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.
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March 2020
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Native electrospray ionization mass spectrometry revealed coronaviral polyprotein processing by the viral protease and subsequent complex formation. For further information, see the article by Kirchel and colleagues (pp. 1009–1019) in this issue. The image was in parts created by Anne Rupprecht (Rostock, Germany) and compiled by Boris Krichel. Image provided by Charlotte Uetrecht.
Research Article|
March 04 2020
Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity
Sunil Singh
;
Sunil Singh
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
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J. Sivaraman
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore
Correspondence: J. Sivaraman (dbsjayar@nus.edu.sg)
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Publisher: Portland Press Ltd
Received:
January 10 2020
Revision Received:
February 03 2020
Accepted:
February 10 2020
Accepted Manuscript online:
February 10 2020
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2020
Biochem J (2020) 477 (5): 905–923.
Article history
Received:
January 10 2020
Revision Received:
February 03 2020
Accepted:
February 10 2020
Accepted Manuscript online:
February 10 2020
Citation
Sunil Singh, J. Sivaraman; Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity. Biochem J 13 March 2020; 477 (5): 905–923. doi: https://doi.org/10.1042/BCJ20200027
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