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J Proteome Res. 2015 Dec 4;14(12):5048-62. doi: 10.1021/acs.jproteome.5b00903. Epub 2015 Nov 18.

Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1.

Author information

1
Institute of Molecular Pathology , Dr.-Bohr-Gasse 7, 1030 Vienna, Austria.
2
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna , Währingerstr. 42, 1090 Vienna, Austria.
3
Institute of Molecular Biotechnology, Austrian Academy of Sciences , Dr.-Bohr-Gasse 3, 1030 Vienna, Austria.

Abstract

The HOP2-MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2-MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL-MS). The final XL-MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC-MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS(2)G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2-MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution.

KEYWORDS:

BS2G; DSS; EDC; HOP2–MND1; SEC; XL−MS; comparative modeling; cross-linking

PMID:
26535604
DOI:
10.1021/acs.jproteome.5b00903
[Indexed for MEDLINE]

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