In Vitro Evolution of Recognition Specificity Mediated by SH3 Domains Reveals Target Recognition Rules

Abstract

We have designed a repertoire of 10 7 different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain. This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPP-PLPP and LSSRPLPTLPSP, which are peptide ligands for the human Abl or Src SH3 domains, respectively. By characterizing the isolates, we have observed that as few as two or three amino acid substitutions lead to dramatic changes in recognition specificity. We propose that the ability to shift recognition specificity with a small number of amino acid replacements is an important evolutionary characteristic of protein binding modules. Furthermore, we have used the information obtained by these in vitro evolution experiments to generate a scoring matrix that evaluates the probability that any SH3 domain binds to the peptide ligands for the Abl and Src SH3 domains. A table of predictions for the 28 SH3 domains of baker's yeast is presented.

Src kinase activation by direct interaction with the integrin β cytoplasmic domain

“…When added to CHO cell lysates, LSSRPLPTLPSP and KGGRSLRPLPPLP-PPG blocked interaction of ␣IIb␤3 with GST-c-Src SH3 (Fig. 3F), but KGELRLRNYYYDVV or a polyproline peptide selective for c-Abl SH3 (APTYPPPLPP) (20) did not. Thus, despite the absence of a polyproline sequence in the ␤3 tail (Fig.…”

“…3D). Interaction between GST-c-Src SH3 and the ␤3 tail was blocked by a 14-aa peptide from the carboxyl terminus of ␤3 (␤3 748-762) and by optimized polyproline peptides selective for c-Src SH3 (LSSRPLPTLPSP) (20) or Src family SH3 domains (KGGRSLRPLPPLPPPG) (ref. 14; see also Fig.…”

Computational Tools and Databases for the Study and Characterization of Protein Interactions

“…Firstly, accurate predictions require high sequence similarities between interologs (~70%) (Mika and Rost 2006) thus limiting its range of applicability. Secondly, even at high sequence identity level, in some cases small variations in protein sequence at the interface have been shown to dramatically change PPI specificity, thus redefining complex protein networks and leading to important phenotypic differences (Panni et al 2002;Kiemer and Cesareni 2007).…”

Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains

“…Degenerate DNA sequences encoding the designed HLH and bHLHZip domain repertoires were synthesized by PCR and cloned in the display vector D4 as fusions to the D capsid protein C terminus (8). Following in vitro packaging, ϳ2 ϫ 10 6 and ϳ1 ϫ 10 6 pfu were obtained for the HLH and bHLHZip libraries, respectively.…”

“…The DNA sequence encoding Max bHLHZip was cloned into the three filamentous phage vectors pC89, pC178, and pHEN⌬, to obtain N-terminal fusions to pVIII or pIII coat proteins (14,15) and into the display vector 4 (D4) to display fusions to the D-protein C terminus (8,16). We asked which vector would efficiently display Max bHLHZip and allow its binding to a natural dimerization partner, the GST fusion protein Mad (2).…”