An intramolecular energetic network regulates ligand recognition in a SH2 domain

Abstract

In an effort to investigate the molecular determinants of ligand recognition of the C‐terminal SH2 domain of the SHP2 protein, we conducted extensive site‐directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in‐depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site‐directed variants of C‐SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C‐SH2 binding specificity. Results highlighted non‐optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C‐SH2 domain.

Folding and Binding Kinetics of the Tandem of SH2 Domains from SHP2

“…The possibility of subtle interdomain communication between the SH2 domains of SHP2 protein might be of key importance in regulating the activation of the enzyme [41,47], the NSH2 domain being reported to be a highly dynamic system in isolation [48], and in tandem with the CSH2 domain [32], where the intrinsic structural plasticity of the construct has been proposed to be fundamental for the function of Shp2. In analogy to what has been described for other isolated SH2 domains [43,49], this phenomenon might be due to underlying energetic networks driving domain functions.…”

“…The binding mechanism of the isolated NSH2 and CSH2 domains has been extensively characterized in the recent past. In particular, our group, through a combination of site-directed mutagenesis and kinetic binding experiments, investigated the binding of the two domains with peptides mimicking different portions of Gab2 protein (Grb2 associated binder) [ 33 , 34 , 43 ]. Here, we resorted to comparing the binding properties of the NSH2-CSH2 tandem of SHP2 with those described for the isolated domains, in order to pinpoint possible changes in affinity and kinetics in the context of the tandem construct.…”

Addressing the Binding Mechanism of the Meprin and TRAF-C Homology Domain of the Speckle-Type POZ Protein Using Protein Engineering

“…This finding is of particular importance, given that the SPOP MATH shows negligible conformational changes upon binding, and residues that are critical to stabilize the complex cannot be necessarily deduced from structural studies only. Notably, these kinds of sparse energetic networks have been previously observed on other protein-protein interaction domains, such as SH2 [26], and PDZ [27] domains, indicating that they may likely represent a general property of this class of proteins. Moreover, the analysis of the LFER plots reveal that, whilst both ligands conform to a linear dependence, there is a detectable change in the contribution of mutations to k on and k off to the stability of the complex with the two different peptides.…”