Key Takeaways & Executive Findings
- •• AlphaFold2 predictions reveal that the LTM motif modulates the HOIP-UBA structure, affecting LUBAC stability. • GeoPPI analysis shows that the LTM motif reduces binding affinity between UBL domains and HOIP, decreasing complex stability. • HOIP (629‒695) and HOIP-UBA cooperatively bind to HOIL-1L-UBL, forming a stable elongated domain (HOIP 466‒695). • Molecular dynamics, SPR, and ITC confirm the structural and functional significance of the HOIP-HOIL-1L interaction in LUBAC.
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.
1. Introduction
The process of covalent conjugation between proteins and several small ubiquitin (Ub) molecules is known as ubiquitination, which is a type of posttranslational modification that plays a crucial role in biological processes [1]. Ubiquitination is a three-step catalytic reaction process sequentially catalyzed by a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3) [2,3]. The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ligase complex which is composed of the catalytic subunit HOIP and two accessory subunits, HOIL-1L and SHARPIN [4]. In LUBAC, the catalytic subunit HOIP interacts with the ubiquitin-like (UBL) domains of SHARPIN and HOIL-1L to maintain the stability of the structure and function of LUBAC [5]. Dysfunction or impairment of any of the three subunits of LUBAC can lead to inflammation, immune deficiency, and even death in animals and humans [6].
HOIP is an E3 ligase of the RBR (RING-between-RING) type [7]. Its full length consists of 1072 residues, and its structure mainly includes an N-terminal PNGase/UBA or UBX-containing protein (PUB) domain, followed by a B-box type zinc finger (ZF), a typical ZF, two Nlp4-like ZF domains (NZF1 and NZF2), an intermediate atypical Ub-associated domain (UBA), and a C-terminal RBR domain conjugated with a unique linear Ub chain determining domain (LDD) [8]. The RBR and LDD regions together form the catalytic core of HOIP, which is responsible for linear Ub chain assembly [9]. Free HOIP exists in a partially autoinhibited state, where the UBA domain of HOIP cannot recognize Ub chains, and the biological activity of the RBR-LDD domain is inhibited [10]. SHARPIN and HOIL-1L are accessory subunits that form LUBAC with HOIP. Binding of HOIL-1L or SHARPIN to HOIP releases the autoinhibition of HOIP [11].
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Chenchen Wang, Chunying Gu, Ying Lv, Hongyu Liu, Yanan Wang, Yongmei Zuo, Guangyu Jiang, Lili Liu, Jiafu Liu (2026). AlphaFold2 assists in providing novel mechanistic insights into the interactions among the LUBAC subunits. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2024047
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Frequently Asked Questions
What is the role of the LTM motif in LUBAC stability?
The LTM motif reduces the interaction between the UBL domains of SHARPIN and HOIL-1L with HOIP, leading to decreased structural stability of LUBAC.
How was the LUBAC structure predicted in this study?
AlphaFold-Multimer was used to predict the structure of the LUBAC core domain without the LTM motif, and GeoPPI was employed to calculate binding free energy changes.
What experimental methods validated the computational predictions?
Gromacs molecular dynamics simulations, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) were used to confirm the interactions.
What is the significance of the HOIP (466‒695) structure?
The elongated domain formed by HOIP (629‒695) and HOIP-UBA interacts with HOIL-1L-UBL to form a structurally stable complex, highlighting cooperative binding.
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