Structural insights into a HECT-type E3 ligase AREL1 and its ubiquitination activities in vitro

Singh, Sunil; Ng, Joel; Nayak, Digant; Sivaraman, J.

doi:10.1074/jbc.RA119.010327

Structural insights into a HECT-type E3 ligase AREL1 and its ubiquitination activities in vitro

Journal Article · Fri Nov 15 00:00:00 EST 2019 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.RA119.010327· OSTI ID:1591895

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National Univ. of Singapore (Singapore)

The HECT E3 ligase family comprises three subfamilies: NEDD4 E3 ubiquitin protein ligase (NEDD4), HECT and RLD domain–containing E3 ubiquitin protein ligase (HERC), and "other.” Most previous studies have focused on the NEDD4 subfamily. Apoptosis-resistant E3 ligase 1 (AREL1) belongs to “other” subfamily HECT that inhibits apoptosis by ubiquitinating and degrading proapoptotic proteins. In this paper, we report the crystal structure of the extended HECT domain of AREL1 (amino acids (aa) 436–823) at 2.4 Å resolution and its ubiquitination of the proapoptotic protein second mitochondria-derived activator of caspase (SMAC). We found that the extended HECT domain adopts an inverted, T-shaped, bilobed conformation and harbors an additional loop (aa 567–573) absent in all other HECT members. We also show that the N-terminal extended region (aa 436–482) preceding the HECT domain is indispensable for its stability and activity and that without this region, the HECT domain becomes inactive. AREL1 ubiquitinated SMAC, primarily on Lys⁶² and Lys¹⁹¹. We solved the crystal structure of the tetrameric form of SMAC to 2.8 Å resolution, revealing the Lys⁶² and Lys¹⁹¹ locations. The AREL1 HECT domain assembled Lys³³-, Lys⁴⁸-, and Lys⁶³-linked polyubiquitin chains. Moreover, E701A substitution in the AREL1 HECT domain substantially increased its autopolyubiquitination and SMAC ubiquitination activity, whereas deletion of the last three amino acids at the C terminus completely abrogated AREL1 autoubiquitination and reduced SMAC biquitination. Finally, an AREL1-specific ubiquitin variant inhibited SMAC ubiquitination in vitro. Our findings may assist in the development of AREL1 inhibitors that block its anti-apoptotic activity in cancer.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Ministry of Education, Singapore; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1591895

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 52 Vol. 294; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
E3 ubiquitin ligase
HECT domain
apoptosis
autopolyubiquitination
cancer apoptosis-resistant E3 ligase 1 (AREL1)
crystal structure
post-translational modification (PTM)
protein degradation
second mitochondria-derived activator of caspase (SMAC)
structural biology
structure-function
ubiquitylation (ubiquitination)

Structural insights into a HECT-type E3 ligase AREL1 and its ubiquitination activities in vitro

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