Dataset Information

Crosslinking mass spectrometry (CLMS) analysis of monoubiquitinated FANCD2-FANCI complex

ABSTRACT: Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. In this study, monoubiquitinated FANCD2-FANCI complex was characterized using crosslinking mass spectrometry in order to provide in sights into the 3D structure of the complex.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Gallus Gallus (chicken)

SUBMITTER: Zhuo Chen

LAB HEAD: Juri Rappsilber

PROVIDER: PXD017020 | Pride | 2020-03-09

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	FA_ubD2I_CLMS_BS3_PSM_Rappsilber.csv	Csv
	FA_ubD2I_CLMS_BS3_Rappsilber.raw	Raw
	recal_FA_ubD2I_CLMS_BS3_Rappsilber.mgf	Mgf

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Publications

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair.

Alcón Pablo P Shakeel Shabih S Chen Zhuo A ZA Rappsilber Juri J Patel Ketan J KJ Passmore Lori A LA

Nature structural & molecular biology 20200217 3

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conforma ...[more]

PMID: 32066963

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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.