Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch

Abstract

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to WW1 domain in the context of WW1-WW2 tandem module of WWOX tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of WW1-WW2 tandem module to an array of putative PPXY ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically-relevant manner, the WW2 domain does not. Moreover, ligand binding to WW1 domain in the context of WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of WW2 domain with WW1 blocks access to ligand. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of WW2 orphan domain to chaperone physiological action of WW1 domain within the context of the WW1-WW2 tandem module of WWOX.

Tandem WW/PPxY motif interactions in WWOX: the multifaceted role of the second WW domain

“…Beyond the main model presented in Figure 7 however, WWOX is functional as a dynamic ensemble of different conformations, as suggested from our NMR experiments, as well from previous studies (25,36). While the models generated by AlphaFold2 converge, models that we generated using additional protocols (including Rosetta ab initio folding with GREMLIN constraints (37), TrRosetta (38) and RoseTTAFold (39), data not shown) exhibit a range of additional possible relative orientations between WW1 and WW2, with predominantly similar conformation of the individual WW domains, but a wide variability for the linker and the tails.…”

“…Previous studies have reported no detectable binding of isolated WW2 to WWOX-binding peptides derived from different proteins in vitro and in cells (17,18,21,22,25,32). Indeed, we too find that WW2 will not bind a single PPxY ligand independently, and that in tandem it is outcompeted by WW1.…”

“…Of note, our model differs from a model suggested in a previous study, which was generated using molecular dynamics simulations starting from the template structure of FBP21 (25,36). That model suggested that the WW1 peptide-binding site is occupied by the WW2 domain and consequently, binding of peptide substrates would involve major domain rearrangement to allow access of the peptide to the binding site.…”

“…WWOX contains two WW domains (residues 16-49 and 57-91, respectively), and an SDR domain (residues 122-383; domain position annotations according to InterPro (24)). Isolated WW1 is unstable (dashed lines) but binds to PPxY motifs (filled crescent), while WW2 is stable (solid lines), but was not reported to bind to PPxY motifs with measurable affinity (dashed crescent(25)). WWOX partner ErbB4 is cleaved upon maturation, releasing the C-terminal cytoplasmic region (residues 676-1308) that contains a kinase domain (positions 718-974) followed by an extended disordered region (gray curve) that contains several recognition motifs, including the three WWOX binding PPxY motifs investigated here (PY1 1032-1035, PY2 1053-1056 and PY3 1298-1301; black ovals), and several phosphorylated tyrosine residues (diamonds).…”

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

“…WWOX contains two WW domains (residues 16–50 and 57–91, respectively) and an SDR domain (residues 122–383; domain position annotations according to InterPro ( 52 )). Isolated WW1 is unstable ( dashed lines ) but binds to PPxY motifs ( filled crescent ), whereas WW2 is stable ( solid lines ) but was not reported to bind to PPxY motifs with measurable affinity ( dashed crescent ( 32 )). WWOX partner ErbB4 is cleaved upon maturation, releasing the C-terminal cytoplasmic region (residues 676–1308) that contains a kinase domain (positions 718–974) followed by an extended disordered region ( gray curve ) that contains several recognition motifs, including the three WWOX-binding PPxY motifs investigated here (PY1 1032–1035, PY2 1053–1056, and PY3 1298–1301; black ovals ).…”