Dataset Information

Comprehensive XL-MS reveals parallel orientation and versatile 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. The HOP2/MND1 dimer forms via the central, split coiled coils (CC1 and CC2) present in both proteins, and the resulting complex contains several flexible regions such as the hinge between the coils. To investigate the conformational flexibility of the heterodimer, important for understanding mechanistic details of HOP2/MND1 function, we studied the spatial relation of the complex partners and their domains in solution. We performed chemical cross-linking in combination with mass spectrometry (XL-MS) to generate distance restraints, which were subsequently used for molecular modeling. The final XL-MS workflow encompassed the use of cross-linkers with varying spacer arm lengths, quenching, digestion, size exclusion enrichment and HCD based LC-MS/MS detection prior to data evaluation. We applied and systematically tested two different homobifunctional amine-reactive crosslinkers (DSS-11.4 Å and BS2G 7.7 Å) and one zero-length heterobifunctional crosslinker (EDC). Crosslinked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for crosslink guided molecular modeling. Our miniaturized SEC approach reduced the required starting material and led to a high amount of crosslinked peptides allowing the analysis of replicates. The majority of the identified crosslinks was found in the coiled coil domains, indicating a parallel orientation of the interaction partners. Furthermore, flexibility of the C-terminal head domains of HOP2 and MND1 was observed. The experimentally derived distance constraints combined with an iterative comparative modeling approach not only confirm the elongated, open conformation predicted by the crystal structure of the Giardia lamblia Hop2-Mnd1 heterodimer, but further suggest the coexistence of a closed complex conformation in solution.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Otto Hudecz

LAB HEAD: Karl Mechtler

PROVIDER: PXD001538 | Pride | 2015-11-05

REPOSITORIES: Pride

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			Action	DRS
	20140603_QEx3_RSLC4_Rampler_Mechtler_IMP_shotgun_BS2G12.raw	Raw
	20140603_QEx3_RSLC4_Rampler_Mechtler_IMP_shotgun_BS2G22.raw	Raw
	20140603_QEx3_RSLC4_Rampler_Mechtler_IMP_shotgun_BS2G32.raw	Raw
	20140603_QEx3_RSLC4_Rampler_Mechtler_IMP_shotgun_BS2G42.raw	Raw
	20140603_QEx3_RSLC4_Rampler_Mechtler_IMP_shotgun_DSS12.raw	Raw

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Publications

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

Rampler Evelyn E Stranzl Thomas T Orban-Nemeth Zsuzsanna Z Hollenstein David Maria DM Hudecz Otto O Schlögelhofer Peter P Mechtler Karl K

Journal of proteome research 20151118 12

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 HC ...[more]

PMID: 26535604

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