CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation

Abstract

An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting.

Structure of CRL7FBXW8 reveals coupling with CUL1–RBX1/ROC1 for multi-cullin-RING E3-catalyzed ubiquitin ligation

“…6d,e ). Rather, CRL7 FBXW8 partnering with neddylated CUL1–RBX1 may share some conceptual parallels with canonical neddylated CRLs partnering with ARIH-family RBR E3s such that one E3 (the CRL) recruits substrate while the other (the ARIH-family member) mediates ubiquitination 36 , 41 , 52 .…”

“…All proteins are of human origin and were cloned and verified by standard molecular biology techniques. Full-length CUL1, CUL1 K720R , glutathione S -transferase (GST)-TEV site (TEV)-RBX1 (residue 5 to C terminus), CUL5, GST-TEV-RBX2 (residue 5 to C terminus) and GST-TEV-UBA1, were expressed in Trichoplusia ni High-Five insect cells, with CUL and corresponding RBX protein, being coexpressed 35 , 41 , 52 . CUL1 N-terminal region (residues 1 to 410 encompassing CUL1’s CR1, CR2 and CR3 domains), UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2N, UBE2Q2, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2V1, UBE2V2 and the neddylation components UBE2M and NAE1-UBA3 were expressed in E. coli Rosetta (DE3) cells as GST-thrombin or TEV fusion proteins.…”

Regulation of Hedgehog Signaling Through Arih2-Mediated Smoothened Ubiquitination and Endoplasmic Reticulum-Associated Degradation

“…The ER-localized Arih2β isoform fully rescues the Smo phenotypes while the nuclear-localized form does not. Most work on Arih2 focuses on Arih2α function as part of the cullin-5 complex (Hüttenhain et al, 2019; Kelsall et al, 2013; Kostrhon et al, 2021). Complete loss of Arih2 in mouse leads to embryonic lethality and increased inflammatory responses (Lin et al, 2013).…”

Roles of Cullin-RING Ubiquitin Ligases in Cardiovascular Diseases

“…As a result, neddylation converts the CRL from an open conformation, which benefits the engagement of E2~Ub and diverse substrates, to a closed and compact conformation, which promotes the transfer of the donor ubiquitin to the acceptor lysine and triggers efficient substrate ubiquitination [185]. Furthermore, cullin neddylation enables the ARIH family of RBR E3 ligases to bind CRLs-ARIH1 binds neddylated CUL•RBX1, whereas ARIH2 binds neddylated CUL5•RBX2-and the ARIH E3 ligase accelerates the transfer of Ub from an E2 to the CRL-bound protein substrate [186][187][188].…”