Multivalent Binding and Facilitated Diffusion Account for the Formation of the Grb2–Sos1 Signaling Complex in a Cooperative Manner

Abstract

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although Grb2 adaptor can potentially bind to all four PXψPXR motifs — designated herein S1, S2, S3 and S4 — located within the Sos1 guanine nucleotide exchange factor, the formation of Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of Grb2 homodimer to only two out of a four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that out of a possible six pairwise combinations in which S1–S4 motifs may act in concert for the docking of Grb2 homodimer through bivalent binding, only S1/S3, S1/S4, S2/S4 and S3/S4 do so, while S1/S2 and S2/S3 pairwise combinations appear to only afford monovalent binding. This salient observation implicates the role of local physical constraints in fine tuning the conformational heterogeneity of Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor

“…Notably, the molar mass of the PR domain was estimated to be around 28kD from our SAXS data. Given that the theoretical molar mass of PR domain calculated from its amino acid sequence alone is 22kD, our SAXS analysis indicates that the PR domain largely exists as a monomer in solution in agreement with our previous static light scattering measurements (23). In short, our SAXS analysis suggests that the PR domain is likely to adopt an extended random coil-like conformation in solution.…”

“…Additionally, a tryptophan (W) residue was added to both the N- and C-termini of the PR domain to aid in the quantification of protein concentration using spectrophotometry. The recombinant protein was expressed in Escherichia coli BL21* (DE3) bacterial strain and purified on a Ni-NTA affinity column followed by size-exclusion chromatography (SEC) on a Hiload Superdex 200 column using standard procedures as described previously (23). Final yield was typically between 5–10mg protein of apparent homogeneity, as judged by SDS-PAGE analysis (Figure 1c), per liter of bacterial culture.…”

Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands

“…These findings are in contrast to work by McDonald and coworkers, who showed that PxxPxR motifs were critical for the binding of GRB2 to the PRR or SOS1. However, these investigators used a truncated PRR for SOS1 that had an affinity of only 7 µM for GRB2 [33]. The SOS1 PRR used in this study bound to GRB2 with an affinity of 350 nM, which is nearly identical to the affinity of GRB2 for full length SOS1 [14].…”

Characterization of GRB2 and SOS1 binding downstream of TCR activation

“…Using ITC, they examined the binding of the proline-rich region of SOS1 to full-length Grb2. However, their construct lacked the 24 amino acids preceding Site 1 and the 19 amino acids following Site 4 (McDonald et al, 2012). They observed an affinity for the interaction of Grb2 with this construct of approximately 7 µM, or about 20-fold weaker than our observed Kd for SOS1WT or our mutant lacking all of the PXXPXR binding sites, SOS1X1234Z (Table 5) (McDonald et al, 2012).…”

“…However, their construct lacked the 24 amino acids preceding Site 1 and the 19 amino acids following Site 4 (McDonald et al, 2012). They observed an affinity for the interaction of Grb2 with this construct of approximately 7 µM, or about 20-fold weaker than our observed Kd for SOS1WT or our mutant lacking all of the PXXPXR binding sites, SOS1X1234Z (Table 5) (McDonald et al, 2012). This is excellent evidence in support of our hypothesis that the sequence surrounding these consensusbinding motifs is essential for the binding of these proteins.…”