Dataset Information

Photocrosslinking Activity-Based Probes to Capture the Dynamics of Ubiquitin RING E3 Ligase Interactions

ABSTRACT: Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provide the specificity for ubiquitination, with the largest class among them, the RING E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses a modified ubiquitin carrying a photoactivatable crosslinker that once stably linked to the active site of an E2, creates an activity based probe (ABP) to monitor interactions with E3 ligases. Using this, regions of interaction between ubiquitin and a selection of different RING E3 were determined, which not only confirmed existing structures of E2-Ub-RING-E3 complexes but was also used to assess new Ub-E2-E3 models generated in absence of existing structures.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mike Tatham

LAB HEAD: Ronald T. Hay

PROVIDER: PXD075613 | Pride | 2026-06-08

REPOSITORIES: Pride

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Photocross-linking activity-based probes to capture the dynamics of ubiquitin RING E3 ligase interactions.

Chandler Sarah F SF Tatham Michael H MH Branigan Emma E Nakasone Mark A MA Makukhin Nikolai N Ciulli Alessio A Hay Ronald T RT

The Biochemical journal 20260701 7

Almost all cellular processes are influenced by ubiquitination. A large family of enzymes known as E3 ligases provides the specificity for ubiquitination, with the largest class among them, the Really Interesting New Gene (RING) E3s, comprising over 600 members in humans. RING E3s facilitate transfer of ubiquitin (Ub) to substrates by constraining the highly dynamic E2-Ub thioester linkage to be primed for attack from the substrate nucleophile. We have established a workflow that uses an N-malei ...[more]

PMID: 42112908

Similar Datasets

Project description:Proteolysis Targeting Chimeras (PROTACs) hijack the ubiquitin proteasome system to ubiquitinate target proteins, targeting them for degradation by the proteasome. An understudied process that is essential to the PROTAC mechanism of action is ubiquitination of the target protein and ubiquitin (Ub) chain elongation. These phenomena are difficult to study structurally due the instability of the thioester-linked ubiquitin (Ub)-E2 conjugates that rapidly discharges ubiquitin onto the target protein, catalysed by a Cullin-RING E3 Ubiquitin Ligase (CRL). To overcome this, stable isopeptide-linked Ub-E2 conjugates were generated using an active site mutant of the chain elongation E2 enzyme UBE2R1 and used in structural studies of a model PROTAC system consisting of neddylated CRL2VHL, MZ1 and BRD4(BD2)-Ub. This approach resulted in multiple novel cryo-EM structures of the active CRL in complex with the model substrate and MZ1. To validate the conformation of the Ub-Ube2R1-Rbx1 within these structures, a ubiquitin-directed photoreactive probe (UDPRP) was designed. The UDPRP consists of a stable-isopeptide linked Ub-Ube2R1 conjugate as used in the except the ubiquitin is site-specifically labelled with the photocrosslinker, N-maleimido-diazirine. The diazirine based photocrosslinker is site-specifically labelled to ubiquitin through cysteine-maleimide chemistry. Upon exposure to UV light (365 nM) forms a reactive carbene intermediate which either rapidly inserts into X-H bonds (including O-H, N-H, S-H and C-H bonds) and can therefore covalently trap proteins in close-proximity. In a photocrosslinking assay containing neddylated-CRL2VHL and the UDPRP. The UDPRP crosslinked to Rbx1, the RING portion of the multi-subunit CRL E3. To map the site of crosslinking the photo-crosslinked product was excised from the gel and analysed by mass-spectrometry. Four inter-protein crosslinked peptides from ubiquitin-rbx1 were detected, with an andromeda score >90, linking ubiquitin E34C to the last three C-terminal residues of Rbx1.

Project description:Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.

Project description:Ubiquitination is a much-studied post-translational modification (PTM) playing crucial roles in regulating cellular processes across eukaryotes. Canonical ubiquitination relies on a cascade of E1-, E2-, and E3 enzymes which conjugate the Gly76 C-terminus of ubiquitin (Ub) to the lysine side chains in proteins creating a stable isopeptide bond(1). However, it became evident that Ub can also be conjugated to non-lysine residues in proteins to form a thioester bond with cysteine(2, 3) and an oxyester bond with serine or threonine(4-6). Beyond protein modification, recent discoveries reveal ubiquitination on several non-protein substrates, such as lipopolysaccharide(7), oligosaccharide(8), phosphatidylethanolamine(9), nucleic acids(10, 11), as well as on another PTM, ADP-ribose (ADPr) (12, 13). ADP-ribosylation is an ancient modification which is conserved across all kingdoms of life. This modification relies on ADP-ribosyltransferase enzymes which utilise NAD+ and transfer ADP-ribose onto proteins and nucleic acids(14, 15). Crosstalk between ubiquitination and ADP-ribosylation occurs in mammals where for instance poly-ADP-ribose (PAR) chains direct subsequent ubiquitination events in DNA damage responses(16) and ADPr-dependent ubiquitination in immunity responses (17). Such crosstalk is also cleverly utilised by bacteria, which themselves lack Ub, to disrupt host Ub signaling by modifying it with ADPr, as shown by Legionella and Chromobacterium(18, 19). These are prime examples where the already complex ubiquitination code is made even more diverse upon crosstalk with ADPr. In humans, the family of Deltex RING E3 Ub ligases (DTX1, DTX2, DTX3, DTX3L, DTX4) catalyse Ub conjugation to the 3’-hydroxyl group of ADPr, which results in a hybrid ADPr-3’-Ub modification (Fig. 1A)(12, 20). It was first reported that the heterodimeric complex of DTX3L/PARP9 conjugates ADPr to the Gly76 residue of Ub(21). This unique reaction is catalysed by all members of Deltex E3 ligases through their conserved RING and DTC domains, which interacts with Ub-loaded E2 (E2~Ub) and NAD+/ADPr, respectively(20). The DTC domains of Deltex E3 ligases bind to NAD+ and ADPr, and can recruit ADP-ribosylated proteins for ubiquitination(22). However, instead of ADP-ribosylation of Ub which occurs on the C1” of nicotinamide ribose, Deltex E3 ligases catalyse the ubiquitination of ADPr on the 3’-hydroxyl of adenosine ribose (Fig 1A) (12). To support these reactions, the DTC domains of Deltex E3 ligases harbour a conserved Glu-His diade to deprotonate the 3’-hydroxyl of the proximal adenosine ribose in ADPr to attack the thioester E2~Ub conjugate, resulting in a covalent ester bond between Ub Gly76 and the ADPr 3’-hydroxyl group(12). This allows formation of dual hybrid modification composed of ubiquitin and ADPr (ADPr-3’-Ub) either on protein and nucleic acids substrates(12, 13). However, the biological function of such Deltex-catalysed hybrid ADPr-3’-Ub-substrates remains unknown. In this work, we wondered if there are modules involved in reading this new expansion of the ubiquitin code, which would establish a first indication regarding the biological consequence and the underlying molecular mechanisms of the hybrid ADPr-3’-Ub. One of the major challenges in studying the ADPr-3’-Ub modification is the inherent lability of the ester bond. Here we synthesised a non-hydrolysable tool that mimics the formed ubiquitinated ADP-ribose (further annotated as ADPr3’-Ub) to perform an unbiased pulldown from cell lysate and start exploring its interactome. We identified RNF114 as one of the top binders for ADPr3’-Ub. RNF114 was previously suggested to bind mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) through its Zinc finger (ZnF) domains (23, 24). Here, we discovered that RNF114 mediate low micromolar binding to ADPr3’-Ub through its tandem ZnF2+ZnF3 and Ub-interacting motif (UIM) domains. We further showed that RNF114 can engage ADPr3’-Ub and elongate it with K11-linked polyubiquitin chains forming a novel mixed Ub chain linkage product. Finally, the tandem ZnF2+ZnF3+UIM domains of RNF114 are indispensable for its recruitment to the sites of DNA damage in cells, which suggest RNF114 as a reader of ADPr-3’-Ub signal during response to DNA damage.

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