title: 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 the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the 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.
authors.affiliation: Department of Biochemistry and Molecular Biology and USylvester Braman Family Breast Cancer Institute, Leonard Miller School of Medicine, University of Miami, Miami, Florida 33136, United States.
authors.forename: Caleb B
authors.initials: CB
authors.lastname: McDonald
authors.forename: Jordan E
authors.initials: JE
authors.lastname: Balke
authors.forename: Vikas
authors.initials: V
authors.lastname: Bhat
authors.forename: David C
authors.initials: DC
authors.lastname: Mikles
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Deegan
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Seldeen
authors.forename: Amjad
authors.initials: A
authors.lastname: Farooq
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055459:Facilitated Diffusion; D051380:GRB2 Adaptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020664:Son of Sevenless Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; C485509:SOS2 protein, human; D020664:Son of Sevenless Proteins