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Structure of a meiosis-specific complex central to BRCA2 localization at recombination sites

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Abstract

Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2–BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2–BRCA2–MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2–BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.

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Fig. 1: BRCA2 is sandwiched between two MEILB2 homodimers.
Fig. 2: Hydrophobic MEILB2–MEILB2 and ionic MEILB2–BRCA2 interfaces drive formation of the 4:2 complex.
Fig. 3: The MEILB2–BRCA2 assembly falls apart in the presence of mutations at the MEILB2–BRCA2 interface.
Fig. 4: Mutations at the MEILB2–BRCA2 interface compromise recruitment of both proteins to recombination nodules in mouse spermatocytes.
Fig. 5: Model for BRCA2 recruitment to sites of meiotic HR.

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Data availability

Coordinates and structure factors of the MEILB283-end–BRCA2MBD crystal structure are deposited in the Protein Data Bank (PDB) under accession code 7LDG. Source data are provided with this paper.

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Acknowledgements

We thank J. S. Brunzelle at the Life Sciences Collaborative Access Team (LS-CAT) beamline of the Argonne National Laboratory for help with X-ray diffraction data collection and initial processing, the University of Michigan Structural Biology Supergroup for helpful suggestions, V. M. Tesmer of the Nandakumar laboratory for help with collection of crystals and input on figures, and the members of the Nandakumar laboratory for critical feedback on the manuscript. This work was supported by NIH grants R01-AG050509 (J.N.) and R01-GM120094 (J.N.), American Cancer Society Research Scholar grant RSG-17-037-01-DMC (J.N.), an American Heart Association predoctoral fellowship award ID 830111 (R.A.), Assar Gabrielssons Foundation grant FB-20-57 (J.Z.), European Research Council grant StG-801659 (H.S.), Swedish Research Council grant 2018-03426 (H.S.), Cancerfonden grant 2018/326 (H.S.) and the Knut och Alice Wallensbergs Stiftelse KAW2019.0180 (H.S.).

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Author notes

  1. Devon F. Pendlebury

    Present address: Pharmaceutical Sciences, University of California Irvine, Irvine, CA, USA

  2. These authors contributed equally: Devon F. Pendlebury, **g**g Zhang.

Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Devon F. Pendlebury, Ritvija Agrawal & Jayakrishnan Nandakumar

  2. Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA

    Devon F. Pendlebury & Jayakrishnan Nandakumar

  3. Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

    **g**g Zhang & Hiroki Shibuya

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  1. Devon F. Pendlebury
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Contributions

J.N., D.F.P. and H.S. designed the study. Crystallography was performed by D.F.P. and J.N. R.A. and D.F.P. performed in vitro validation of the structural interface. J.Z. performed the in vivo validation of the structural interface, with help from H.S. All authors were involved in data analysis. J.N., D.F.P. and H.S. wrote the manuscript, with critical feedback from the remaining authors.

Corresponding authors

Correspondence to Hiroki Shibuya or Jayakrishnan Nandakumar.

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Peer review information Nature Structural & Molecular Biology thanks Mark Glover, John Weir and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Electron density for BRCA2MBD.

The sandwich between MEILB283-end a and B subunits, and BRCA2MBD is shown in cartoon representation along with 2Fo-Fc electron density map encompassing BRCA2MBD contoured at 1.0 σ.

Extended Data Fig. 2 MEILB2-BRCA2 quaternary structure.

(A) B-factor heat map (blue→red indicates low→high B-factors) for the MEILB283-end-BRCA2MBD complex highlighting the ordered nature of the MEILB2-MEILB2 and MEILB2-BRCA2 interfaces. (B) Back view and (C) side view (right) of the human MEILB283-end-BRCA2MBD complex (relative to depictions in Fig. 1) shown in cartoon representation. Note in panel C how the NCS two-fold axis of MEILB283-end homodimerization is oblique (that is, neither parallel nor perpendicular) with respect to the crystallographic two-fold axis of 4:2 complex formation. (D) Head-to-tail anti-parallel alignments of the N terminus of one BRCA2MBD chain (aa 2276–2282; head) and the C terminus of another BRCA2MBD chain (aa 2335 and upstream residues; tail) in the crystal lattice results in the formation of a triangular 6:3 MEILB283-end-BRCA2MBD assembly from three 2:1 complexes around the three-fold crystallographic axis. (E) Further inclusion of the symmetry mates related by the two-fold crystallographic axis results in a 12:6 assembly in the crystal lattice. (F) SEC-MALS analysis of MEILB283-end - BRCA2MBD ΔPLILVGE strongly suggests formation of a 4:2 complex. (G) Anti-FLAG co-immunoprecipitation (co-IP) analysis of HEK 293T cell lysates containing transiently transfected FLAG-tagged MEILB283-end, myc-tagged MEILB283-end and either eGFP-myc-vector control, eGFP-myc-tagged BRCA2MBD wild type, or eGFP-myc-tagged BRCA2MBD ΔPLILVGE constructs. Data are representative of three replicates.

Source data

Extended Data Fig. 3 MEILB2 ARM repeat domain structure and homodimerization is an adaptation of the extensive ARM repeat structure of β-catenin.

(A) Overlay of 9 different ligand-bound β-catenin structures in the PDB showing the ligands tracing the concave track formed by tandem ARM repeats. β-catenin and its binding partners from the various structures are shown in different shades of green and blue, respectively. (B, C) Closeup of the partner-bound concave surfaces at the N-terminal (B) and C-terminal (C) ARM repeats of β-catenin. PDB IDs: 1qz7, 1jdh, 3ouw, 1jpp, 1g3j, 2gl7, 1i7x, 1i7w, and 1jpw. (D) Alignment of the MEILB283-end B subunit with its top DALI server alignment hit, β-catenin (PDB ID: 1i7w). (E) Alignment of MEILB283-end B with β-catenin as in panel D but also showing the homodimeric partner MEILB283-end A subunit that was not used in the alignment. (F) The hairpin connecting two helices of β-catenin that align with the N-terminal helices of MEILB283-end A and B is structurally replaced by the disulfide linkage between the MEILB283-end subunits. (G) The helices of the N-terminal ARM repeat of MEILB283-end A superimpose with β-catenin regions but the polarity of the helices (n→c) is reversed. Curved lines indicate the superimposed helices of β-catenin and MEILB283-end A. Arrowhead indicates disulfide linkage.

Extended Data Fig. 4 Schematic showing the polar interactions between the BRCA2MBD and its two MEILB2 binding partners.

Ionic (salt-bridge; left) and H-bonding (right) interactions along the entire BRCA2MBD chain is shown schematically. MEILB2 and BRCA2 color-coding is retained from structural depictions in main and other supplementary figures.

Extended Data Fig. 5 Salt-bridges dominate the MEILB2-BRCA2 interface.

(A–H) 2Fo-Fc electron density map contoured at 1.0 σ and stick representations for the indicated interactions at the MEILB2-BRCA2 interface. (I, J) Consurf analysis of MEILB283-end (I) and BRCA2MBD (J) showing the high level of conservation of the MEILB2-MEILB2 (blue asterisks) and MEILB2-BRCA2 (red asterisks) interface residues.

Extended Data Fig. 6 The MEILB2-MEILB2 interface is bolstered by BRCA2.

(A) Co-IP analysis of MEILB283-end – BRCA2MBD in the presence of MEILB2 mutations including the D268R/D326H double mutant. (B) Anti-FLAG co-immunoprecipitation (co-IP) analysis of HEK 293T cell lysates containing the indicated transiently transfected with wild type myc-tagged MEILB283-end and indicated FLAG-tagged MEILB283-end constructs harboring mutations in the MEILB283-end homodimerization interface. (C) Co-IP analysis of MEILB2-MEILB2 association in the absence and presence of BRCA2MBD. (D) Co-IP of MEILB283-end homodimerization mutants in the presence of wild type myc-tagged MEILB283-end. WT: wild type. Data are representative of at least three replicates.

Source data

Extended Data Fig. 7 Biochemical analysis of single-mutations in each half of the MEILB2-binding segments of BRCA2MBD.

(A) Size-exclusion profiles of purified human MEILB283-end -BRCA2MBD complexes containing either wild type, D2294R or D2317R BRCA2MBD subunits. (B–D) Coomassie-stained SDS-PAGE analysis of fractions for the size-exclusion analysis shown in panel A. (E) SEC-MALS of purified human MEILB283-end-BRCA2MBD D2294R rules out complete dissociation of MEILB283-end -BRCA2MBD or dissociation into smaller (that is, 2:1) complexes; dRI: differential Refractive Index. MEILB283-end-BRCA2MBD D2317R could not be analyzed by SEC-MALS because of closely eluting BRCA2MBD-complexed and free MEILB2 peaks (seen in panel A). WT and mutant complexes were expressed and purified in the same experiment. WT complex has been purified for more than three times while the two mutant complexes were purified once.

Source data

Extended Data Fig. 8 BRCA2MBD and MEILB2 interaction is indispensable for their recombination nodule localization.

(A) Immunoblot of testis extracts after electroporating GFP-BRCA2MBD wild type (WT) or indicated mutants, probed with the indicated antibodies. Asterisks indicate non-specific bands. GFP and actin blots in each lane correspond to the same processed sample run on the same gel but blotted sequentially after strip**. (B) Immunostaining of WT and Meilb2−⁄− mouse spermatocytes expressing GFP-MEILB2 R204D mutant protein. Note that axis-associated foci seen in WT were not detectable in Meilb2−⁄− spermatocytes even after the intensification of the signals. Scale bars: 5 μm in the main panel and 1 μm in the magnified panel. Data are representative of two repeats.

Source data

Extended Data Fig. 9 Protein expression and localization of the MEILB2 dimerization mutants.

(A) and (D) Immunostaining of WT spermatocytes expressing indicated GFP-MEILB2 WT and mutant proteins. (B) and (E) Quantification of GFP foci intensities represented in panel A and panel D, respectively, normalized with the average value of WT. n shows the analyzed foci number pooled from four cells from one electroporated mouse. All analyses used two-tailed t-tests. ****p < 0.0001 (WT vs. Δ1–87: 1.81285 ×10−14). Scale bars: 5 μm. (C) and (F) Immunoblots of testis extracts after electroporating indicated GFP-MEILB2 WT and mutant proteins, blotted with the indicated antibodies (repeated two times). GFP and actin blots in each lane correspond to the same processed sample run on the same gel but blotted sequentially after strip**.

Source data

Supplementary information

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Source Data Fig. 1

Unprocessed gel.

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Unprocessed western blots.

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Spreadsheet for quantitation.

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Unprocessed western blots.

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Unprocessed gels.

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Unprocessed western blots.

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Unprocessed western blots.

Source Data Extended Data Fig. 9

Spreadsheet for quantitation.

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Pendlebury, D.F., Zhang, J., Agrawal, R. et al. Structure of a meiosis-specific complex central to BRCA2 localization at recombination sites. Nat Struct Mol Biol 28, 671–680 (2021). https://doi.org/10.1038/s41594-021-00635-0

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