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Differences in Flexibility Underlie Functional Differences in the Ras Activators Son of Sevenless and Ras Guanine Nucleotide Releasing Factor 1


Tanya S. Freedman, Holger Sondermann, Olga Kuchment, Gregory D. Friedland, Tanja Kortemme, and John Kuriyan


Structure 17, 41-53, January 14, 2009 (local copy)

Abstract / Figures from the paper / Supplementary Information


Abstract:

The Ras-specific nucleotide exchange factor Son of sevenless (Sos) is inactive without Ras bound to a distal allosteric site. In contrast, the catalytic domain of Ras guanine nucleotide releasing factor 1 (RasGRF1) is active intrinsically. By substituting residues from RasGRF1 into Sos, we have generated mutants of Sos with basal activity, partially relieved of their dependence on allosteric activation. We have performed molecular dynamics simulations showing how Ras binding to the allosteric site leads to a bias toward the active conformation of Sos. The trajectories show that Sos fluctuates between active and inactive conformations in the absence of Ras and that the activating mutations favor conformations of Sos that are more permissive to Ras binding at the catalytic site. In contrast, unliganded RasGRF1 fluctuates primarily among active conformations. Our results support the premise that the catalytic domain of Sos has evolved an allosteric activation mechanism that extends beyond the simple process of membrane recruitment.

Illustrations from the paper.

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Figure 1. Comparison of Sos and RasGRF1
(A) Schematic diagram comparing the activation of Ras by Sos and RasGRF1.
(B) Interface between Ras and nucleotide exchange factors, and residues mutated in this study. A view down the helical hairpin of Sos highlights the conformational change that occurs upon Ras binding to the allosteric site. The Ras molecule is modeled into the structure of inactive Sos (PDB ID code 2II0; Freedman et al., 2006) from the crystal structure of active Sos (PDB ID code 1NVV; Margarit et al., 2003). When Ras binds to the allosteric site of Sos, the Rem domain is pivoted downward to maintain this interaction. The helical hairpin is also pivoted outward to open the catalytic site. Residues V805, V964, and T968, which comprise an interface between the Rem domain, flap1, and the helical hairpin, are highlighted in pink. RasGRF1 (PDB ID code 2IJE; Freedman et al., 2006) assumes an active conformation in the absence of bound Ras.



Figure 2. Engineering Intrinsic Activity into Sos (A and B) Nucleotide release assays show that RasGRF1 (Cdc25 domain) has a high basal activity compared to Sos (Rem and Cdc25 domains). Substituting residues from the flap1/helical hairpin interface of RasGRF1 into Sos increases the basal activity of Sos. The error bars represent the standard deviation of the fit rates of at least three independent experiments. (C) Two mutants have different responses to allosteric Ras binding. The single mutant T968I (Sos[TI]) is more active than wild-type Sos at all concentrations of RasY64A, a variant of Ras that binds selectively to the allosteric site. The triple mutant V805F+V964I+T968I (Sos[VFVITI]) has the highest basal activity, but fails to respond to allosteric Ras binding and has a dramatically impaired maximal activity.



Table 1. Molecular Dynamics Simulations The starting structure for RasGRF1 comes from the crystal structure of RasGRF1Cdc25 (PDB ID code 2IJE; Freedman et al., 2006), the starting structure for active Sos comes from the crystal structure of Soscat (PDB ID code 1NVV; Margarit et al., 2003) with two bound Ras molecules, and the starting structure for inactive Sos comes from the crystal structure of apo-Soscat (PDB ID code 2II0; Freedman et al., 2006). Mutations were created in the wild-type crystal structures using PyMOL (DeLano, 2002). Rascat, catalytic-site Ras; Rasallo, allosteric-site Ras; Sos, Soscat (Rem + Cdc25 domains); RasGRF1, RasGRF1Cdc25.



Figure 3. Confirmation of the Helical Hairpin in Molecular Dynamics Trajectories of Active Sos and RasGRF1
(A) The average conformation over the six GRF trajectories is similar to that observed in the crystal structure of RasGRF1 (pink and purple cartoons, respectively). The light surface reflects the range of sampled conformations, including the average structures for 500 ps windows over all the trajectories and eight instantaneous structures representing the extremes of conformation with respect to active and inactive Sos (determined individually by Cα rmsd of the helical hairpin or the Rem domain with respect to comparable regions of active Sos or of inactive Sos. The instantaneous structures with the highest and lowest rmsd values for both regions with respect to both crystal structures represent the diversity of conformations achieved during the trajectories). The dark surface surrounds the six structures that represent the average conformation of each individual simulation and thus reflects the heterogeneity among different simulations.
(B) In the GRF simulations, the helical hairpin is in a position more similar to active Sos (red) than to inactive Sos (blue). The helical hairpin samples conformations, however, that would clash with Ras bound to the catalytic site.
(C) Ras•SosActive simulations are more limited in the range of conformations sampled by the helical hairpin, avoiding clashes with Ras at the catalytic site.



Figure 4. Active-Site Occlusion by the Helical Hairpin during the Simulations (A-E) The number of clashes (Cα-Cα contacts closer than 2.2 Å) between the helical hairpin and a Ras molecule modeled into the active site is counted every 10 ps along the trajectory of each simulation. The solid line indicates one backbone clash between the helical hairpin and catalytic-site Ras. Six simulations are concatenated in each panel, and the dotted lines represent the boundaries between them. (F) The average number of clashes over each trajectory over time is plotted as a square. Points with similar y axis values are spaced horizontally for clarity. The horizontal bars represent the overall average number of clashes for all simulations. According to an ANOVA analysis, RasGRF1, Ras•SosActive•Ras (see Figure S6), and Ras•SosActive simulations are not significantly different in their extents of active-site occlusion. All other pairs of simulations in this figure have significantly different numbers of clashes between the helical hairpin and active-site Ras (p > .0001).



Figure 5. Rem Domain and Helical Hairpin Conformations in Sos Trajectories The crystal structures of active and inactive Sos are depicted in red and blue, respectively. In the left column is the result of one trajectory (of the type indicated) with the lowest average number of clashes with Ras modeled into the active site. In the right column is depicted the trajectory with the greatest number of clashes. The fraction of related simulations represented by each panel is indicated (for instance, 5/6 means that five simulations of the six performed have a similar degree of active-site occlusion to the one shown). The light surface reflects the range of conformations sampled within the simulation, including the conformations with highest and lowest helical-hairpin and Rem-domain rmsd with respect to active and inactive Sos as well as the average structures for every 500 ps of the simulation. Simulations not shown have intermediate degrees of active-site occlusion and are depicted in Figures S7 and S8.



Figure 6. Dynamic Fluctuations and Heterogeneity within Simulations The rms fluctuation value (related to a crystallographic B factor) of each Cα residue is indicated by color. Individual replicates of each simulation are overlaid after alignment on the rigid core of the Cdc25 domain.



Figure 7. Rem-Domain Position in Wild-Type and Mutant Sos Average conformation over all simulations. Mutated residues are colored in pink.



Figure 8. Interface Created by the Rem Domain, Helical Hairpin, and flap1 in the Simulations of Sos Average conformation over all simulations. Residues participating in the interface of the Rem domain, the helical hairpin, and flap1 are indicated in the surface. Mutated residues are colored in pink. Reference structures for active and inactive Sos are shown in red and blue, respectively.



Supplementary Information

Supplementary Figures