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The structure of the cohesin ATPase elucidates the mechanism of SMC–kleisin ring opening

Nature Structural & Molecular Biology volume 27, pages 233–239 (2020)Cite this article

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Abstract

Genome regulation requires control of chromosome organization by SMC–kleisin complexes. The cohesin complex contains the Smc1 and Smc3 subunits that associate with the kleisin Scc1 to form a ring-shaped complex that can topologically engage chromatin to regulate chromatin structure. Release from chromatin involves opening of the ring at the Smc3–Scc1 interface in a reaction that is controlled by acetylation and engagement of the Smc ATPase head domains. To understand the underlying molecular mechanisms, we have determined the 3.2-Å resolution cryo-electron microscopy structure of the ATPγS-bound, heterotrimeric cohesin ATPase head module and the 2.1-Å resolution crystal structure of a nucleotide-free Smc1–Scc1 subcomplex from Saccharomyces cerevisiae and Chaetomium thermophilium. We found that ATP-binding and Smc1–Smc3 heterodimerization promote conformational changes within the ATPase that are transmitted to the Smc coiled-coil domains. Remodeling of the coiled-coil domain of Smc3 abrogates the binding surface for Scc1, thus leading to ring opening at the Smc3–Scc1 interface.

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Fig. 1: Cryo-EM structure of the cohesin ATPase head module.
Fig. 2: Conformational changes in the engaged cohesin head module lead to remodeling of the NScc1–Smc3 interface.
Fig. 3: The Smc3 and Smc1 ATPase sites are structurally distinct.

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Data availability

Cryo-EM reconstructions are deposited in the Electron Microscopy Data Bank (EMDB accession number EMD-4614). The Smc3–Scc1–Smc1 and CtSmc1–CScc1 structures are deposited in the Protein Data Bank (PDB accession numbers 6QPW and 6QPQ, respectively). Source Data for Fig. 2g and Extended Data Fig. 1c,e are available with the paper online.

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Acknowledgements

We thank M. Hons and M. Karuppasamy (European Molecular Biology Laboratory (EMBL)) for training K.W.M. in cryo-EM, M.W. Bowler (EMBL) for X-ray data collection, S. Cusack (EMBL) and D. Barford (MRC-LMB) for their support, and P.V. Sauer (University of California, Berkeley) for critical reading of the manuscript. This work was funded by EMBL.

Author information

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble, France

    Kyle W. Muir, Yan Li & Daniel Panne

  2. MRC Laboratory of Molecular Biology, Cambridge, UK

    Kyle W. Muir

  3. European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany

    Felix Weis

  4. Department of Molecular and Cell Biology, Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, UK

    Daniel Panne

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Contributions

K.W.M., Y.L. and D.P. conceived the study. K.W.M. and Y.L. designed c-link. K.W.M. and D.P. devised the crosslinking strategy. Y.L. crystallized CtSmc1–CScc1. K.W.M. and F.W. collected cryo-EM data. K.W.M. processed cryo-EM data. Molecular models were built and refined by K.W.M. and D.P. K.W.M. and D.P. wrote the manuscript with input from Y.L. and F.W.

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Correspondence to Kyle W. Muir or Daniel Panne.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cohesin domain organization and purification of c-link complexes.

a, Domain organization and cartoon depiction of the cohesin complex. b, SDS-PAGE analysis of a representative c-link purification. Bands corresponding to each subunit are indicated. c, Size-exclusion chromatography analysis of c-link prior to (orange) and following 3C-mediated cleavage (yellow) of the NScc1–CtCSmc1 linker. Elution volume of molecular weight standards are shown. d, SDS-PAGE analysis of indicated fractions corresponding c, (c-link left of marker; 3C-treated c-link to the right). e, ATPase assays of wild-type (Wt) and Walker B (EQ) mutant c-link. A single experiment was performed at the indicated protein concentration. Data for the graphs in c and e are available as source data.

Source data

Extended Data Fig. 2 Cryo-EM data processing and validation.

a, Following initial 2D classification, several iterations of 3D classification were conducted. Classes are presented as 3D volumes. Percentages of particles sorted into each class are displayed below. The selected final 3D class is boxed, and was proceeded by a further round of 2D classification prior to masked global consensus refinement. b, A representative micrograph is shown. c, Angular distribution plot of final 3D consensus refinement. d, Fourier shell correlation plot. Final overall resolution is 3.2 Å (when FSC=0.143). e, Local resolution of the EM density map (Å) as computed by ResMap. f, EM density and modelled residues corresponding to catalytic motifs of the CtSmc1 ATPase domain, and the coiled-coils (RMSD 2.5). g, EM density and modelled residues corresponding to catalytic motifs of the Smc3 ATPase domain, and the coiled-coils (RMSD 2.5).

Extended Data Fig. 3 Comparative structural analysis of the cohesin ATPase.

a, Structural alignment-based superposition of the RecA N-lobes of apo CtSmc1–CScc1 (red) and ATPγS-bound ySmc1–CScc1 complex (grey; PDB code 1W1W). Cα root-mean-square deviation [RMSD] = 0.98 Å. b, The Smc3–NScc1 ATPγS complex (PDB code 4UX3). c, Relative motions of α-helices within the ctSmc1 ATPase upon ATPγS binding and head heterodimerization. d, Relative motions of α-helices within the ySmc3 ATPase upon ATPγS binding and head heterodimerization. e, The cross-links are positioned in loops between secondary structural elements. f, Structural details around the cross-linked disulfides. Smc3 N1204 and CtSmc1 L1160 are closely apposed in the modeled heterodimer (grey). Replacement of these residues by cysteine allows cross-linking without major distortions in the Smc heterodimer. The cystine disulfide bonds are indicated in yellow.

Extended Data Fig. 4 Nucleotide-induced conformational changes in SMC ATPases.

a, Structural alignment based on ATPγS-bound CtSMC1, the nucleotide-free form of Bacillus subtilis (Bs) SMC and the ATPγS-bound form of Geobacillus stearothermophilus (Gs) SMC. b, Nucleotide free (grey) and bound (green) Pyrococccus furiosis (Pf) Rad50 conformations. Nucleotide binding induces an ~35 °C-lobe rotation. c, Nucleotide free (teal) form of Chaetomium thermophilium (Ct) Smc2 and ATPγS-bound form of CtSmc1 (red). d, Nucleotide free (blue) form of CtSmc4 and ATPγS-bound form of CtSmc1 (red). All structural superpositions were done using the SMC N-lobe.

Supplementary information

Reporting Summary

Supplementary Video 1

Conformational changes of Smc1–Smc3 on binding of ATPγS and heterodimerization. Smc1 in the absence of nucleotide and Smc3 bound to ATPγS and NScc1 (PDB 4UX3) were superimposed and movies generated by morphing between states in PyMOL56. The movie shows how ATPγS binding and Smc heterodimerization lead to remodeling of Smc1 (red), Smc3 (blue) and NScc1 (green) displacement on Smc head engagement. ATPγS is shown as sticks and Mg2+ as gray spheres. For clarity, CScc1 is omitted

Supplementary Video 2

Conformational changes in the Smc1–Smc3 heterodimerization interface. Structures were superimposed and the movie generated as in Supplementary Video 1. The movie shows how ATPase head engagement results in remodeling of ATPase site 2. The Smc3 loop connecting the signature helix α11 and α7 moves toward the signature-coupling helix α4, thus apparently contributing to its displacement and Smc3 coiled coil rearrangement. ATPγS is shown as sticks

Source data

Source Data Fig. 2

Uncropped gel images Fig. 2g

Source Data Fig. 2

Source Data for the graph in Fig. 2g

Source Data Extended Data Fig. 1

Source Data for the graph in Extended Data Fig. 1e and Extended Data Fig. 1c

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Muir, K.W., Li, Y., Weis, F. et al. The structure of the cohesin ATPase elucidates the mechanism of SMC–kleisin ring opening. Nat Struct Mol Biol 27, 233–239 (2020). https://doi.org/10.1038/s41594-020-0379-7

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