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

• DOI: 10.1021/acs.jproteome.5b00903
• Publication Date: 2015-11-04T19:55:17Z
• Authors: Evelyn Rampler; Thomas Stranzl; Zsuzsanna Orban-N...; David Maria Holle...; Otto Hudecz; Peter Schlögelhofer; Karl Mechtler
• Keywords: Models, Molecular; 0301 basic medicine; 106037 Proteomik; 104002 Analytische Chemie; Protein Conformation; 106002 Biochemie; HOP2 PROTEIN; 106037 Proteomics; Succinimides; LIGAND INTERACTIONS; Workflow; comparative modeling; 03 medical and health sciences; 104002 Analytical chemistry; XL¯MS; Tandem Mass Spectrometry; MECHANISTIC INSIGHTS; MEIOTIC RECOMBINATION; HOP2-MND1; STRUCTURAL PROTEOMICS; XL-MS; DSS; MND1; COMPLEX; Arabidopsis Proteins; Phosphotransferases; 106002 Biochemistry; STRAND ASSIMILATION; SEC; LINKED PEPTIDES; BS G; Carbodiimides; Cross-Linking Reagents; Multiprotein Complexes; DNA-REPAIR; Chromatography, Gel; BS(2)G; Protein Multimerization; cross-linking; EDC

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.