WWOX-substrate communication and functionality: Tracking the cooperation between an unstable binding and a stable non-binding WW domain

WW domains occur predominantly as tandem repeats, and their target proteins often contain multiple PY motifs, establishing multiple domain-peptide combinations. The interplay of WW/peptide interactions is not always intuitive: As an example, WW domain-containing oxidoreductase (WWOX) contains a partially unfolded, binding WW1 domain and a non-binding, stable WW2 domain thought to act as chaperone.

To investigate the evolution of this special arrangement, we identified positions that deviate from the WW consensus: Beyond the previously reported WW2 Y85 mutation that abolishes substrate binding, we focus here on WW1 A35, predicted to reduce WW1 stability. Using the wt and a back to consensus mutant (WW1_A35V) we pinpointed details of structure and binding that characterize the WWOX WW1-WW2 tandem domain.

CD, NMR and ITC experiments revealed the interplay between WW2, WW1 and peptide substrate. While no defined orientation between the two domains is evident, specific regions are highlighted. The tandem domain binds with significantly increased affinity, and substrates containing two PP.Y motifs bind engaging both the WW1 and WW2 peptide-binding grooves. The mutant significantly increased the stability of WW1. While binding affinity of the mutant tandem domain is only marginally affected by this mutation, binding specificity changes, as revealed by selection experiments for peptide binders from a peptide library covering the human unstructured proteome.

Thus, using two point mutations, WWOX has evolved its specific character. Experimental phenotypic characterization of cellular location, as well as cellular proliferation is currently underway to elucidate the functional importance of the WWOX A35 deviations from consensus.