Biophysical Basis of the Binding of WWOX Tumor Suppressor to WBP1 and WBP2 Adaptors

Abstract

The WWOX tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WBP1 and 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 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, introduction of E66R/Y85W double-substitution within the WW2 domain not only results 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.

Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks

“…Intriguingly, complexes pulled down via GST-WW1,2 were much less than those by GST-WW1 alone, suggesting that WW2 might have an inhibitory effect on the interaction ability of WWOX. In agreement with these observations, McDonald et al (37) FIGURE 6. ITCH mediates Lys-63-linked polyubiquitination of WWOX.…”

“…This impaired ability was suggested to be due to the presence of Glu-66/Tyr-85 residues within the WW2 domain binding pocket as compared with Arg-25/Trp-44 within the WW1 domain. Introduction of an E66R/Y85W double substitution within the WW2 domain of WWOX results in gain of function and restores binding to WBP1 and WBP2 (37). Furthermore, the Y85W substitution within the WW2 domain restores its binding to PPXY motifs of ErbB4 in a manner very similar to the binding of the WW1 domain of WWOX.…”

WWOX: A fragile tumor suppressor

“…Initially, Wwox was thought to belong to Group I after demonstrating binding of proteins harboring PP x Y motifs (where P is proline, Y is tyrosine and x is any amino acid) through its first WW domain. 4,5,6 However, a recent study by Abu-Odeh et al employed an MS-based screen which confirmed previous PP x Y protein interactions, but also demonstrated that the majority of Wwox interacting proteins did not contain PY motifs. 7 Rather, these proteins exhibited PP x F or LP x F motifs (where F is phenylalanine and L is leucine), suggesting that perhaps WW1 domain of WWOX binds non-canonical proline-rich motifs.…”

“…5,6,7 After showing that the second WW domain, WW2, contains two distinct amino acid residues within the WW binding pocket, compared to WW1, McDonald et al proposed that the WW2 domain serves as a chaperone for augmenting physiological binding of WW1. 4 …”

Roles of the WWOX in pathogenesis and endocrine therapy of breast cancer

“…WBP2 can activate the ER transactivation pathway and promote cell proliferation. WWOX physically interacts with WBP2 and suppresses ER transactivation pathways 48,49 ( Figure 1). Thus, loss of WWOX expression can result in tamoxifen resistance by up-regulating ER and Her2 transcriptional activities.…”