期刊
JOURNAL OF PROTEOME RESEARCH
卷 14, 期 12, 页码 5048-5062出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.5b00903
关键词
HOP2-MND1; XL-MS; SEC; cross-linking; comparative modeling; DSS; EDC; BS(2)G
资金
- Austrian Science Fund [SFB F3402, P2465-B24, TRIP 308-N15, I1468-B16]
- European Commission via the Seventh Framework Programme (FP7)
- project PRIME-XS [262067]
- IMP
- IMBA
- Rijk Zwaan
- MEIOsys project [222883-2]
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.
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