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. 2021:20:100019.
doi: 10.1074/mcp.RA120.002414. Epub 2021 Jan 6.

The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases

Ryan J Lumpkin  1 , Alla S Ahmad  1 , Rachel Blake  1 , Christopher J Condon  1 , Elizabeth A Komives  2
Affiliations

The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases

Ryan J Lumpkin et al. Mol Cell Proteomics. 2021.

Abstract

Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

Keywords: hydrogen-deuterium exchange mass spectrometry; post-translational modifications; protein complex; ubiquitin ligase.

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Conflict of interest statement

Conflicts of interest Authors declare no competing interests.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Schematics of the ubiquitylation cascade as mediated by the ASB9 CUL5 E3 ligase.A, ubiquitin is activated through a three-step enzymatic cascade. The ubiquitin-activating enzyme (E1) binds ATP and catalyzes adenylation of ubiquitin. The active-site cysteine on E1 attacks the Ub-AMP complex to form a thioester bond. Through a trans-thioesterification reaction, ubiquitin is transferred to the active-site cysteine on a ubiquitin-conjugating enzyme (E2). Ubiquitin ligases (E3) facilitate the highly specific covalent attachment of activated ubiquitin (Ub) to bound substrate proteins through an isopeptide bond on an exposed lysine residue. B, schematic showing the states of the ASB9-CRL explored in this work: unactivated, activated by NEDD8, and activated by NEDD8 with ARIH2 present. The protein abbreviations and colors are consistent throughout the manuscript: creatine kinase brain-type (CKB, two-tone teal); ankyrin and SOCS-box protein 9 (ASB9, yellow); elongins B and C (ELOB/C, purple); cullin 5 (CUL5, salmon), ring box protein 2 (RBX2, olive); the ubiquitin conjugating enzyme bound to RBX2 (UBE2D1/2, magenta); ubiquitin (Ub, tan); Ariadne RBR E3 ubiquitin protein ligase 2 Ring between Ring ligase (ARIH2 RBRL, orange); the ARIH2 RBRL ubiquitin conjugating enzyme (UBE2L3, gray). The Ub is transferred from UBE2L3 to ARIH2 prior to transfer to the substrate.
Fig. 2
Fig. 2
In vitro ubiquitylation assays showing AriH2-induced ubiquitylation.A, Coomassie Blue–stained gel of various ubiquitylation reactions. All reactions contained MgATP, UBE1, and Ub. Lanes 1, 3, 5, 7, 9 contain CKB-ASB9-ELOB/C-CUL5-RBX2. Lanes 2, 4, 6, 8, 10 contain CKB-ASB9-ELOB/C-CUL5(NEDD8)-RBX2. Lane-specific additional components are UBE2D2 (lane 1, 2), UBE2R1 (lanes 3, 4), ARIH2-UBE2L3 (lanes 5, 6), ARIH2-UBE2L3-UBE2D2 (lanes 7, 8), and ARIH2-UBE2L3-UBE2R1 (lanes 9, 10). B, anti-CKB blot for the reactions as in panel A showing the increase in CKB migrating at high molecular weight. C, image J analysis of the Anti-CKB blot in B. The high-molecular-weight CKB ratioed against CKB reveals that the most efficient combination of components for polyubiquitylation of CKB is ARIH2 with UBE2R1. Subsets of this experiment (biological replicates) were repeated three times.
Fig. 3
Fig. 3
Model of the ASB9-CRL showing sites of ubiquitylation on CKB.A, molecular docking was used to place ARIH2 residues 11 to 38 into the basic cleft in CUL5. Acidic residues on ARIH2 are shown in red and CUL5 basic residues previously shown to be critical for the interaction (21) are shown in blue. B, a structural model of CKB-ASB9-ELOB/C-CUL5(NEDD8)-RBX2-E2D1/2∼Ub-ARIH2-UBE2F∼Ub built from the published structure of ASB9-ELOB/C-CUL5-RBX2-UBE2D1/2 (16) by addition of ARIH2-UBE2F∼Ub based on the docked structure from A). Homologous structural information is not available until residue 57 of ARIH2, so the absolute position of ARIH2 relative to the ASB9-CRL is speculative. The four CKB lysines (K45, K101, K107, and K381) on one subunit of the monomeric CKB that were observed to be ubiquitylated (red side chains) are the closest lysines to ARIH2. The protein colors are consistent throughout the manuscript: CKB, aquamarine, dark cyan; ASB9, yellow; ELOB/C, orchid, purple; CUL5, rosy brown, RBX2, olive; NEDD8, chartreuse; UBE2D1/2, magenta; Ub on UBE2D1/2, khaki; ARIH2, orange, UBE2L3, gray; Ub on UBE2F, blue.
Fig. 4
Fig. 4
HDX-MS reveals flexible tethers on ARIH2 and RBX2. HDX-MS shows residues 46 to 58 of ARIH2 are highly exchanging when ARIH2 is alone (green) or bound to the ASB9-CRL (magenta). This segment of ARIH2 (orange) is colored in red in the structural model of the ASB9-CRL (expanded view). Similarly, RBX2 residues 41 to 47 are highly exchanging in the ASB9-ELOB/C-CUL5-RBX2 complex (brown) as well as in the neddylated complex (black). This segment of RBX2 (olive) is also colored red in the structural model of the ASB9-CRL (expanded view).
Fig. 5
Fig. 5
HDX-MS reveals long-range allostery upon neddylation of CUL5. Comparison of the HDX-MS deuterium uptake into CKB-ASB9-ELOB/C-CUL5-RBX2 complex (brown symbols in plots) with the CKB-ASB9-ELOB/C-CUL5(NEDD8)-RBX2 complex (black symbols in plots) revealed that neddylation of CUL5 causes increased exchange in CUL5 residues (red) and decreased exchange in RBX2 residues (blue).
Fig. 6
Fig. 6
HDX-MS reveals transient protection of CUL5 by the N terminus of ARIH2. Comparison of the HDX-MS deuterium uptake into ELOB/C-CUL5(NEDD8)-RBX2 complex (magenta symbols in plots) with the ELOB/C-CUL5(NEDD8)-RBX2-ARIH2 complex (black symbols in plots) revealed that the CUL5 regions that form the basic cleft and the surrounding residues are transiently protected upon ARIH2 binding (blue regions in structure).
Fig. 7
Fig. 7
HDX-MS of AriH2 reveals the mechanism for relief of autoinhibition. Comparison of the HDX-MS deuterium uptake into ARIH2 alone (red symbols in the plots) versus in complex with the ASB9-CRL (black symbols in the plots) shows marked increase in exchange (red regions in the structure) of the helical regions surrounding the ARIH2 active site upon binding to ASB9-CRL.
Fig. 8
Fig. 8
Schematic diagram of the CRL E3 ligase mechanism based on the results of this study. Our data support a mechanism by which RBX2 initially engages UBE2F to neddylate CUL5. Neddylation alters the RBX2 to decrease exchange at its binding site for UBE2F. Neddylation also alters the conformation of CUL5 opening a cleft of basic residues to which the acidic C terminus of ARIH2 can bind with high affinity. ARIH2 is autoinhibited, but upon binding to CUL5, its active site increases exchange via a long-range allosteric mechanism. The increased exchange likely indicates opening of the ARIH2 active site and relief of autoinhibition. After initial Ub transfer by ARIH2 to specific lysines on CKB that are close to the ARIH2 active site, polyubiquitylation on those same lysines can occur by a number of different routes including continued ubiquitylation by ARIH2 or contributions from UBE2D2 or UBE2R1 bound to RBX2.
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