Dataset Information

Structural basis of PROTAC-induced target ubiquitination mechanism - verification of ubiquitin UBE2R1 (C93K) loading assay

ABSTRACT: 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. To generate the stable isopeptide-linked Ub-E2 conjugates, the active site cysteine (C93) of UBE2R1 was mutated to a lysine (C93K). Then in a reaction using recombinant human E1 at high pH, ubiquitin was conjugated to UBE2R1. As the ubiquitin E1 transfers ubiquitin to the active site cysteine of UBE2R1 (in unmodified systems), the preferred site of ubiquitin modification should theoretically be on K93. However, other lysine residues can also be modified so to ensure that the primary site of ubiquitin modification occurred at the active site (residue K93), ubiquitin-modified UBE2R1 was analysed by mass spectrometry and a search for GG-K modified peptides was performed. This search provided good evidence that the main site of modification occurred on residue K93 of the UBE2R1 C93K mutant. Verifying this as a suitable structural mimetic of the thioester-linker ubiquitin loaded UBE2R1.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sarah Chandler

LAB HEAD: Ronald T Hay

PROVIDER: PXD049050 | Pride | 2025-05-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220620_MT_SC_Ube2R1_Ub_Loading_01.raw	Raw
	20220620_MT_SC_Ube2R1_Ub_Loading_02.raw	Raw
	20220620_MT_SC_Ube2R1_Ub_Loading_03.raw	Raw
	20220620_MT_SC_Ube2R1_Ub_Loading_04.raw	Raw
	20220620_MT_SC_Ube2R1_Ub_Loading_05.raw	Raw

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Publications

Mechanism of degrader-targeted protein ubiquitinability.

Crowe Charlotte C Nakasone Mark A MA Chandler Sarah S Craigon Conner C Sathe Gajanan G Tatham Michael H MH Makukhin Nikolai N Hay Ronald T RT Ciulli Alessio A

Science advances 20241011 41

Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4<sup>BD2</sup> towa ...[more]

PMID: 39392888

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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.