title: Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1.
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.
authors.affiliation: Institute of Molecular Pathology , Dr.-Bohr-Gasse 7, 1030 Vienna, Austria.|Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna , Währingerstr. 42, 1090 Vienna, Austria.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Rampler
authors.affiliation: Institute of Molecular Pathology , Dr.-Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stranzl
authors.affiliation: Institute of Molecular Pathology , Dr.-Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Orban-Nemeth
authors.forename: David Maria
authors.initials: DM
authors.lastname: Hollenstein
authors.affiliation: Institute of Molecular Biotechnology, Austrian Academy of Sciences , Dr.-Bohr-Gasse 3, 1030 Vienna, Austria.
authors.forename: Otto
authors.initials: O
authors.lastname: Hudecz
authors.forename: Peter
authors.initials: P
authors.lastname: Schlögelhofer
authors.affiliation: Institute of Molecular Pathology , Dr.-Bohr-Gasse 7, 1030 Vienna, Austria.|Institute of Molecular Biotechnology, Austrian Academy of Sciences , Dr.-Bohr-Gasse 3, 1030 Vienna, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D002234:Carbodiimides / Q000737:chemistry; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D057188:Workflow
chemical_list: C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; C117967:AHP2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D002234:Carbodiimides; D003432:Cross-Linking Reagents; C514180:MND1 protein, Arabidopsis; D046912:Multiprotein Complexes; D013388:Succinimides; D010770:Phosphotransferases; C019358:disuccinimidyl suberate
keywords: BS2G; DSS; EDC; HOP2–MND1; SEC; XL−MS; comparative modeling; cross-linking