Distinct Peptide Binding Specificities of Src Homology 3 (SH3) Protein Domains Can Be Determined by Modulation of Local Energetics across the Binding Interface

Maryna Gorelik,Alan R Davidson

doi:10.1074/jbc.m111.330753

Abstract

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Highlights

Two SH3 domains investigated are cross-reactive in peptide binding, yet display highly divergent specificities
We found that the Nbp2p SH3 (NbpSH3) and BemSH3b domains bound to all of these sites, some sites were bound with strong affinities, whereas other sites were bound with remarkably different affinities by the two domains [23]
We identified a residue within the BemSH3b domain (Lys14), which serves to prevent high affinity binding to certain sites (e.g. Bck1) and plays a general role in maintaining the BemSH3b domain specificity required for its optimal function [23]

Summary

Background

Two SH3 domains investigated are cross-reactive in peptide binding, yet display highly divergent specificities. The similarity in binding specificities of the NbpSH3 and BemSH3b domains was first detected when they were found to both bind the same PXXP-containing motif in Ste20p [20] To analyze this cross-reactivity further, we previously tested binding of the NbpSH3 and BemSH3b domains to peptides from six different yeast proteins that are either proven or likely biologically relevant target proteins for these domains (Ste20p, Cla4p, Pbs2p, Bck1p, Skm1p, and Boi2p). These peptides share a class I consensus sequence, ⌿XPXRXAPXXP (where ⌿ is a hydrophobic residue), which was derived for the NbpSH3 domain using phage display [22]. Our analysis allowed us to identify previously unrecognized sites in yeast proteins that are highly selective for the BemSH3b domain

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION