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A Ras-induced Conformational Switch in the Ras Activator Son of Sevenless

Tanya S. Freedman*, Holger Sondermann*, Gregory D. Friedland, Tanja Kortemme, Dafna Bar-Sagi, Susan Marqusee and John Kuriyan

* The authors contributed equally to this work.

PNAS 103(45): 16692-16697   local copy

Abstract / Figures / Supporting Data / PDB Coordinates


Abstract: The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.


Figures (Click on the small image to view the bigger one):


Figure 1. Sos and RasGRF1 catalyze Ras nucleotide-exchange. (a) Domain structure of human Sos1 and murine RasGRF1. Sos and RasGRF1 both contain Rem domains (yellow) and Cdc25 homology domains (gray) that include a helical hairpin motif (HH; blue in Sos, red in RasGRF1). Together, the Sos Rem and Cdc25 domains are referred to as Soscat. Other domains in Sos and RasGRF1 contribute to localization and regulation: DH, Dbl homology; PH, pleckstrin homology; IQ, motif for Ca2+/calmodulin binding; and PxxP, motif for SH3 binding. (b) Nucleotide-exchange cycles of Sos and RasGRF1. Sos stimulates nucleotide exchange from Ras when its Rem and Cdc25 domains engage a nucleotide-bound Ras molecule at an allosteric site distal to the catalytic site. The Cdc25 domain of RasGRF1 is sufficient for Ras nucleotide-exchange activity.


Figure 2. Nucleotide-exchange assays. Nucleotide release from Ras is followed by a loss in fluorescence emission of mant-dGDP. RasGRF1Cdc25 increases the rate of nucleotide release from Ras relative to a control reaction. In contrast, the Sos Cdc25 domain alone and a mutant of Sos with the Rem and Cdc25 domains deficient in binding Ras at the allosteric site, Soscat W729E, lack substantial activity. Wild-type Soscat also is essentially inactive in the absence of allosteric activator. When GMPPNP-bound RasY64A, a mutant of Ras that interacts only with the Sos allosteric site, is added at a saturating concentration, Soscat becomes maximally active. These reactions are carried out by using 0.1 µM substrate Ras·mant-dGDP, a concentration at which Ras·GDP does not interact significantly with the allosteric site of Sos


Figure 3. Crystal structures of Soscat and RasGRF1Cdc25. (a) Sos activation occurs through coordinated rotation of the helical hairpin and the Rem domain upon Ras binding to the allosteric site. The structures of uncomplexed Sos and Ras-bound Sos are superposed on the Cdc25 domain core, excluding the helical hairpin, extended loops, and termini. Upon allosteric activation by Ras, the helical hairpin and the Rem domain pivot outward by 10°. (b and c) The Cdc25 domain of RasGRF1 has a conformation more similar to that of active Sos than that of inactive Sos.


Figure 4. Inward rotation of the helical hairpin toward the catalytic Ras binding site. (a) A cutaway view of the catalytic site of uncomplexed Sos shows that when Ras is docked in its binding site, it clashes extensively with the inward-rotated helical hairpin. The placement of Ras in the catalytic site is modeled from the Ras-bound Sos structure with Tyr-64 of Ras oriented correctly in its binding pocket. (b) Upon allosteric Ras binding, the Sos helical hairpin rotates outward, relieving the steric clashes with Ras at the catalytic site (1NVV). The helical hairpin pivots around residue Tyr-915 from the Sos Cdc25 domain core, which hydrogen-bonds through its hydroxyl group to the amide nitrogen of Sos Phe-929 in the helical hairpin. (c) RasGRF1Cdc25 achieves a helical hairpin position compatible with Ras binding to the catalytic site and lacks the anchor/pivot point interaction for helical hairpin rotation, substituting Leu-1164 for Sos Tyr-915.


Figure 5. The clamping of the helical hairpin. (a) View of RasGRF1 showing the helical hairpin (red), flap1, and flap2 (both gray). (b) A cutaway view through the catalytic Ras binding site of RasGRF1. A tight interface between flap1 and the helical hairpin of RasGRF1 is formed by bulky, hydrophobic residues (Phe-1052, Phe-1051, and Tyr-1048 in flap1, Ile-1214, and Ile-1210 in the helical hairpin). A salt-bridge network and hydrophobic interactions connect the helical hairpin with flap2 (Met-1181 and Phe-1188 bury Asp-1185 in the helical hairpin, bridging to Arg-1160 and Arg-1165 in flap2). (c) In the active conformation of Sos, the helical hairpin (dark blue) is similar in position to that of RasGRF1, but the interface with flap1 is not well packed (Val-805, Leu-804, and Pro-801 in flap1, Thr-964 and Val-968 in the helical hairpin). (d) In the absence of allosteric Ras binding, the helical hairpin of uncomplexed Sos (light blue) collapses inward to interact more closely with flap1. Neither active nor inactive Sos helical hairpins form close interactions with flap2 (Lys-939, Ile-932, and Asn-936 in the helical hairpin do not form contacts with His-911 and Leu-916 in flap2).


Figure 6. Computational study of the effects of swapping residues from RasGRF1 and Sos. The number of times a given residue accumulated a conformation-stabilizing mutation in low-energy sequences from 100 separate Monte Carlo simulations is described by the substitution frequency. (a and b) Cα positions for buried residues that are swapped with high frequency are indicated (spheres) for Sos (a) and RasGRF1 (b). (c and d) Several Sos residues that substitute with high frequency are located in the flap1-helical hairpin interface (see also Fig. 5). (c) Wild-type Sos. (d) Substitutions from RasGRF1.


Supporting Figures:


Figure 7. Alignment of the Cdc25 domain sequences of Sos and RasGRF from mouse (m), human (h), rat (r), and fruit fly (d). The Cdc25 domains of mRasGRF1 and hSos1 (numbered) are 30% identical. Sequence elements that may be responsible for differences between Sos and RasGRF1 are highlighted. These residues are specific to RasGRF1 or Sos and are conserved in RasGRF1 and Sos sequences from different organisms.


Figure 8. Soscat and SosCdc25 have similar denaturation profiles and CD spectra. (a) Soscat displays two cooperative transitions upon equilibration in increasing amounts of urea. The CD spectrum in the absence of urea (Inset) reflects predominantly helical secondary structure, which disappears upon addition of urea to 9 M. (b) The cooperative denaturation profile and helical CD spectrum of SosCdc25 are similar to those of Soscat, indicating that SosCdc25 well folded.


Figure 9. Distance difference matrices show regions of change in intramolecular Ca positions in pairs of Sos and RasGRF1 Cdc25 domains. (a) Inactive and active Sos structures differ the most in helical hairpin position relative to the Cdc25 domain core. The base of the helical hairpin (hh base) similarly changes position with respect to the Cdc25 domain core. The flap1 region extending from the Cdc25 domain core to abut one side of the helical hairpin also changes position with respect to the helical hairpin and the rest of the Cdc25 domain core. (b) Like the comparison between inactive and active Sos, the comparison between inactive Sos and RasGRF1 shows different positioning of the helical hairpin and the flap region relative to the rest of the Cdc25 domain. (c) The position of the helical hairpin in RasGRF1 relative to the rest of the Cdc25 domain is more similar to that of active Sos.


Figure 10. Changes in the accessibility of the Ras Tyr-64 binding pocket in uncomplexed and Ras-bound Soscat. (a) Ras (from the active Sos structure, 1NVV) is docked into the catalytic site of uncomplexed Sos in a proper orientation with respect to the helical hairpin (as opposed to the best binding position of Tyr-64 shown in Figure 4). In this orientation, catalytic site Ras clashes extensively with the core of the Cdc25 domain. Combination of both methods for docking Ras (from this view and that of Figure 4) shows that binding of Tyr-64 and interaction with the helical hairpin, both critical for nucleotide exchange from Ras by Sos, are mutually exclusive in inactive Sos. (b) Upon allosteric Ras binding, steric clashes with Ras at the catalytic site are relieved.


Figure 11. b-Sheets couple the position of the helical hairpin to the position of the Rem domain in Sos and Epac2. (a) Two b-strands in the turn of the inactive Sos helical hairpin (light blue) and two strands in the Rem domain (light purple) form a four-stranded b-sheet across the two domains. Hydrogen-bond distances are indicated. (b) A similar b-sheet is formed by strands in the helical hairpin of active Sos (dark blue) and the Rem domain (yellow) despite significant motion of the Rem domain upon allosteric Ras binding. The hydrogen-bond distances indicate that this interaction is equally close in the active and inactive states. (c) Similar b-interactions between the helical hairpin (green) and Rem domain (orange) of Epac2 suggest that this mechanism of communication between the Rem and Cdc25 domains is conserved among other exchange factors.


Figure 12. The Cdc25 domain of Epac2 adopts an autoinhibited conformation similar to inactive Sos. (a) Relative to active Sos, the helical hairpin of autoinhibited Epac2 (from the full-length structure, green) is pivoted inward toward the catalytic site, with a conformation resembling inactive Sos (b).


Figure 13. Computational analysis of inactive Sos Cdc25 domain structure. As in Figure 6, a computational analysis was performed where Sos or RasGRF1 residues could be exchanged at each position, this time using the inactive Sos backbone conformation. Results for inactive Sos are similar to those for active Sos except for the loss of several substitutions in the flap1-helical hairpin interface and an increase in lower-frequency substitutions to RasGRF1 residues in the Cdc25 domain core.


Supporting Table: Table 1


PDB Coordinates:

PDB files: RasGRF1: 2IJE; Sos: 2II0
Links in PDB site: RasGRF1: 2IJE; Sos: 2II0