Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2鈥揗ND1

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• 作者：Evelyn Rampler ; Thomas Stranzl ; Zsuzsanna Orban-Nemeth ; David Maria Hollenstein ; Otto Hudecz ; Peter Schloegelhofer ; Karl Mechtler
• 刊名：Journal of Proteome Research
• 出版年：2015
• 出版时间：December 4, 2015
• 年：2015
• 卷：14
• 期：12
• 页码：5048-5062
• 全文大小：706K
• ISSN：1535-3907

文摘

The HOP2鈥揗ND1 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鈥揗ND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL鈥揗S). The final XL鈥揗S workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC鈥揗S/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS²G) 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鈥損rotein 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鈥揗ND1 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.