Insights into human Lck SH3 domain binding specificity: Different binding modes of artificial and native ligands

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lek SH3 ligands using phage display. In addition, we analyzed Lek SH3 binding sites within known natural Lek SH3 binding proteins using an Lek specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lek SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lek SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands- which are not due to significant K-D value differences as shown by calculating Lek SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lek S143 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions.