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

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. Abbreviations Grb2 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 Cell survival, 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. Grb2 is not an enzyme, instead, its multiple domains and flexible linkers provide the ability to bind to multiple partners in the cell. Grb2 is a pivotal intermediate in the

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding

“…24 This domain is represented by the residues between 59 and 153 of the sequence and folds into a central β-sheet with five antiparallel β-strands surrounded by two α-helices, one on each side. 25 Even though the domains are highly conserved in function, each GRB2 SH3 domain plays a different role in regulating early signaling complexes and signaling downstream. The N-SH3 domain establishes the primary protein−protein interaction with SOS1, a cytoplasmic Guanine Exchange Factor (GEF) protein that promotes RAS activation and, consequently, the MAPK signaling pathway downstream.…”

Structural dynamics of the N‐terminal SH2 domain of PI3K in its free and CD28‐bound states

“…In contrast to the BG loop, the BC loop conformation has rarely been studied [25,27,28]. In the crystal structure of the nSH2-pY_CD28 complex [3], the BC loop was located adjacent to the pY of the bound pY_CD28 peptide.…”

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect

“…The docking technique was not capable of supplying direct information on the dynamics of the peptide, but it suggested a molecular conformation that favors the stabilization of a helix-like conformation based on the possibility of forming hydrophobic interactions driven by residues Trp191, Ile195, and Leu198 in HcTnI-C27. Hydrophobic surfaces are important in forming encounter complexes ( 45 ), or recently for the complex between a protein and a peptide ( 46 ). Accordingly, our docking findings suggest that the Arg192His mutation in HcTnI-C27-H peptide would disrupt the key binding region on the peptide surface, disfavoring the association with tropomyosin.…”