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

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

SUBMITTER: Mukherjee M

PROVIDER: S-EPMC4162166 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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

Mukherjee Manjeet M Jing-Song Fan F Ramachandran Sarath S Guy Graeme R GR Sivaraman J J

The Journal of biological chemistry 20140729 37

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 ...[more]

PMID: 25074933

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Dataset Information

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

Publications

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

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