Master of Science (MS)
Biochemistry and Molecular Biology (Medicine)
Date of Defense
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
WW-domain containing proteins such as the antagonistic pair, YAP2 transcriptional regulator and WWOX tumor suppressor, mediate a multitude of homeostatic cellular processes including cell growth, proliferation, and apoptosis through specific interactions with shared PPXY-containing cognate ligands, which help transmit signals from the plasma membrane through the cytosol into the nucleus. Although there are numerous cell and mouse studies describing the signaling cascade events of such protein-protein interactions, the molecular basis of WW-PPXY interactions has yet to be explored. In addition, the ability of WW domains to act alone or in tandem, such as those found in YAP2 and WWOX, may uncover additional mechanisms of communication between WW domains. Therefore, we provide here a detailed biophysical analysis of the binding of WW domains of YAP2 and WWOX, acting alone and in the context of tandem domains, to shared PPXY-containing cognate ligands to uncover the molecular basis for the antagonistic action of these two proteins. Our data show that while both the WW1 and WW2 domains of YAP2 bind PPXY motifs of the ErbB4 receptor tyrosine kinase in a physiologically relevant matter, only the WW1 domain of WWOX is able to bind ErbB4. While replacement of consensus residues in the PPXY sequence eliminates binding of WW domains to ErbB4, non-consensus residues are not essential for WW-PPXY binding integrity. The lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX. Interdomain communication between tandem WW domains of YAP2 and WWOX was explored using a myriad of shared cognate ligands. Our data suggest that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands due to unfavorable entropic contribution to the overall free energy of binding relative to isolated WW domains. YAP2 WW tandem domains also adopt a fixed spatial orientation such that the WW1 domain curves outward and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both domains. In contrast, tandem WW domains of WWOX demonstrate binding with enhanced affinity via allosteric communication such that the physical association of WW2 domain with WW1 blocks access to ligand. Consequently, ligand binding to WWOX WW1 domain not only results in the displacement of WW2 lid but also disrupts the WW domain interaction in the liganded conformation. Overall, these studies explore WW-PPXY interactions and uncover novel modes of communication between the tandem WW domains of YAP2 transcriptional regulator and WWOX tumor suppressor to describe the biophysical basis of their cellular antagonism.
YAP2; WWOX; biophysics; WW domain; tandem; thermodynamics
Schuchardt, Brett J., "Biophysical Basis of the Antagonistic Action of YAP Transcriptional Regulator and WWOX Tumor Suppressor" (2015). Open Access Theses. 543.