Molecular insight into the systematic affinity and selectivity of partner recognition sites between the WW1 and WW2 domains of human KIBRA neuroprotein.

Human KIBRA, a member of the WWC family proteins, is an upstream regulator of the Salvador/Warts/Hippo (SWH) signaling pathway and predominately expressed in nervous system. The protein has two functionally regulatory domains WW1 and WW2 at N-terminal region, which recognize and bind to the PY-motif segments of their partner proteins to serve as a signaling scaffold role in the SWH pathway. The two domains are highly conserved, but their downstream ligands and biological functions may not be fully consistent. In this study, we attempted to systematically profile the PY-motif affinity to and selectivity between KIBRA WW1 and WW2 domains involved in partner recognition sites. Ontology mining was used to enrich the KIBRA-interacting proteins in literature libraries, from which a variety of PY-motif peptide segments were identified, and their binding behavior to each domain was then analyzed by integrating computational modeling and experimental assay. Most PY-motif peptides were found to interact potently with WW1 and WW2, but they generally only exhibit a moderate or modest selectivity between the two domains. Subsequently, several representative peptides were further examined in detail to elucidate the molecular mechanism underlying their affinity and selectivity. It is revealed that the middle motif region of PY-motif peptides is primarily responsible for the affinity and stability of peptide binding, but only contributes marginally to peptide selectivity. Instead, the N-terminal region and, particularly, C-terminal region of PY-motif peptides play a crucial role in the selectivity. Hydrophobic contacts and hydrogen bonds confer stability and specificity to the domain-peptide interaction, respectively.