Loops Govern SH2 Domain Specificity by Controlling Access to Binding Pockets

Abstract

Cellular functions require specific protein-protein interactions that are often mediated by modular domains that use binding pockets to engage particular sequence motifs in their partners. Yet, how different members of a domain family select for distinct sequence motifs is not fully understood. The human genome encodes 120 Src homology 2 (SH2) domains (in 110 proteins), which mediate protein-protein interactions by binding to proteins with diverse phosphotyrosine (pTyr)-containing sequences. The structure of the SH2 domain of BRDG1 bound to a peptide revealed a binding pocket that was blocked by a loop residue in most other SH2 domains. Analysis of 63 SH2 domain structures suggested that the SH2 domains contain three binding pockets, which exhibit selectivity for the three positions after the pTyr in a peptide, and that SH2 domain loops defined the accessibility and shape of these pockets. Despite sequence variability in the loops, we identified conserved structural features in the loops of SH2 domains responsible for controlling access to these surface pockets. We engineered new loops in an SH2 domain that altered specificity as predicted. Thus, selective blockage of binding subsites or pockets by surface loops provides a molecular basis by which the diverse modes of ligand recognition by the SH2 domain may have evolved and provides a framework for engineering SH2 domains and designing SH2-specific inhibitors.

Mechanism of Phosphorylation-induced Activation of Phospholipase C-γ Isozymes

“…2 and 3). Similar to many SH2 domains, the BG loop of the cSH2 domain of PLC-␥1 forms part of the groove needed to bind phosphotyrosine-containing peptides (31). Perhaps not surprisingly then, the BG loop of the cSH2 domain of PLC-␥1 alters its conformation upon peptide engagement, and different peptides stabilize distinct conformations of the BG loop (23,32,33).…”

Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav

“…Besides Syk and SLP-76, Vav1 SH2 has been shown to have the ability to associate with the phosphorylated regions from several other proteins, such as ZAP-70, 3BP2, c-Cbl, CD19, HS1, BLNK, and SHP-1 (7,10,24,32,33,38,39,46). The phosphopeptide library screening results also indicate binding by diverse sequences, and the importance of the interaction between the EF and BG loops and the pTyr ϩ3 residue (30,36). Conformational flexibility of the critical regions can facilitate Vav1 SH2 recognition of these different targets, as bound-state conformations are populated to greater extent in the unligated state.…”

Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

“…2A). We provide the first crystal structure of the Arg SH2 domain and show that Leu 207 is located near the P ϩ 1 binding pocket and Thr 233 is on the EF loop previously shown to mediate access to the P ϩ 3 binding pocket (18). Finally, we show that although these residues do not affect Arg localization to the cell periphery, they are critical for mediating actin-based cell edge protrusion in fibroblasts. )…”

“…In particular, three residues C-terminal to the phosphotyrosine determine SH2 domain binding specificity (16,17). Within the SH2 domain itself, there are three binding pockets that confer specificity for each of these binding target residues as well as several loops, which govern binding pocket accessibility (18,19).…”