Biophysical studies and NMR structure of YAP2 WW domain - LATS1 PPxY motif complexes reveal the basis of their interaction.

YES-associated protein (YAP) is a major effector protein of the Hippo tumor suppressor pathway, and is phosphorylated by the serine/threonine kinase LATS. Their binding is mediated by the interaction between WW domains of YAP and PPxY motifs of LATS. Their isoforms, YAP2 and LATS1 contain two WW domains and two PPxY motifs respectively. Here, we report the study of the interaction of these domains both in vitro and in human cell lines, to better understand the mechanism of their binding. We show that there is a reciprocal binding preference of YAP2-WW1 with LATS1-PPxY2, and YAP2-WW2 with LATS1-PPxY1. We solved the NMR structures of these complexes and identified several conserved residues that play a critical role in binding. We further created a YAP2 mutant by swapping the WW domains, and found that YAP2 phosphorylation at S127 by LATS1 is not affected by the spatial configuration of its WW domains. This is likely because the region between the PPxY motifs of LATS1 is unstructured, even upon binding with its partner. Based on our observations, we propose possible models for the interaction between YAP2 and LATS1.

Dimeric switch of Hakai-truncated monomers during substrate recognition: insights from solution studies and NMR structure.

Hakai, an E3 ubiquitin ligase, disrupts cell-cell contacts in epithelial cells and is up-regulated in human colon and gastric adenocarcinomas. Hakai acts through its phosphotyrosine-binding (HYB) domain, which bears a dimeric fold that recognizes the phosphotyrosine motifs of E-cadherin, cortactin, DOK1, and other Src substrates. Unlike the monomeric nature of the SH2 and phosphotyrosine-binding domains, the architecture of the HYB domain consists of an atypical, zinc-coordinated tight homodimer. Here, we report a C-terminal truncation mutant of the HYB domain (HYB(ΔC)), comprising amino acids 106-194, which exists as a monomer in solution. The NMR structure revealed that this deletion mutant undergoes a dramatic structural change caused by a rearrangement of the atypical zinc-coordinated unit in the C terminus of the HYB domain to a C2H2-like zinc finger in HYB(ΔC). Moreover, using isothermal titration calorimetry, we show that dimerization of HYB(ΔC) can be induced using a phosphotyrosine substrate peptide. This ligand-induced dimerization of HYB(ΔC) is further validated using analytical ultracentrifugation, size-exclusion chromatography, NMR relaxation studies, dynamic light scattering, and circular dichroism experiments. Overall, these observations suggest that the dimeric architecture of the HYB domain is essential for the phosphotyrosine-binding property of Hakai.

NMR structure of Carcinoscorpius rotundicauda thioredoxin-related protein 16 and its role in regulating transcription factor NF-κB activity.

Thioredoxins (Trxs), which play a key role in maintaining a redox environment in the cell, are found in almost all organisms. Trxs act as potential reducing agents of disulfide bonds and contain two vicinal cysteines in a CXXC motif at the active site. Trx is also known to activate the DNA binding activity of NF-κB, an important transcription factor. Previously, Trx-related protein 16 from Carcinoscorpius rotundicauda (Cr-TRP16), a 16-kDa Trx-like protein that contains a WCPPC motif, was reported. Here we present the NMR structure of the reduced form of Cr-TRP16, along with its regulation of NF-κB activity. Unlike other 16-kDa Trx-like proteins, Cr-TRP16 contains an additional Cys residue (Cys-15, at the N terminus), through which it forms a homodimer. Moreover, we have explored the molecular basis of Cr-TRP16-mediated activation of NF-κB and showed that Cr-TRP16 exists as a dimer under physiological conditions, and only the dimeric form binds to NF-κB and enhances its DNA binding activity by directly reducing the cysteines in the DNA-binding motif of NF-κB. The C15S mutant of Cr-TRP16 was unable to dimerize and hence does not bind to NF-κB. Based on our finding and combined with the literature, we propose a model of how Cr-TRP16 is likely to bind to NF-κB. These findings elucidate the molecular mechanism by which NF-κB activation is regulated through Cr-TRP16.