Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

I 类 WW 结构域存在于许多具有各种功能的蛋白质中，并通过与短的线性 PPxY 基序结合来介导蛋白质相互作用。串联 WW 结构域通常与具有多个 PPxY 基序的肽结合，但 WW-肽相互作用的相互作用并不总是直观的。含有 WW 结构域的氧化还原酶 (WWOX) 包含两个 WW 结构域：能够结合 PPxY 的不稳定 WW1 和不能结合 PPxY 的稳定 WW2。WW2 域已被建议充当 WW1 域伴侣，但其伴侣活性的潜在机制仍有待揭示。在这里，我们结合 NMR、等温量热法和结构建模来阐明两个 WW 结构域在 WWOX 与其含有 PPxY 的底物 ErbB4 结合中的作用。使用核磁共振，我们确定了这两个结构域之间的相互作用表面，该表面支持与肽底物结合兼容的 WWOX 构象。等温量热法和 NMR 测量还表明，虽然与单个 PPxY 基序的结合亲和力在 WW2 存在下略有增加，但对双基序肽的亲和力增加了 10 倍。此外，我们发现 WW2 可以使用其规范结合位点直接结合双基序肽。最后，诱变实验中肽的差异结合与与 WW1-WW2 串联结构域结合时平行的 N 到 C 端 PPxY 串联基序方向一致，验证了相互作用的结构模型。总之，我们的结果揭示了串联 WW 域组织和底物结合的复杂性，