The dynamics of free and phosphopeptide-bound Grb2-SH2 reveals two dynamically independent subdomains and an encounter complex with fuzzy interactions

Karoline Sanches,Fabio C. L. Almeida,Fernando A. Melo,Icaro P. Caruso

doi:10.1038/s41598-020-70034-w

Abstract

The growth factor receptor-bound protein 2 (Grb2) is a key factor in the regulation of cell survival, proliferation, differentiation, and metabolism. In its structure, the central Src homology 2 (SH2) domain is flanked by two Src homology 3 (SH3). SH2 is the most important domain in the recognition of phosphotyrosines. Here, we present the first dynamical characterization of Grb2-SH2 domain in the free state and in the presence of phosphopeptide EpYINSQV at multiple timescales, which revealed valuable information to the understanding of phophotyrosine sensing mechanism. Grb2-SH2 presented two dynamically independent subdomains, subdomain I involved in pY recognition and subdomain II is the pY + 2 specificity pocket. Under semi-saturated concentrations of pY-pep we observed fuzzy interactions, which led to chemical exchange observed by NMR. This information was used to describe the encounter complex. The association with pY-pep is dynamic, involving fuzzy interactions and multiple conformations of pY-pep with negative and hydrophobic residues, creating an electrostatic-potential that drives the binding of pY-pep. The recognition face is wider than the binding site, with many residues beyond the central SH2 binding site participating in the association complex, which contribute to explain previously reported capability of Grb2 to recognize remote pY.

Highlights

Abbreviations Grb[2] Growth factor receptor-bound protein 2 SH2 Src homology 2 mitogen-activated protein kinases (MAPKs) Mitogen activated protein kinase NMR Nuclear magnetic resonance CPMG Carr–Purcell–Meiboom–Gill pulse train chemical shift perturbation (CSP) Chemical shift perturbation root mean square deviation (RMSD) Root mean square deviation root mean square fluctuation (RMSF) Root mean square fluctuation
This is the first description of the dynamics of growth factor receptor-bound protein 2 (Grb2)-SH2 domain in the presence and absence of phosphopeptide
We measured the 15N nuclear spin relaxation parameters R1, R2 and 1H-15N-heteronuclear NOE (Figure S1), which showed that Grb2-SH2 domain is mostly rigid, displaying only a few residues involved in thermal motion

Summary

Introduction

Abbreviations Grb[2] Growth factor receptor-bound protein 2 SH2 Src homology 2 MAPK Mitogen activated protein kinase NMR Nuclear magnetic resonance CPMG Carr–Purcell–Meiboom–Gill pulse train CSP Chemical shift perturbation RMSD Root mean square deviation RMSF Root mean square fluctuation. Control, proliferation, differentiation, and metabolism are mediated by a variety of well-orchestrated series of events In this context, the growth factor receptor-bound protein 2 (Grb2) has demonstrated to be a key factor in regulating many cellular events. The phosphopeptide binds to SH2 as an extended linear conformation at the domain surface that spans from one α-helix to the other An exception for this linear conformation is the interaction with the SH2 domain of Grb[2], in which the W121 (site II) is a barrier, hindering the pY + 3 interaction. The domain-swapped dimer is able to bind phosphopeptides with different affinities when compared to the monomeric state, sometimes with higher affinity and others with lower affinitiy[14,15], contributing as another degree of freedom for Grb[2] plasticity and ability of regulation. The only structure available of the full-length Grb[2] is not s wapped[17]

Methods

Results

Conclusion