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,...

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Bibliographic Details
Published in:The Journal of biological chemistry 2014-09, Vol.289 (37), p.25611-25623
Main Authors: Mukherjee, Manjeet, Jing-Song, Fan, Ramachandran, Sarath, Guy, Graeme R, Sivaraman, J
Format: Article
Language:English
Subjects:
Binding Sites
Cadherins - chemistry
Cadherins - metabolism
Crystallography, X-Ray
Epithelial Cells - chemistry
Epithelial Cells - metabolism
Humans
Ligands
Magnetic Resonance Spectroscopy
Phosphotyrosine - chemistry
Protein Binding
Protein Conformation
Protein Structure and Folding
Solutions - chemistry
src Homology Domains
Ubiquitin-Protein Ligases - chemistry
Ubiquitin-Protein Ligases - metabolism
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M114.592840