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Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry

ABSTRACT: [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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli

SUBMITTER: Adam Belsom

LAB HEAD: Juri Rappsilber

PROVIDER: PXD051661 | Pride | 2024-10-28

REPOSITORIES: Pride

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			Action	DRS
	18276_B240123_HF_AB_B3_7_A1_B1_comb_1.mzid.gz	Mzid
	18277_B240123_HF_AB_B3_7_D1_E1_comb_1.mzid.gz	Mzid
	18346_B240302_HF_AB_B3_14_A1_B1_comb_1.mzid.gz	Mzid
	18350_B240302_HF_AB_B3_14_M1_N1_comb_1.mzid.gz	Mzid
	B240123_01_HF_AB_DE_120_B3_7_A1_1.raw	Raw

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Project description:The HOP2/MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes, and promotes the repair of DNA double strand breaks. The HOP2/MND1 dimer forms via the central, split coiled coils (CC1 and CC2) present in both proteins, and the resulting complex contains several flexible regions such as the hinge between the coils. To investigate the conformational flexibility of the heterodimer, important for understanding mechanistic details of HOP2/MND1 function, we studied the spatial relation of the complex partners and their domains in solution. We performed chemical cross-linking in combination with mass spectrometry (XL-MS) to generate distance restraints, which were subsequently used for molecular modeling. The final XL-MS workflow encompassed the use of cross-linkers with varying spacer arm lengths, quenching, digestion, size exclusion enrichment and HCD based LC-MS/MS detection prior to data evaluation. We applied and systematically tested two different homobifunctional amine-reactive crosslinkers (DSS-11.4 Å and BS2G 7.7 Å) and one zero-length heterobifunctional crosslinker (EDC). Crosslinked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for crosslink guided molecular modeling. Our miniaturized SEC approach reduced the required starting material and led to a high amount of crosslinked peptides allowing the analysis of replicates. The majority of the identified crosslinks was found in the coiled coil domains, indicating a parallel orientation of the interaction partners. Furthermore, flexibility of the C-terminal head domains of HOP2 and MND1 was observed. The experimentally derived distance constraints combined with an iterative comparative modeling approach not only confirm the elongated, open conformation predicted by the crystal structure of the Giardia lamblia Hop2-Mnd1 heterodimer, but further suggest the coexistence of a closed complex conformation in solution.

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Structural analysis of the HypCD apo- and holoproteins by crosslinking mass spectrometry

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