The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

Qi Zhang, Zhongduo Wang, Ming-Feng Hou, Rachel Harding, Xinyi Huang, Aiping Dong, John R. Walker, Yufeng Tong

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13 Citations (Scopus)


Background 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. Methods 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. Results 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. Conclusions 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. General significance 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.

Original languageEnglish
Pages (from-to)3095-3105
Number of pages11
JournalBiochimica et Biophysica Acta - General Subjects
Issue number1
Publication statusPublished - Jan 2017
Externally publishedYes


  • Crystallography
  • Deubiquitinase
  • E3 Ubiquitin ligase
  • Menadione
  • Protein-protein interaction
  • Small molecule inhibitors

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