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Publication Abstract from PubMed

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(DeltaC)), 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(DeltaC). Moreover, using isothermal titration calorimetry, we show that dimerization of HYB(DeltaC) can be induced using a phosphotyrosine substrate peptide. This ligand-induced dimerization of HYB(DeltaC) 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.

Dimeric switch of Hakai-truncated monomers during substrate recognition: insights from solution studies and NMR structure.,Mukherjee M, Jing-Song F, Ramachandran S, Guy GR, Sivaraman J J Biol Chem. 2014 Sep 12;289(37):25611-23. doi: 10.1074/jbc.M114.592840. Epub, 2014 Jul 29. PMID:25074933^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.