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A folded conformation of MukBEF and cohesin

Nature Structural & Molecular Biology volume 26, pages 227–236 (2019)Cite this article

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Abstract

Structural maintenance of chromosomes (SMC)–kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC–kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head–kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.

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Fig. 1: Folded conformation of MukBEF and cohesin.
Fig. 2: Elbow positions revealed by cross-linking and mass spectrometry.
Fig. 3: Structure of the MukB elbow.
Fig. 4: In vivo cross-linking of Pds5 to the Smc1 hinge.
Fig. 5: Conservation of the SMC elbow.
Fig. 6: Models for conformational changes that involve the SMC elbow.

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Code availability

The Xi software suite is available at https://github.com/Rappsilber-Laboratory/XiSearch. Custom code for statistical analysis is available upon request.

Data availability

Crystallographic structure factors and model coordinates have been deposited in the Protein Data Bank (PDB) with accession code 6H2X. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository88 with the dataset identifiers PXD012370 (MukBEF) and PXD012377 (cohesin). Source data for Fig. 5 are available with the paper online. Other data are available upon request.

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Acknowledgements

We are grateful to D. Ciziene for help with crystallography and M. Yu for help with X-ray data collection. We thank X. Deng, F. Coscia and G. Cannone for help with electron microscopy. We thank J. Fredens for advice on recombineering and for the gift of the pheS*-hygR cassette. We thank G. Fisher and D. Sherratt for help with initial complementation experiments and gift of the neoR marker. We thank the staff of beamline I04-1 at Diamond Light Source for assistance during crystallographic data collection. We thank S. Gruber and A. Durand for comments on the manuscript. F.B. is funded by an EMBO Long-Term Fellowship (EMBO ALTF 1151-2017). This work was funded by the Medical Research Council (U105184326 to J.L.), the Wellcome Trust (202754/Z/16/Z to J.L. and 202062/Z/16/Z to B.H.), the DFG (25065445 to J.R.), and the Wellcome Trust through a Senior Research Fellowship to J.R. (103139). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149).

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  1. Stanislau Yatskevich

    Present address: MRC Laboratory of Molecular Biology, Cambridge, UK

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Frank Bürmann, Byung-Gil Lee & Jan Löwe

  2. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

    Thane Than & Bin Hu

  3. Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany

    Ludwig Sinn, Francis J O’Reilly & Juri Rappsilber

  4. Department of Biochemistry, University of Oxford, Oxford, UK

    Stanislau Yatskevich & Kim Nasmyth

  5. Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK

    Juri Rappsilber

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Contributions

F.B. and B.-G.L. purified proteins. F.B. and B.-G.L. performed electron microscopy experiments. L.S. and F.J.O. performed mass spectrometry experiments and identified cross-links. F.B. performed CLMS data analysis and bioinformatics. F.B. performed X-ray crystallography experiments. F.B. and J.L. analyzed X-ray diffraction data. F.B. constructed E. coli strains. T.T. constructed yeast strains and performed in vivo cross-linking experiments. S.Y. conceived the paper model. F.B. prepared the manuscript with input from all authors. J.R., B.H., K.N. and J.L. supervised the work.

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Correspondence to Jan Löwe.

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Integrated supplementary information

Supplementary Figure 1 EM analysis of MukBEF.

a, Negative stain 2D class averages for the folded conformation of native E. coli MukBEF using a circular mask of 640 Å. b, SDS-PAGE analysis of purified Desulfovermiculus halophilus MukBEF. The gel was stained with Coomassie. c, Cartoon of intermediate particle shapes of D. halophilus MukBEF indicating the presence of a coiled-coil elbow in different conformations. d, Cryo-EM imaging of D. halophilus MukBEF in unsupported vitreous ice. Contrast was enhanced by use of a Volta phase plate and high total electron dose. Typical fields of view are shown on the left, examples of single particle images are shown on the right. We estimate that approximately 35 % of particles may adopt a fully folded conformation under the conditions used. Low particle abundance and sample heterogeneity prevented further structural analysis.

Supplementary Figure 2 Cross-linking and mass spectrometry of MukBEF and cohesin.

a, SEC profiles of native co-expressed MukBEF (blue), BS3 treated co-expressed MukBEF (orange), singlet MukBEF (MukBEFS) reconstituted in buffer containing 40 mM NaCl, 2 mM MgCl2 (red) and doublet MukBEF (MukBEFD) reconstituted in buffer containing 200 mM NaCl (green). Reconstitution was similar to protocols established previously (J. Biol. Chem. 281, 34208–34217, 2006). b, SDS-PAGE analysis of a purified cohesin complex containing Smc1, Smc3, Scc1 and Scc3. The gel was stained with Coomassie. c, SEC profiles of the cohesin complex containing Smc1, Smc3, Scc1 and Scc3 before and after treatment with BS3 (see Fig. 1h). d, Inter-subunit cross-links of a cohesin complex containing Smc1, Smc3, Scc1, Scc3 and Scc2. As in Fig. 2a. e, Kernel density estimates for the position of cross-link sites mapped onto the partial structure of the H. ducreyi MukBEF head module (PDB ID 3EUH) and the cohesin Smc1–Scc1 cWHD interface (PDB ID 1W1W). f, Kernel density estimates for long-distance cross-links at the MukB hinge. Probability density for MukB cross-links to MukB sites located at least 500 aa away (left) or to MukEF (middle). The cartoon (right) illustrates an explanation for the observed cross-linking pattern. g, Cross-link midpoint analysis for MukB performed as in Fig. 2c but using random resampling without replacement before data processing. h, Cross-link midpoint analysis for various cohesin datasets (as in Fig. 2). Peak density for human cohesin corresponds to residues 375 and 813 (Smc1) and 379 and 811 (Smc3).

Supplementary Figure 3 Conservation analysis and mutagenesis of the MukB elbow.

a, Sequence alignment of the N-terminal (left) and C-terminal (right) parts of the MukB elbow. Residues chosen for mutagenesis are highlighted by triangles. Eco, Escherichia coli; Mmo, Morganella morganii; Tmo, Thioflavicoccus mobilis; Emo, Endozoicomonas montiporae; Tau, Tolumonas auensis; Osp, Oceanimonas sp. GK1; Btr, Bibersteinia trehalosi; Hdu, Haemophilus ducreyi. b, Sequence conservation (Jensen-Shannon divergence) was mapped onto the structure (high conservation is purple, low conservation is cyan). c, Growth of strains containing point mutations at the elbow in the endogenous mukB gene. d, Construction of a functional mukB-HaloTag allele. e, Protein levels of elbow mutants fused to a HaloTag. Extracts were labelled with a HaloTag-TMR substrate and were analyzed by in-gel fluorescence (top) and Coomassie staining (bottom) after SDS-PAGE. WT, wild-type.

Supplementary Figure 4 BPA-dependent expression of Smc1(K620BPA).

Strains were grown either in the absence or presence of 1 mM BPA, and extracts were analyzed by Western blotting.

Supplementary Figure 5 Locations of coiled-coil discontinuities in bacterial and archaeal Smc proteins.

a, Aggregate coiled-coil probability profile (same as in Fig. 5) and single-sequence profiles for B. subtilis Smc (bacterial) and Pyrococcus yayanosii Smc (archaeal). Positions of coiled-coil discontinuities experimentally determined by X-ray crystallography (Mol Cell 67, 334-347.e5, 2017) or disulfide cross-linking (Proteins 83, 1027–1045, 2015) are highlighted in red. b, The elbow region of P. yayanosii Smc. The predicted coiled-coil probability from aggregate analysis (see a and Fig. 5) is mapped onto the crystal structure of a central arm fragment (PDB ID 5XG2). Positions of the predicted and crystallographically determined discontinuities are shown.

Supplementary Figure 6 Bending of SMC dimers.

a, An SMC dimer with C2 symmetry. Monomers and their body-frame coordinate systems are shown in black or blue. The symmetry axis of the dimer is shown in purple. b, Symmetry breaking upon elbow bending. Option 1: monomers bend into opposite directions; Option 2: monomers twist and bend into the same direction. Orientations of the relevant body-frame coordinate axes are shown at the bottom.

Supplementary Figure 7 Inchworm models for DNA and translocation and loop extrusion.

a, DNA translocation model requiring a regulated grapple DNA binding site and a sliding anchor DNA binding site. DNA binding may or may not involve a DNA entrapping ring that could be used to enhance processivity. b, Loop extrusion using a second anchor site. DNA binding may or may not involve a DNA entrapping ring that could be used to enhance processivity.

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Bürmann, F., Lee, BG., Than, T. et al. A folded conformation of MukBEF and cohesin. Nat Struct Mol Biol 26, 227–236 (2019). https://doi.org/10.1038/s41594-019-0196-z

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