Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner

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.