Characterization of substrate binding of the WW domains in human WWP2 protein

WW domains harbor substrates containing proline‐rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif‐containing substrates only and does not recognize other amino acids and phospho‐residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class‐I WW domains for the first time. The results from alanine‐scanning mutagenesis and modeling further point to functionally conserved residues in WW2.