title: The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear.
abstract: Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically.
  
  
  
  We used X-ray crystallography, peptide array, bioinformatic analysis, and biophysical techniques to characterize the structure and interaction of SIAHs with deubiquitinases and literature reported compounds.
  
  
  
  We solved the crystal structures of SIAH1 in complex with a USP19 peptide and of the apo form SIAH2. Phylogenetic analysis revealed the SIAH/USP19 complex is conserved in evolution. We demonstrated that menadione destabilizes both SIAH1 and SIAH2 non-specifically through covalent modification.
  
  
  
  The SBDs of SIAH E3 ligases are structurally similar with a subtle stability difference. USP19 is the only deubiquitinase that directly binds to SIAHs through the substrate binding pocket. Menadione is not a specific inhibitor for SIAH2.
  
  
  
  The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: Qi
authors: Q
authors: Zhang
authors: Fisheries College, Guangdong Ocean University, Zhanjiang, Guangdong 524025, China.
authors: Zhongduo
authors: Z
authors: Wang
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: Feng
authors: F
authors: Hou
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: Rachel
authors: R
authors: Harding
authors: Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: Xinyi
authors: X
authors: Huang
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: Aiping
authors: A
authors: Dong
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
authors: John R
authors: JR
authors: Walker
authors: Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5G 1L7, Canada. Electronic address: yufeng.tong@utoronto.ca.
authors: Yufeng
authors: Y
authors: Tong
mesh_terms: D000595:Amino Acid Sequence; D063646:Carcinogenesis / Q000378:metabolism / Q000473:pathology; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010802:Phylogeny; D011485:Protein Binding; D000072417:Protein Domains; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D000090063:Proto-Oncogene Mas; D013379:Substrate Specificity / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D024483:Vitamin K 3 / Q000494:pharmacology; D000098563:Seven in Absentia Proteins
chemical_list: C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D024483:Vitamin K 3; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D010450:Endopeptidases; C559917:USP19 protein, human
keywords: Crystallography; Deubiquitinase; E3 Ubiquitin ligase; Menadione; Protein-protein interaction; Small molecule inhibitors