AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits

Chenchen Wang; Chunying Gu; Ying Lv; Hongyu Liu; Yanan Wang; Yongmei Zuo; Guangyu Jiang; Lili Liu; Jiafu Liu

doi:10.3724/abbs.2024047

AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits

Acta Biochim Biophys Sin (Shanghai). 2024 Apr 24. doi: 10.3724/abbs.2024047. Online ahead of print.

Authors

Chenchen Wang¹, Chunying Gu², Ying Lv³, Hongyu Liu⁴, Yanan Wang⁵, Yongmei Zuo⁶, Guangyu Jiang⁵, Lili Liu⁵, Jiafu Liu⁵

Affiliations

¹ College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
² Department of Medical Laboratory Science and Technology, Harbin Medical University-Daqing, Daqing 163319, China.
³ College of Life Sciences, Northeast Agricultural University, Harbin 150030, China.
⁴ Preventive and Control Center for Animal Disease of Heilongjiang Province, Harbin 150069, China.
⁵ College of Basic Medical Sciences, Harbin Medical University-Daqing, Daqing 163319, China.
⁶ Heilongjiang Institute of Animal Health Inspection, Harbin 150006, China.

PMID: 38655618
DOI: 10.3724/abbs.2024047

Abstract

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex in which the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L interact with HOIP to determine the structural stability of LUBAC. The interactions between subunits within LUBAC have been a topic of extensive research. However, the impact of the LTM motif on the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP remains unclear. Here, we discover that the absence of the LTM motif in the AlphaFold2-predicted LUBAC structure alters the HOIP-UBA structure. We employ GeoPPI to calculate the changes in binding free energy (ΔG) caused by single-point mutations between subunits, simulating their protein-protein interactions. The results reveal that the presence of the LTM motif decreases the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to a decrease in the structural stability of LUBAC. Furthermore, using the AlphaFold2-predicted results, we find that HOIP (629‒695) and HOIP-UBA bind to both sides of HOIL-1L-UBL, respectively. The experiments of Gromacs molecular dynamics simulations, SPR and ITC demonstrate that the elongated domain formed by HOIP (629‒695) and HOIP-UBA, hereafter referred to as the HOIP (466‒695) structure, interacts with HOIL-1L-UBL to form a structurally stable complex. These findings illustrate the collaborative interaction between HOIP-UBA and HOIP (629‒695) with HOIL-1L-UBL, which influences the structural stability of LUBAC.

Keywords: HOIL-1L-UBL; HOIP-UBA; LTM motif; LUBAC; SHARPIN-UBL.