PXD001538

PXD001538 is an original dataset announced via ProteomeXchange.

Title	Comprehensive XL-MS reveals parallel orientation and versatile conformations of plant HOP2/MND1
Description	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.
HostingRepository	PRIDE
AnnounceDate	2015-11-05
AnnouncementXML	Submission_2015-11-05_09:29:17.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Otto Hudecz
SpeciesList	scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2014-11-25 05:25:03	ID requested
⏵ 1	2015-11-05 09:29:18	announced

Rampler E, Stranzl T, Orban-Nemeth Z, Hollenstein DM, Hudecz O, Schl, ö, gelhofer P, Mechtler K, Comprehensive Cross-Linking Mass Spectrometry Reveals Parallel Orientation and Flexible Conformations of Plant HOP2-MND1. J Proteome Res, 14(12):5048-62(2015) [pubmed]

ProteomeXchange project tag: PRIME-XS Project

curator keyword: Biological, Technical

submitter keyword: XL HOP2 MND1 cross link

Karl Mechtler
contact affiliation	Mass Spectrometry and Protein Chemistry Institute of Molecular Pathology Institute of Molecular Biotechnology
contact email	mechtler@imp.univie.ac.at
lab head
Otto Hudecz
contact affiliation	Protein Chemistry Facility
contact email	hudecz@imp.univie.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD001538
     1. Label: PRIDE project
     2. Name: Comprehensive XL-MS reveals parallel orientation and versatile conformations of plant HOP2/MND1