In vitro evolution of recognition specificity mediated by SH3 domains reveals target recognition rules.
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, APTYPPPLPP 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.
Panni
Simona
S
Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, Rome 00133, Italy.
Dente
Luciana
L
Cesareni
Gianni
G
D000465:Algorithms; D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D019143:Evolution, Molecular; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D018909:src Homology Domains
D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins