Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity.

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  • Author(s): Singh S;Singh S; Sivaraman J; Sivaraman J
  • Source:
    The Biochemical journal [Biochem J] 2020 Mar 13; Vol. 477 (5), pp. 905-923.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Published by Portland Press on behalf of the Biochemical Society Country of Publication: England NLM ID: 2984726R Publication Model: Print Cited Medium: Internet ISSN: 1470-8728 (Electronic) Linking ISSN: 02646021 NLM ISO Abbreviation: Biochem J Subsets: MEDLINE
    • Publication Information:
      Original Publication: London, UK : Published by Portland Press on behalf of the Biochemical Society
    • Subject Terms:
      Protein Domains/*physiology ; Ubiquitin-Protein Ligases/*chemistry ; Ubiquitin-Protein Ligases/*metabolism ; Ubiquitination/*physiology; Crystallization/methods ; Humans ; Protein Structure, Secondary ; Ubiquitin-Protein Ligases/genetics
    • Abstract:
      The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the 'other'. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the 'other' subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744-1083) with an additional fifty N-terminal amino acids (aa 693-743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693-743) preceding the UBE3C HECT domain as well as a loop region (aa 758-762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2-E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.
      (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
    • Contributed Indexing:
      Keywords: E3-ubiquitin ligase; HECT type domain; autoubiquitination; cancer; protein degradation; structure
    • Accession Number:
      EC 2.3.2.26 (UBE3C protein, human)
      EC 2.3.2.27 (Ubiquitin-Protein Ligases)
    • Publication Date:
      Date Created: 20200211 Date Completed: 20201021 Latest Revision: 20201021
    • Publication Date:
      20231215
    • Accession Number:
      10.1042/BCJ20200027
    • Accession Number:
      32039437