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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / metabolism
  • Carbodiimides / chemistry
  • Chromatography, Gel
  • Cross-Linking Reagents / chemistry
  • Models, Molecular
  • Multiprotein Complexes / chemistry
  • Phosphotransferases / chemistry*
  • Phosphotransferases / metabolism
  • Protein Conformation
  • Protein Multimerization
  • Succinimides / chemistry
  • Tandem Mass Spectrometry
  • Workflow

Substances

  • 1-ethyl-3-(3-(diethylamino)propyl)carbodiimide
  • AHP2 protein, Arabidopsis
  • Arabidopsis Proteins
  • Carbodiimides
  • Cross-Linking Reagents
  • MND1 protein, Arabidopsis
  • Multiprotein Complexes
  • Succinimides
  • Phosphotransferases
  • disuccinimidyl suberate