kalbitzer (42) was used, which corresponds to 85% external phosphoric acid contained in a spherical bulb.

Luciferase Reporter Gene Assay

Rabbit kidney epithelial-like RK-13 cells were grown to 25% confluence on 6-cm dishes and then transfected with DNA by the calcium phosphate co-precipitation method. 2 μg of the reporter constructs (E74₃-tk80-luc or tk80-luc), 0.5 μg of a β-galactosidase expression vector (pEQ176), 1.5 μg of ERK-1 expression vector (43), 1.5 μg of an Ets protein expression vector (44), and 1.5 μg of pcDNA3 expression plasmid alone or pcDNA3 expression plasmid containing a Raf mutant were used for each transfection. When indicated, 80 ng of the RSV-Ras G12V or RSV-Ras wild type plasmid were additionally transfected. 36 h after transfection, cells were harvested and lysed, and luciferase and β-galactosidase activities were determined as described (45). Relative luciferase activity was obtained by normalizing the luminescence to the β-galactosidase activity. ERK inhibitor was purchased from Calbiochem (catalogue number 328007; see supplemental Fig. 1).

Computational Design of Raf-RBD Mutants with Higher Ras·GppNHp Association Rates

Among the Ras binding domains studied so far, the Raf-RBD association rate is the fastest with Ras, whereas the RBDs of other effectors associate much more slowly (46). We have previously shown that optimizing electrostatic steering on this system by using the computer program PARE (31, 32) resulted in a significant increase in the association rate constant k_on. In the present work, we aimed to further increase k_on of Ras/effector association, particularly to determine if this would affect differently the activity of the GDP- versus GTP-bound form of Ras. For the design, Raf-RBD residues located at the edge of the Ras-Raf interface (Asn⁵⁶, Asp⁸⁰, and Glu¹²⁵) and located within the interface (Thr⁶⁸, Val⁶⁹, Met⁸³, and Ala⁸⁵) were mutated. Lysine residues were introduced at these positions into the crystal structure of the Raps/Raf-RBD complex (Protein Data Bank entry 1GUA; Ras homologue Raps loaded with the non-hydrolyzable GTP analogue GppNHp and complexed with the Raf-RBD wild type) using SwissPdbViewer (37). The relative increase of the association rate constants for the single lysine mutants as well as for the multiple mutations were calculated using the protein design algorithm HyPare (33). The results for the calculations of Ras·GppNHp binding to Raf-RBD mutants are listed in Table 1. The calculations suggested a smaller effect for the lysine mutants situated at the edge of the binding interface than for the residues within the binding interface. The former, N56K, D80K, and E125K, were calculated to increase the association rate constant by a factor between 1.8 and 2.8 (relative to wild type), whereas the latter, T68K, V69K, M83K, and A85K, were predicted to increase the association rate constant by a factor between 2.3 and 5.6. The multiple lysine mutants were calculated to increase association by a factor between 6.3 and 45.6.

Experimental Definition of the Binding Characteristics of Raf-RBD Mutants

To test the computational results, the mutations mentioned above were introduced to Raf-RBD, and the corresponding proteins were purified as described under “Experimental Procedures.” The measurements of the association rate constants were performed under pseudo-first order conditions, and the observed rate constants k_obs were plotted versus Raf-RBD concentration. Linear regression yields the association rate constants k_on (Fig. 1A and Table 1). The increase of the association rate constant was not as pronounced as calculated using HyPare. The lysine substitutions of Asn⁵⁶ and Glu¹²⁵ in the proximity of the binding interface and of Val⁶⁹ and Met⁸³ within the interface showed no change in the association rate constant. The lysine mutations at the positions Thr⁶⁸, Asp⁸⁰, and Ala⁸⁵ showed a small but significant increase of the association rate constants from 29 to 67, 52, and 50 μm⁻¹ s⁻¹, respectively (Table 1). The association rate constants of all multiple mutations harboring A85K were in the range of 80 μm⁻¹ s⁻¹. Except for E125K and its combination with A85K, the trend of the change in rate constant is as predicted by the calculation, whereas the values differed significantly (Fig. 1B). The largest increase that could be reached was only 3-fold above the wild type value. As was discussed before, it seems that an upper limit for association rate increase through electrostatic steering is reached in these cases (32).

Having the Raf-RBD mutants with the potential for faster and stronger binding at hand, we measured now the affinity for Ras. To this end, the Raf-RBD-Ras·GppNHp interaction was quantified using isothermal titration calorimetry (Table 1 and Fig. 1C). Surprisingly, the only modest increase in the association rate constants is not reflected by the complex stability. Raf-RBD with the mutation A85K alone and in combination with the other lysine substitutions increased the affinity constant (reciprocal K_d value) of the complex with Ras·GppNHp by factors between 3 and 12. Because all other single mutations did not contribute significantly to an increase of complex stability, A85K in Raf-RBD was identified as the most prominent mutation to increase the association rate constant and the stability of the complex with Ras·GppNHp.

Raf-RBD Interaction with Ras·GDP

Having identified Raf mutants that bind Ras·GppNHp around 10-fold tighter than wild type, we were curious about their impact on Ras·GDP affinity toward the Raf-RBD mutants and on a possible change of specificity of Ras·GppNHp versus Ras·GDP. To address this issue, mutants binding more tightly to Ras·GppNHp (namely Raf-RBD A85K, Raf-RBD A85K/E125K, Raf-RBD N56K/A85K, and Raf-RBD N56K/D80K/A85K were evaluated as to their binding to Ras·GDP. The dissociation constants (K_d) of Ras·GDP and Ras·GppNHp were determined using the GDI assay, because it allows us to define precisely Ras/effector interactions over a range of K_d values of more than 4 orders of magnitude. This assay takes advantage of the observation that binding of effector protein inhibits nucleotide dissociation from Ras. The dissociation of the fluorescent nucleotides, mantGppNHp or mantGDP (40), leads to a decrease of fluorescence intensity, and the obtained time traces (Fig. 2A) can be fitted using a single exponential equation. The plot of the observed rate constants versus the concentration of the RBDs can be fitted using a quadratic equation (21, 35). The dissociation constants of the Raf-RBD interaction with either Ras·mantGDP (Fig. 2, B–F) or Ras·mantGppNHp (data not shown) were determined at 37 °C and at buffer conditions identical to the previous kinetic and thermodynamic measurements. The lysine mutation at position 85 decreased the dissociation constant of the Ras·GDP-Raf-RBD interaction from 46 to 1.9 μm. A similar change was also observed for all multiple mutations harboring the lysine at position 85 (Table 2). The changes in the stability of the complexes with Ras·GppNHp and Ras·GDP, respectively, can be described in terms of the changes in free energy of binding, ΔΔG (Fig. 3A), which are listed in Table 2. The magnitude of change was larger for the Ras·GDP complexes than for Ras·GppNHp, which was also evident from the smaller ratios of the mutant K_d values of the Ras·GDP versus Ras·GppNHp complexes (Fig. 3B). This indicates that for the Raf mutants, the improvement in binding is more pronounced for Ras·GDP interaction than for Ras·GppNHp (Fig. 3B); thus, the specificity of binding of Raf-RBD to Ras·GppNHp and Ras·GDP was shifted.

Structural Requirements for Interaction Analyzed by ³¹P NMR Spectroscopy

By ³¹P NMR spectroscopy, we wanted to investigate possible structural changes in the Ras-Raf binding interface evoked by the Raf mutation. Changes of Ras switch I conformation are reported by shifts of ³¹P NMR resonances (47–49). Wild type Raf-RBD binds tightly only to the GppNHp-bound state of Ras, where the switch I region of Ras (residues 30–38) adopts an appropriate conformation favoring effector binding. This positioning of switch I is established by Thr³⁵ hydrogen bonds with the γ-phosphate of the nucleotide, and this conformation (state 2) is shown to be stabilized by effector binding (47–49). After hydrolysis and loss of the γ-phosphate, Ras·GDP relaxes to other conformations of switch I, leading to much lower effector binding affinity.

Above we have identified the lysine substitution at position 85 of the Raf-RBD that is able to bind not only the activated Ras, Ras·GTP, but also the inactive, GDP-loaded Ras more tightly. The ³¹P NMR spectrum of Ras·GDP alone shows two resonance lines with chemical shifts at −10.6 ppm and −2.0 ppm, corresponding to the α- and β-phosphates of Ras·GDP with a slight inhomogeneity in line shape of the β-phosphate resonance, as shown previously (Fig. 4A) (50). The resonance lines of the bound nucleotide are assigned by their chemical shift values because the α-phosphate groups of free metal bound nucleotide are very similar (Table 3). The chemical shift values obtained are −10.6 ppm of the Ras·GDP complex and −11.1 ppm for Ras·GppNHp T35A. The addition of Raf-RBD A85K to Ras·GDP leads to the appearance of a second resonance line of the α-phosphate at −11.2 ppm, which means a chemical shift difference of −0.6 ppm, which is similar to the Δδ in the GppNHp-bound wild type with −0.5 ppm between state 1 and the effector-bound state 2 (Table 3). The new signal becomes larger with higher concentration of Raf-RBD A85K, whereas the resonance at −10.6 ppm decreases and disappears completely at a molar ratio of 4:1 for Raf-RBD A85K and Ras·GDP (Fig. 4A). In contrast, with wild type Raf-RBD, the new α-phosphate peak at −11.2 ppm assignable to the Raf complex can only be observed at much higher concentrations (Fig. 4B). Even at a molar ratio of 6:1, the resonance at −10.6 ppm still predominates, whereas it has entirely vanished in favor of −11.2 ppm in the case of the A85K mutant. The observed chemical shift change by binding of wild type Raf is again −0.6 ppm (Table 3), indicating that the local conformational change induced by binding is similar in the two cases. The NMR spectra are typical for a slow two-site exchange. From the slow exchange condition |2π Δν τ_e| ≫ 1, a lower limit of the exchange correlation time τ_e of 1.3 ms can be obtained (51).

In order to support our hypothesis on the potential of Raf-RBD A85K to stabilize state 2 of Ras, we used the GppNHp-bound form of Ras T35A. This mutant is known to be arrested in state 1, leading to decreased effector affinity (40, 48). The ³¹P NMR spectrum of Ras·GppNHp T35A is shown in the bottom panel of Fig. 4C, where all resonance lines correspond to state 1. The addition of high concentrations of wild type Raf-RBD and its binding to Ras·GppNHp T35A leads to small changes in chemical shift values but no shift toward lines corresponding to state 2 (48). In contrast, the addition of Raf-RBD A85K leads to the appearance of a second set of resonance lines that correspond to state 2. This is most clearly visible for the γ-phosphate resonance of Ras·GppNHp at −3.29 ppm (Fig. 4C). As described for Ras·GDP above, the up-field-shifted line at −3.3 ppm increases with Raf-RBD A85K concentration, and at a molar ratio of 3:1 it strongly dominates over the line at −2.6 ppm, which represents state 1 of Ras·GppNHp T35A. For the wild type Ras, the shift of the β-resonance is indistinguishable in the two states and is not influenced by Raf binding. The same is true for the binding of Raf-RBD A85K to Ras·GppNHp T35A.

The resonance position of the α-phosphate group is shifted slightly downfield in the presence of Raf-RBD A85K. Although for the γ-phosphate resonance, clearly slow exchange conditions prevail because two separate lines are observable, this is not clear for the resonance of the α-phosphate, since the line widths are here too large. If fast exchange conditions would prevail then at complete saturation, a much larger upfield shift would correspond to the Ras·Raf complex. Summarizing, the ³¹P NMR measurements show that Raf-RBD A85K binding to Ras leads to a conformational change of switch I in Ras·GDP as well as in Ras·GppNHp T35A, which could not be achieved by wild type Raf-RBD in the concentration range studied.

Biological Activity of Mutant Complexes Using a Reporter Gene Assay

To determine the biological consequences of the loss of specificity toward the GTP-bound state of Ras when using the tighter binding Raf-RBD mutations, we followed the level of expression of a reporter gene. RK13 cells were transfected with the luciferase reporter gene driven by three E74 binding sites. The E74 is a high affinity binding site for Ets transcription factors and has been shown to specifically mediate transcription upon activation of the Ras/Raf/MEK/ERK signaling pathway (45). This Ras/Raf/MEK/ERK-dependent signaling has been shown to correlate quantitatively with the affinity of the Ras-Raf interaction in vitro (43, 52).

Here, we addressed the activation of the Ras/Raf/MEK/ERK signaling cascade by the Raf-RBD A85K mutant binding more tightly to Ras·GTP and Ras·GDP. To examine the tighter binding of Raf-RBD mutants to Ras·GTP, the cells were co-transfected with the constitutively active Ras mutant, Ras G12V, and Raf kinase wild type and mutants, respectively. Fig. 5A shows immunoblots of transfected Raf mutants demonstrating similar concentration levels of the proteins in the cells. In order to compare quantitatively the luciferase activity that reports the degree of Raf activation, the luminescent readout was normalized to the enzymatic activity of co-transfected β-galactosidase. Inhibition of ERK phosphorylation by the addition of 40 μm inhibitor results in abolishment of luciferase activity (53) (supplemental Fig. 1), demonstrating that Raf activation leads to transcription of the luciferase gene mediated by the ERK pathway.

The activities from each experiment were set into relation to the activity of the co-transfection of Ras G12V with Raf wild type (100%) (Fig. 5B, blank columns). The transfection with the constitutively active Ras mutant G12V in the absence of Raf was done as a negative control, showing that the activity increase is due to the presence of the Raf kinase (43). All proteins harboring the A85K mutation showed an increased reporter gene activity in the presence of Ras G12V compared with the Raf wild type.

To test if the stronger Ras·GDP binding to Raf A85K leads also to higher activation, the cells were co-transfected with Ras wild type (Fig. 5B, hatched columns) or without Ras (Fig. 5B, gray columns). Interestingly, the activity of the reporter gene was almost the same with and without the co-transfection of Ras wild type, indicating the interaction of Raf kinase with the endogenous Ras·GDP. All of the Raf kinase mutants harboring the A85K mutation had an increased reporter gene activity relative to the Raf kinase wild type. In addition, in this type of experiment, instead of Ras WT, we co-transfected Ras S17N, which is known not to be activated, to reside constitutively in the GDP-bound state, and to suppress activation of endogenous Ras. A pull-down experiment demonstrates that no Ras GTP is detectable when Ras S17N is co-transfected (data not shown). Ras S17N leads to a similar low level of luciferase activity (16–18%) as after co-transfection with Ras WT, and Raf A85K leads to a 4-fold increase for both Ras WT and Ras S17N co-transfection (supplemental Fig. 1).

Because the reporter gene assay is known for the correlation of its activities with the affinities determined using in vitro methods like the GDI assay (43), we plotted the reporter gene activities of the Ras G12V and Ras wild type transfection versus log K_d values of the respective Ras·Raf complexes (Fig. 5C). The plot shows a clear correlation between the dissociation constants determined using the GDI assay and the reporter gene activity with a correlation coefficient of −0.97. This linear dependence identifies the affinity as the major factor of the activation of the Ras/MEK/ERK signaling cascade.

Improving Ras-Raf Binding Affinity

The first part of the project presented here aimed at improving Ras-Raf association, challenging the upper limit of Ras/effector binding affinity. We have previously shown that RalGDS furnished with some appropriate mutations, identified using the computer algorithm PARE, may reach the Ras binding strength of Raf (32). These calculations are based on Coulomb energies contributed by charged residues near or within the binding interface areas of the two interacting proteins. The most promising mutations proposed were lysine residues, which were introduced into the sequence of Raf-RBD. In contrast to the case of Ras/RalGDS with good agreement between predicted and experimentally determined changes of the association rate constants (32), the comparison of theoretical and experimental results obtained here with Raf-RBD shows large discrepancies. Basically, the changes observed experimentally are much smaller than the calculated values, whereas the trend is predicted correctly. This is not due to the use of HyPare instead of PARE (data not shown) and may suggest that for Raf there is not much space for improvement of the association rate. Natural evolution seems to have exploited the complementary electrostatics on the two protein surfaces to reach almost an optimum rate of association. The upper limit of the rate of binding for Ras/effector association can be defined to k_on ≤ 10⁸ m⁻¹ s⁻¹.

The largest increase of the k_on value by a single mutation is realized by A85K (1.7-fold), T68K (2.3-fold), and D80K (1.8-fold), whereas the others show less pronounced and insignificant changes. Intriguingly and in contrast to RalGDS in our previous study (32), the changes of the k_on values do not increase the binding affinity of Ras-Raf in the manner expected from pure electrostatic steering effects. Although the K_d value for D80K is similar to that for the wild type protein, it is 2 times higher for T68K and almost 5-fold lower for A85K. The contradictory effect of lysine 68 on association rate and affinity can be rationalized by a positive contribution to electrostatic steering but a negative consequence for the affinity, since this residue is located in the binding interface and leads to the loss of original contacts. In contrast, the improvement of binding affinity by lysine 85 is even stronger, as expected from its increased k_on value. An increased binding of Ras and Raf A85K was observed earlier when using radioactive in vitro assays (54). As pointed out by Fridman et al. (54), this lysine may reach into the interface area and may form a direct contact to Ras, whereas the wild type alanine side chain is too short for this. Altogether, not only electrostatic steering effects are responsible for changes in affinities of our Raf mutations. The interaction between Ras and Raf is significantly improved by A85K, and with the assistance of N56K and D80K the K_d value is decreased up to 10-fold.

Raf with High Affinity for Ras·GDP and Lower Nucleotide Specificity

The interaction between Ras and Raf leads to the activation of one of many possible signal pathways. The affinity of a protein·protein complex in the context of network formation with various binding partners is one side of the coin, whereas the specificity meaning relative affinities is the other. Therefore, we wondered about the impact of our Raf mutations on the interaction with the “non-active” form of Ras (i.e. bound to GDP). Compared with Ras·GppNHp, a 1000-fold higher K_d value was reported for the interaction between Raf-RBD and Ras·GDP (40). Here a 350-fold difference was measured using a buffer with a salt concentration close to the physiological ionic strength. We focused our attention on the Raf mutations containing A85K, which show higher affinity to Ras. In Table 2, the K_d values obtained using the GDI method are listed for the GppNHp- and GDP-bound form of Ras, respectively, together with the corresponding changes of free binding energy. Surprisingly, the positive effect of these mutations on binding affinity is even larger for Ras·GDP than for Ras·GppNHp. The single mutation A85K shows a very large change already (23-fold), which becomes slightly larger in the presence of N56K and D80K (37-fold). The larger increase in affinity toward the GDP form results in a reduction of specificity relative to the GTP-bound form, which drops by a factor of 5 (i.e. the ratio of K_d values GDP/GppNHp is only 100-fold for Raf A85K and 25-fold for the triple mutant). Finally, it is worthwhile to mention that these lysine mutations of Raf have reached an affinity for Ras·GDP similar to the affinity of other Ras effectors like RalGDS for Ras·GppNHp.

High Affinity of Raf to Ras Is Responsible for Biological Activity, Also in the GDP Form

An earlier alanine scanning analysis of Ras-Raf affinity in relation to the quantitative outcome for the biological activity demonstrated a direct correlation between binding and signaling (43). The assay is based on the quantification of the activity of the luciferase reporter gene, which is controlled by the activity of Raf. The alanine mutations of Raf lead to decreased Ras affinity and therefore lower biological activity. We used the same reporter gene setup in order to answer the question of whether the biological activity of the wild type proteins has reached a maximum value or if our Raf mutations with higher Ras affinity are more active. Intriguingly, the Raf mutants containing A85K led to increased reporter gene activity, namely 40–50% higher than the wild type (Fig. 5B, blank columns). In the same manner as the activity steps down with lower Ras-Raf affinity (43), it goes up with higher affinity. The slope of the correlation of binding strength (log K_d) and biological activity is the same as described earlier for decreased affinity and activity of the alanine mutants (Fig. 5C). Residue 85 of Raf is located in the binding interface with Ras, and A85K is described in the literature only in the context of increased Ras affinity; no increased enzymatic activity was reported. Nevertheless, a possible gain-of-function of Raf harboring the A85K mutation cannot completely be ruled out.

As pointed out, the lysine mutations of Raf have reached a K_d value of 1 μm for Ras·GDP, which is even slightly smaller than observed for Ras·GppNHp and the effector RalGDS (3.5 μm) (19). The question we addressed is obvious. Can Ras·GDP activate Raf A85K? In fact, in contrast to Raf wild type, the lysine mutants of Raf lead to activation (60%) of the reporter gene when Ras wild type (or S17N) is co-transfected rather than Ras G12V (which is constitutively in the active GTP-bound form and which corresponds to 100% activity).

Strikingly, the same activities are observed without co-transfection of Ras wild type, meaning the concentration of endogenous Ras is high enough to activate the transfected Raf mutants, as shown in Fig. 5B, and thus does not influence the outcome of our experiments. The reporter gene activities observed here for the lysine mutants of Raf and Ras·GDP (open squares in Fig. 5C) correspond to the values observed in the earlier study for the alanine mutants of Raf and Ras·GTP with similar K_d values (filled circles in Fig. 5C). Notably, there are high affinity Raf mutants leading to higher reporter gene activation with Ras·GDP compared with low affinity mutants in the presence of Ras G12V. Taking all of these observations together, it seems that the state of Ras, GTP versus GDP, is not relevant for the activation of Raf; only binding affinity counts.

A85K Mutant of Raf Induces the High Affinity Conformation of Ras Switch I

It has been shown earlier that Ras·GppNHp exists in two conformational states, with state 1 a low affinity state for effectors and state 2 a high affinity effector binding state (47–49). Ras mutations of Thr³⁵ lead to a shift of the switch I conformational equilibrium to state 1, resulting in lower affinity for Raf or other effectors, as was shown for different Ras·nucleotide complexes (48, 49, 55, 56). It is assumed that only in state 2 does switch I occur in the correct conformation for effector binding. Intriguingly, although in Ras T35S this equilibrium can be shifted back to state 2 by Raf binding, this is not observed in Ras T35A even at high concentrations of Raf (48). This is rationalized by a stronger shift of the equilibrium in T35A to state 1, which cannot be overcome by Raf binding. Hence, Raf binds to the weak binding state 1 of Ras T35A and therefore shows lower affinity, similar to wild type Ras·GDP (40, 48). In line with this interpretation, the more tightly binding Raf-RBD mutant A85K was found here to force Ras T35A to adopt the effector binding state 2, where switch I is in the high affinity conformation. Hence, a structural switch of Ras evoked by lysine 85 of Raf-RBD rather than an additional, local interaction between two amino acid side chains leads to stronger binding.

The lysine substitution at position 85 of the Raf-RBD is able to bind not only the GTP-activated Ras but also the inactive, GDP-loaded Ras more tightly. Now it was interesting, of course, to understand the molecular mechanism for this observation and for the decrease in specificity of Ras·GTP versus Ras·GDP. Since ³¹P NMR chemical shift changes are mainly due to local structural changes (mainly torsional strain of bonds and polarization effects), they can be qualitatively interpreted in structural terms. Binding of Raf-RBD A85K to Ras·GppNHp T35A leads to chemical shift changes similar to those observed earlier for the binding of wild type Raf-RBD to Ras·GppNHp T35S. The latter chemical shift changes were interpreted as a shift of the conformational equilibrium to state 2. The same interpretation holds probably also for the complex of Raf-RBD A85K with Ras·GppNHp T35A. Different chemical shifts of the resonance of the α-phosphate group of bound GppNHp in the two states indicate different environments (mainly coordination patterns and/or bond polarizations) of the α-phosphate groups in the two states. The same general description holds also for GDP. The ³¹P data suggest that the environment of the β-phosphate group of bound GDP is not changed after Raf binding, but that of the α-phosphate group is clearly changed after binding. The chemical shifts of the α-phosphates of GppNHp as well as GDP are moderately shifted after binding to Ras (Table 3). They are rather similar in Ras·GppNHp T35A (state 1) and Ras·GDP, which suggests that the environment of the α-phosphate group may also be similar in the two complexes. Analogously, the chemical shifts after Raf-RBD binding (state 2 in Ras·GppNHp) are again similar. This could mean that, concerning the α-phosphate, Ras·GDP is predominantly in a state 1-like environment, and after Raf-RBD interaction, it is in a state 2-like environment.

In conclusion, higher affinity between Ras and Raf achieved by mutation led to stronger activation of the mitogen-activated protein kinase pathway. This suggests that nature has evolved the residues within and around the Ras-Raf interface to an optimum as to fast association and strong binding on the one hand and specific recognition of the active state of Ras on the other hand. Raising the affinity (e.g. by Raf mutation A85K) leads to further improvement of the interaction, particularly with Ras·GDP. This is achieved by lysine 85-mediated positioning of switch I in a Ras·GTP-like conformation. Thus, the specificity drops, and the K_d value for Ras·GDP falls into a range that leads to effector activation. Our results show that lysine at position 85 of Raf would be unfavorable for the biological function because signaling requires high specificity and was therefore discarded by evolution. Not strong binding alone but rather a sensitive balance of the affinities of all partner proteins participating in a complex protein network is required to make it operate smoothly.