title: 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(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.
authors: From the Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543 and.
authors: Manjeet
authors: M
authors: Mukherjee
authors: From the Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543 and.
authors: Fan
authors: F
authors: Jing-Song
authors: From the Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543 and.
authors: Sarath
authors: S
authors: Ramachandran
authors: the Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.
authors: Graeme R
authors: GR
authors: Guy
authors: From the Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543 and dbsjayar@nus.edu.sg.
authors: J
authors: J
authors: Sivaraman
mesh_terms: D001665:Binding Sites; D015820:Cadherins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004847:Epithelial Cells / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D015820:Cadherins; D008024:Ligands; D012996:Solutions; D019000:Phosphotyrosine; C471638:CBLL1 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: E3 Ubiquitin Ligase; Nuclear Magnetic Resonance (NMR); Phosphotyrosine; Structural Biology; Zinc Finger