Systematic profiling and identification of the peptide-mediated interactions between human Yes-associated protein and its partners in esophageal cancer.

Human Yes-associated protein (YAP) is involved in the Hippo signaling pathway and serves as a coactivator to modulate gene expression, which contains a transactivation domain (TD) responsible for binding to the downstream TEA domain family (TEAD) of transcription factors and two WW1/2 domains that recognize the proline-rich motifs (PRMs) present in a variety of upstream protein partners through peptide-mediated interactions (PMIs). The downstream YAP TD-TEAD interactions are closely associated with gastric cancer, and a number of therapeutic agents have been developed to target the interactions. In contrast, the upstream YAP WW1/2-partner interactions are thought to be involved in esophageal cancer but still remain largely unexplored. Here, we attempted to elucidate the complicated PMIs between the YAP WW1/2 domains and various PRMs of YAP-interacting proteins. A total of 106 peptide segments carrying the class I WW-binding motif [P/L]Px[Y/P] were extracted from 22 partner candidates, which are potential recognition sites of YAP WW1/2 domains. Structural and energetic analyses of the intermolecular interactions between the domains and peptides created a systematic domain-peptide binding profile, from which a number of biologically functional PMIs were identified and then substantiated in vitro using fluorescence spectroscopy assays. It is revealed that: (a) The sequence requirement for the partner recognition site binding to YAP WW1/2 domains is a decapeptide segment that contains a core PRM motif as well as two three-residue extensions from each side of the motif; the core motif and extended sections are responsible for the binding stability and recognition specificity of domain-peptide interaction, respectively. (b) There is an exquisite difference in the recognition specificity of the two domains; the LPxP and PPxP appear to more prefer WW1 than WW2, whereas the WW2 can bind more effectively to LPxY and PPxY than WW1. (c) WW2 generally exhibits a higher affinity to the panel of recognition site candidates than WW1. In addition, a number of partner peptides were found as promising recognition sites of the two domains and/or to have a good selectivity between the two domains. For example, the DVL1 peptide was determined to have moderate affinity to WW2 and strong selectivity for WW2 over WW1. Hydrogen bonds play a central role in selectivity.