PXD051661-1

PXD051661 is an original dataset announced via ProteomeXchange.

Title	Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry
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.
HostingRepository	PRIDE
AnnounceDate	2024-10-28
AnnouncementXML	Submission_2024-10-28_02:10:41.739.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD051661
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Adam Belsom
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationList	iodoacetamide derivatized residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2024-04-23 06:11:29	ID requested
⏵ 1	2024-10-28 02:10:42	announced

10.1021/JACS.4C09791;

10.6019/PXD051661;

submitter keyword: [NiFe]-hydrogenase assembly, photocrosslinking MS,HypCD

Juri Rappsilber
contact affiliation	Technische Universität Berlin, Chair of Bioanalytics, 10623 Berlin, Germany Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK Si-M/"Der Simulierte Mensch", a Science Framework of Technische Universität Berlin and Charité - Universitätsmedizin Berlin, Berlin, Germany
contact email	juri.rappsilber@tu-berlin.de
lab head
Adam Belsom
contact affiliation	Technische Universität Berlin
contact email	adam.belsom@tu-berlin.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD051661
     1. Label: PRIDE project
     2. Name: Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry