Manjeet Mukherjee, Fan Jing-Song, Sarath Ramachandran, Graeme R. Guy, and J. Sivaraman Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition: INSIGHTS FROM SOLUTION STUDIES AND NMR STRUCTURE J. Biol. Chem. 2014 289: 25611-25623. First Published on July 29, 2014, doi:10.1074/jbc.M114.592840
Abstract:
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 (HYBΔC), 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 HYBΔC. Moreover, using isothermal titration calorimetry, we show that dimerization of HYBΔC can be induced using a phosphotyrosine substrate peptide. This ligand-induced dimerization of HYBΔC 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.
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