Closing the cohesin ring: structure and function of its Smc3-kleisin interface

Abstract

Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.

Figure 1. Crystal structure of the Smc3hdCC:Scc1-N complex

(A) The coiled coil segment of Smc3 (blue) is interrupted by a ‘kink’. The NTD of Scc1, Scc1-N (green), binds to the coiled coil segment of Smc3, leading to a four-stranded helical arrangement. Inset: aberrant homodimer formation of Smc3 head domains in the crystals. (B) A superposition of the Smc3hdCC:Scc1-N crystal structure (blue, green; this work) with Smc1hdCC:Scc1-C (red, yellow; PDB 1W1W) reveals that in addition to the ATPase fold, the position of the coiled coil segments is conserved. Crucially, Scc1 binding is completely different for Smc3 and Smc1. (C) Sequence conservation of Scc1’s NTD. (D) ATP binding leads to sandwich dimer formation of the head domains of Smc1 and Smc3, closing the ring temporarily. According to the ring model, Scc1 more permanently bridges the two head domains, which can be released through separase-mediated cleavage of Scc1 or in a separase-independent pathway through opening of the Smc3:Scc1 gate. Scc1 contains many more residues in the middle domain. Separase cleavage sites, Pds5 (7) and Scc3 (Brunet et al., in press) binding sited highlighted. (E) Detail of the KKD strand whose acetylation by Eco1 reduces separase-independent cohesin release. It is far away from the nucleotide binding site on the head domain but the acetylation state may influence the nucleotide binding site through the helix containing R61.

Figure 2. Testing the Smc3-kleisin crystal structure

(A) Conservation of Smc3’s coiled coil. The surface associated with Scc1 is highly conserved but the solvent-exposed side is not. (B) Thiol-specific cross-linking (bBBr) between α2 and α3 of Scc1-N and Smc3’s coiled coil (CC) after immuno-precipitation of Scc1-HA6. Cross-linking specific to K48C-K1032C was observed in cells expressing C56S (K19796, K19769, K19727, K19732, K19764, K23102, K23103). All mutations were functional and all observed cross-links were dependent on a pair of cysteine substitutions. (C) Scc1-N α2 and α3 helices (green), Smc3 coiled coil (Smc3CC, blue), and substituted residues (yellow).

Figure 3. Functional analysis of the Smc3-kleisin structure

(A) Essential Scc1-N residues (11) situated within the Smc3-kleisin interface. (B) Conserved residues in Scc1-N (D92, Y82) and Smc3CC (R1015, L1019, I1026, L1029) whose mutation disrupts interaction and causes lethality. (C) Abolition of Scc1-N/Smc3CC cross-linking by Scc1L89K (K19796, K23144, K23145) and Smc3L1029R (K23146, K23128, K23129) as measured by cross-linking C56-S1043C with bBBr following immuno-precipitation of Scc1-HA6. Western blots of Smc3-myc9 and Scc1-HA6 are shown. (D) Diploid cells expressing ectopic WT or mutant Smc3-GFP with white arrowhead pointing at the cohesin peri-centromeric barrel structure absent in the case of L1029R and R61Q (K23107-23109). (E) Scc1Y82A is lethal at 32 °C (K699, K16296, K23105-6). (F) A summary of mutations created and characterized. Lethal (red) and temperature sensitive (brown) mutations, both in Scc1 and Smc3, were found. Note the viability of Scc1D95A, K99D, K99A, and D95A K99A mutants (K23111-23117).

Figure 4. Cohesin forms heterotrimeric rings in vivo

(A) In vivo thiol-specific cross-linking (BMOE) between α2 and α3 of Scc1-N and the Smc3 coiled coil (CC) followed by immuno-precipitation of Scc1-HA6. In all shifted bands Smc3 is acetylated (K19796, K19732, K19764, K23103). (B) In vivo thiol-specific cross-linking of the three Smc1/Smc3/Scc1 interfaces followed by immuno-precipitation of Scc1-PK6 and observation of TMR fluorescence associated with Smc3-Halo. A circular form (Ci) only appears when all three interfaces are linked (black arrowheads). Western blotting using an antibody specific for acetylated K113 shows that Ci is acetylated (last lane) (strains K22013-K22020). (C) Cohesin forms heterotrimeric rings but not higher order complexes. Halo-tagged Ci (black arrowhead) associated with PK- and HA-tagged proteins from Scc1-PK6/Scc1-PK6, Smc3-HA6/Smc3-Halo diploids after in vivo cross-linking. No Smc3-Halo is present in form Ci molecules that had been immuno-precipitated with HA antibodies, implying that they are circular trimers, not tetramers (K22590).

Figure 5. Heterotrimeric cohesin rings trap sister DNAs both in vitro and in vivo

(A) Dimers of a 2.3 kb circular minichromosome were isolated using sucrose gradients (T=Top, B=Bottom) from strains containing five (5C) or six (6C) cysteines within the Smc1/Smc3/Scc1 interfaces (K20300, K20279). (B) Electrophoresis of dimers denatured with SDS following isolation from sucrose gradients and cross-linking with bBBr or BMOE. DNAs detected by Southern blotting. (C) Electrophoresis of Scc1-PK6 immuno-precipitated DNAs denatured with SDS after in vivo BMOE crosslinking of cycling cells. DNAs detected by Southern blotting. Monomeric circles (M), catenated monomers (CM), nicked DNA (*), catenated dimers (CD)and cohesin catenated dimers (CCD) (K20280, K20279).