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

Abstract

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 SMACmore » 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.« less

Authors:

Singh, Sunil  ^[1];

• Search DOE PAGES for author "Singh, Sunil"
• Search DOE PAGES for ORCID "0000-0002-2863-5492"

Ng, Joel  ^[1];

• Search DOE PAGES for author "Ng, Joel"
• Search DOE PAGES for ORCID "0000-0003-1797-3071"

Nayak, Digant  ^[1];

• Search DOE PAGES for author "Nayak, Digant"
• Search DOE PAGES for ORCID "0000-0003-3680-0846"

Sivaraman, J.  ^[1]

• Search DOE PAGES for author "Sivaraman, J."
• Search DOE PAGES for ORCID "0000-0001-9781-5326"

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+ Show Author Affiliations

1. National Univ. of Singapore (Singapore)

Publication Date:

Fri Nov 15 00:00:00 EST 2019

Research Org.:

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

Sponsoring Org.:

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

OSTI Identifier:

1591895

Grant/Contract Number:  

P41 GM103403; S10 RR029205; AC02-06CH11357; R-154-000-B03-112 (MoE Tier 2); R154-000-A72-114 (AcRF Tier 1)

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 294; Journal Issue: 52; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

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