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

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.

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.

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.

Figure 1. CryoEM structure of the cohesin ATPase head module.

a, Domain organisation and cartoon depiction of c-link. All construct used are from Saccharomyces cerevisiae unless indicated Chaetomium thermophilium (Ct). b, SDS-PAGE analysis of disulphide crosslinked c-link (’x-linked’). c, Low-pass filtered density map of the cohesin head module (blue: Smc3; Green: CScc1; red: CtSmc1). d, Cryo-EM densities at 2.5 RMSD (gray mesh) are superimposed on the atomic model of the ATPγS–Mg²⁺ molecules. Conserved elements of the ABC ATPase modules are labelled for orientation. e, Cryo-EM density at 2.5 RMSD of the Signature motif helix from CtSmc1. f, Cryo-EM density at 1.5 RMSD of the coiled coil from Smc3. There is no apparent density for NScc1. g, Ribbon diagram of the model of the cohesin head module with each colored according to each subunit.

Figure 2. Conformational changes in the engaged cohesin head module lead to remodeling of the NScc1–Smc3 interface.

a, Conformational changes in the coiled coil of the ATPγS and Mg²⁺ engaged cohesin head module as compared to homodimeric Smc3 (PDB 4UX3) and apo CtSmc1 (both in grey) are indicated with curved arrows. The binding site of NScc1 (green) is indicated. Inset: zoomed view of the ATPase active site. The directions of movements are indicated with arrows in α3, the signature-coupling helix α4 and the N-terminal end of the coiled coil α5 in the C-lobe of CtSmc1 (red) and Smc3 (blue). b, Details of the displacement of α3, the signature-coupling helix α4 and the coiled coil α5 in CtSmc1 (bound and apo forms are depicted in red and grey respectively). c, and d, Details of the displacement of Smc3 coiled-coil and signature-coupling helices upon cohesin head engagement and DNA exit gate release (hetero- and homodimeric forms of Smc3 are depicted in blue and grey respectively). For clarity, NScc1 is omitted from the homodimeric form. e, Surface representation of the Smc3 coiled coil (blue) from the ‘closed’ Smc3–NScc1 gate structure. PDB:4UX3. Amino acid residues from NScc1 (green; left) engage Smc3 through a set of surface pockets lined by conserved hydrophobic amino acid residues of Smc3 (right). f, Surface representation of the rearranged Smc3 coiled coil (blue). The NScc1 binding site is occluded by remodeling of the cognate Smc3 interface. g, C-link complexes, harboring a 3C protease in NScc1, were cross-linked and incubated with 3C prior to immobilization on Ni²⁺-conjugated beads. Quantification of NScc1 retention by c-link complexes in the presence or absence of nucleotide is depicted in a scatter plot. Representative silver-stained SDS-PAGE bands are boxed, and the corresponding proteins indicated. Data from two independent experiments are plotted. Data for the graph and uncropped gel images are available as source data.

Figure 3. The Smc3 and Smc1 ATPase sites are structurally distinct.

a, Amino acid residue interactions in the ATPγS-engaged Smc3–CtSmc1 heterodimer and comparison with the hypothetical apo Smc heterodimer interface. Interactions (i.e. hydrogen bonds and salt bridges) are indicated by yellow dashed lines. b, Mutations which suppress cohesin release (green) cluster at the Smc3 ATPase site. Residues in close proximity to the Smc3 acetyl-lysines and which may influence allosteric regulation of the ATPase are depicted in the top inset. Suppressors within the Smc3 ATPase directly influence hydrolysis (bottom inset). c, Model of the cohesin ATPase cycle (Clockwise from left-hand side). DNA capture is mediated by Scc2–4 and depends on ATP. DNA release is catalyzed by Wapl–Pds5. Heterodimerization and ATP-binding causes conformational changes within the Smc3 coiled-coil domain, stabilized by a ‘latch’-like function of the Q-loop (inset) which lead to Scc1 displacement and cohesin release. ATP hydrolysis permits the cycle to resume. Cohesin is liberated from this cycle by acetylation of Smc3, which counteracts release and establishes cohesion (right-hand side).