PXD049047

PXD049047 is an original dataset announced via ProteomeXchange.

Title	Structural basis of PROTAC-induced target ubiquitination mechanism - identification of ubiquitin-rbx1 crosslinks
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.
HostingRepository	PRIDE
AnnounceDate	2025-05-06
AnnouncementXML	Submission_2025-05-06_12:53:58.735.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sarah Chandler
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-31 07:28:38	ID requested
⏵ 1	2025-05-06 12:53:59	announced

Crowe C, Nakasone MA, Chandler S, Craigon C, Sathe G, Tatham MH, Makukhin N, Hay RT, Ciulli A, Mechanism of degrader-targeted protein ubiquitinability. Sci Adv, 10(41):eado6492(2024) [pubmed]

10.1126/sciadv.ado6492;

submitter keyword: ubiquitin, photocrosslinking, CRL2VHL,Rbx1

Ronald T Hay
contact affiliation	University of Dundee, Scool of Life Sciences, Department of Molecular, Cellular andDevelopmental biology
contact email	r.t.hay@dundee.ac.uk
lab head
Sarah Chandler
contact affiliation	University of Dundee
contact email	2431160@dundee.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD049047
     1. Label: PRIDE project
     2. Name: Structural basis of PROTAC-induced target ubiquitination mechanism - identification of ubiquitin-rbx1 crosslinks