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

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

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Free PMC article

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

Evelyn Rampler et al. J Proteome Res. .
Free PMC article

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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.

Figures

Figure 1
Figure 1
Native PAGE analysis of heterologously expressed HOP2− MND1. The complex was expressed in Rosetta (DE3) pLysS E. coli cells and purified via two rounds of SEC (Superdex 200 16/60 column). 10% resolving gel; S1; 4 μL of 20 μM HOP2−MND1; S2; 2 μL of 20 μM HOP2−MND1; M: molecular weight marker.
Figure 2
Figure 2
Venn diagrams of reactive lysine sites and amine-reactive cross-linking products. (A) Overlap of reactive lysine sites in HOP2− MND1 for EDC, DSS, and BS2G (combined BS2Gd0 and BS2Gd0d6). (B) Identified unique amino acid sites observed for mono-, loop-, and cross-links in HOP2 and MND1.
Figure 3
Figure 3
Gain of unique protein positions per replicates. The plot shows the gain in unique cross-linking sites for each cross-linker and cross-linking product. The given number of unique cross-linking sites is the average over the replicates. For every replicate shown, the average is based on all relevant combinations of the four replicates.
Figure 4
Figure 4
Circos plot of the amine-reactive- (A) and EDC- (B) derived cross-links within the three domains of HOP2 (red) and MND1 (brown). Line thickness corresponds to the number of identified cross-link spectra. Lysine positions are marked in yellow. (A) Amine-reactive links: Gray lines correspond to cross-links derived from DSS, whereas green lines indicate BS2G cross-linking products. (B) EDC links: Black lines correspond to cross-links derived from EDC. MND1_N: N-terminus of MND1 protein. MND1_CC: coiled-coil region of MND1. MND1_C: C-terminal domain of MND1. HOP2_N: N-terminal domain of HOP2. HOP2_CC: coiled-coil region of HOP2. HOP2_C: C-terminus of HOP2.
Figure 5
Figure 5
Comparative modeling workflow to analyze the open conformation of the HOP2−MND1 heterodimer. (A) Modeling of the individual proteins HOP2 and MND1. (B) Protein–protein docking for the HOP2−MND1 model in complex.
Figure 6
Figure 6
Structural models for HOP2−MND1 in A. thaliana. (A) A. thaliana HOP2−MND1 model predicted by HADDOCK. Matching distance restraints are shown as black lines. AtHOP2 is shown in red and AtMND1 in blue. (B) A. thaliana HOP2−MND1 superimposed on the G. lamblia using GlHOP2 as the reference chain. The A. thaliana HOP2 chain is labeled in red, MND1 in dark blue, the Giardia Hop2 shown in orange, and the Mnd1 in light blue. HOP2 and MND1 are organized in an elongated, open complex with two N-terminal winged-helix domains (WHDs). The central region consists of two split coiled coils (coiled coil 1 and 2) in parallel orientation and represent the major interaction site. Coiled coil 3, together with the capping helices, form the C-terminus of the heterodimer.

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