USING MOLECULAR REPERTOIRES TO IDENTIFY HIGH-AFFINITY PEPTIDE LIGANDSOF THE WW DOMAIN OF HUMAN AND MOUSE YAP

The WW domain is a globular protein domain that is involved in mediati ng protein-protein interaction and that ultimately participates in var ious intracellular signaling events. The domain binds to polyproline l igands containing the xPPxY consensus (where x signifies any amino aci d, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associa ted Protein (YAP) and its WBP-1 ligand. To further characterize this m olecular interaction, we used two independent approaches, both of whic h focused on the mutational analysis of the WBP-1 ligand, We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids, In addition, we screened decamer repertoires wi th all permutations of the amino acids which individually increased th e binding to the WW domain of YAP, as compared to the wild type, In a parallel approach, we used a phage-displayed combinatorial peptide lib rary biased for the presence of two consecutive prolines to study liga nd preferences for the WW domain of YAP, Interestingly, these two line s of investigation converged and yielded the core sequence PPPPYP, whi ch is preferred by the YAP-WW domain, This sequence was found within t he p53 (tumor suppressor) binding protein-2, a probable cognate or alt ernative ligand interacting with YAP.