Dataset Information

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

ABSTRACT: crosslinking mass spectrometry results for sulfo-SDA crosslinking of human CUL4-NEDD8/ROC1/DDB1/DCAF1-CtD in complex with SAMHD1 and Vpr protein from simian immunodeficiency virus infecting Cercopithecus cephus (SIVmus Vpr)

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrea Graziadei

LAB HEAD: Juri Rappsilber

PROVIDER: PXD020453 | Pride | 2021-07-20

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	B200602_02_Lumos_AG_CO_130_Cul4A_SDA_Ratio24_frac5_rep01.raw	Raw
	B200602_03_Lumos_AG_CO_130_Cul4A_SDA_Ratio24_frac5_rep02.raw	Raw
	B200602_04_Lumos_AG_CO_130_Cul4A_SDA_Ratio24_frac6_rep01.raw	Raw
	B200602_05_Lumos_AG_CO_130_Cul4A_SDA_Ratio24_frac7_rep01.raw	Raw
	B200602_06_Lumos_AG_CO_130_Cul4A_SDA_Ratio24_frac8_rep01.raw	Raw

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Publications

Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses.

Banchenko Sofia S Krupp Ferdinand F Gotthold Christine C Bürger Jörg J Graziadei Andrea A O'Reilly Francis J FJ Sinn Ludwig L Ruda Olga O Rappsilber Juri J Spahn Christian M T CMT Mielke Thorsten T Taylor Ian A IA Schwefel David D

PLoS pathogens 20210802 8

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation ...[more]

PMID: 34339457

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Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.