The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2-RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health Grants R01-GM083897 (to A.F.) and R01-DK079217 (to Z.N. and R.I.A.), the Israeli Science Foundation Grant # 1331-08 (to R.I.A.), an award from the Israel Cancer Research Fund (to Z.S.), funds from the USylvester Braman Family Breast Cancer Institute (to A.F. and Z.N.), the Pennsylvania Breast Cancer Coalition Grants # 60707 and # 9200903 (to M.S.), and funds from the Geisinger Clinic (to M.S.). C.B.M. is a recipient of a postdoctoral fellowship from the National Institutes of Health (Award # T32-CA119929 ).
- WW domain chaperone
- WW domain engineering
- WW tandem domains
- WW-ligand thermodynamics