Intricate communication between WW domains and PPxY motifs in WWOX-substrate association

WW domains predominantly occur as tandem repeats, and their target proteins often contain multiple PY motifs, thus allowing for the establishment of multiple domain-peptide combinations. In WW domain-containing oxidoreductase (WWOX) protein the interplay of WW/peptide interactions is not always intuitive, where the partially unfolded WW1 is capable of PY motif binding and the stable WW2 domain is not due to Trp mutation to Tyr in residue 85. We have used binding experiments, and structural characterization to elucidate the interplay between WW2, WW1 and peptide substrate. ITC binding affinity measurements to ErbB4-derived peptides reveal that while binding affinity to a single motif is marginally increased in the presence of WW2 (2x), binding affinity to a double PY motif increases substantially (20x). NMR experiments on single and tandem WWOX WW domains bound to single and double motifs allow us to follow the rearrangement of the tandem domain upon peptide binding. They reveal that WW2 is directly involved in the binding of double motif-peptides using the canonical binding site. NMR also showed that WW1 is much stable in the tandem domain context and due to peptide binding. A mutation in Ala 35 to Val, back to consensus, adopt a stable conformation. Overall, our results highlight the contribution of WW2 to both stability and binding of WWOX, and map out an unusual way of evolution of complex functionality within a tandem repeat context with two mutations. Studying the difference in binding specificity of the wt tandem domain and the A35V, is now under investigation.