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Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition

M. Mukherjee,Jing-Song Fan,S. Ramachandran,G. Guy,J. Sivaraman

Published 2014 in Journal of Biological Chemistry

ABSTRACT

Background: The novel phosphotyrosine-binding domain (HYB) of Hakai forms an atypical, zinc-coordinated homodimer. Results: C-terminal truncation of the HYB domain causes dramatic structural changes and it becomes a monomer, but in the presence of substrate, it becomes a dimer to recognize the substrate. Conclusion: HYB dimerization is a unique prerequisite for substrate-binding activity of Hakai. Significance: The HYB domain may orchestrate the function of Hakai in cancer and cell-cell contacts. 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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