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Bivalent binding drives the formation of the Grb2-Gab1 signaling complex in a noncooperative manner.

ABSTRACT: Although the growth factor receptor binder 2 (Grb2)-Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer-dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be structurally disordered, it nonetheless adopts a more or less compact conformation reminiscent of natively folded globular proteins. Importantly, the structurally flexible conformation of the PR domain appears to facilitate the binding of Gab1 to Grb2 with a 1:2 stoichiometry. More specifically, the formation of the Grb2-Gab1 signaling complex is driven via a bivalent interaction through the binding of the C-terminal homology 3 (cSH3) domain within each monomer of Grb2 homodimer to two distinct RXXK motifs, herein designated G1 and G2, located within the PR domain of Gab1. Strikingly, in spite of the key role of bivalency in driving this macromolecular assembly, the cSH3 domains bind to the G1 and G2 motifs in an independent manner with zero cooperativity. Taken together, our findings shed new light on the physicochemical forces driving the assembly of a key macromolecular signaling complex that is relevant to cellular health and disease.

SUBMITTER: McDonald CB

PROVIDER: S-EPMC3365654 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Bivalent binding drives the formation of the Grb2-Gab1 signaling complex in a noncooperative manner.

McDonald Caleb B CB Bhat Vikas V Mikles David C DC Deegan Brian J BJ Seldeen Kenneth L KL Farooq Amjad A

The FEBS journal 20120509 12

Although the growth factor receptor binder 2 (Grb2)-Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer-dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be stru ...[more]

PMID: 22536782

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Project description:Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment.

Dataset Information

Bivalent binding drives the formation of the Grb2-Gab1 signaling complex in a noncooperative manner.

Publications

Bivalent binding drives the formation of the Grb2-Gab1 signaling complex in a noncooperative manner.

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