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A Folded Conformation of Mukbef and Cohesin

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A Folded Conformation of Mukbef and Cohesin ARTICLES https://doi.org/10.1038/s41594-019-0196-z A folded conformation of MukBEF and cohesin Frank Bürmann 1, Byung-Gil Lee1, Thane Than2, Ludwig Sinn 3, Francis J O’Reilly3, Stanislau Yatskevich4,6, Juri Rappsilber 3,5, Bin Hu 2, Kim Nasmyth4 and Jan Löwe 1* 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 discontinu- ity, 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. ll organisms maintain enormous chromosomal DNA mol- along DNA, and some, by doing so, might actively organize chro- ecules whose contour lengths exceed cellular dimensions by mosomes by building up loops32–34. Aorders of magnitude. Hence, both regulation of gene expres- To explore how the core activities of SMC–kleisin complexes sion and accurate chromosome segregation require a high degree of might be implemented on a structural level, we have investigated spatial organization. SMC–kleisin complexes are ancient machines the architecture of two representative complexes that are separated that help to organize chromosome superstructure in bacteria, by a billion years of evolution: MukBEF from E. coli and cohesin archaea and eukaryotes1. They are essential for chromosome seg- from budding yeast. We find that both complexes contain a bend- regation in many bacteria2,3, and indispensable for both mitosis and able coiled-coil discontinuity in their arms that allows them to inter- meiosis in eukaryotes4–8. convert between extended and folded conformations, in the latter At the core of SMC–kleisin complexes is a conserved tripartite bringing the hinge dimerization domain closer to the head–kleisin protein ring composed of an SMC homo- or heterodimer, bridged module. Our findings show that SMC proteins have the capacity for by a kleisin subunit9–12. SMC proteins are elongated molecules con- a large conformational transformation, and provide the basis for taining an ABC-type ATPase head and a hinge dimerization domain investigating long-distance domain movements during DNA load- at opposite ends of an approximately 50 nm long intramolecular and ing and translocation reactions. antiparallel coiled-coil arm9,13–16. The kleisin asymmetrically con- nects the two heads of an SMC dimer and contains binding sites for Results additional KITE or HAWK subunits17,18. A folded conformation of MukBEF and cohesin. MukBEF is a A widely conserved and possibly fundamental aspect of SMC– diverged SMC–kleisin complex that serves as an essential chromo- kleisin activity is the ability to entrap chromosomal DNA within some-organization machine in E. coli11,27,35,36. The complex comprises their ring structure19–21. DNA entrapment is the molecular basis for the SMC protein MukB, the kleisin MukF and the KITE protein sister chromatid cohesion by the cohesin complex, and might also MukE. We co-overexpressed the MukBEF subunits in E. coli and pre- be used by cohesin, condensin and bacterial Smc–ScpAB to organize pared the complex using a multi-step procedure that yielded puri- DNA into large loops. Loading of DNA into the complex is thought fied material without extra residues on any of the subunits (Fig. 1a). to involve transient opening of a ring interface in cohesin22,23, and is The purified complex eluted as a single peak in size-exclusion chro- likely to be mediated by the SMC arms in Smc–ScpAB24. matography (SEC) (Fig. 1b) and was analyzed by negative-stain The second possibly universal aspect of SMC–kleisin activity is electron microscopy (EM) immediately after elution from the col- their translocation along DNA. Cohesin and bacterial SMC–kleisin umn (Fig. 1c,d). Although subject to heterogeneity, most particles complexes associate with chromosomes in a manner that requires had a characteristic double cherry-like shape, composed of a two- ATP binding and they redistribute or translocate from initial load- lobed density (the MukB head–MukEF module) from which a stalk ing sites to adjacent regions dependent on ATP hydrolysis25–28. emerged (the MukB arms). Surprisingly, many particles possessed a Translocation of bacterial Smc–ScpAB coincides with progressive stalk length of about 24 nm, roughly half of what is expected for an linking of distant chromosomal loci in vivo, indicating that the extended MukB arm consisting of canonical coiled-coil segments. complex might actively extrude DNA loops28,29. Recently, ATPase- As evident from partially extended particles, this conformation dependent DNA translocation and loop extrusion reactions have was caused by folding at a kink close to the center of the MukB been reconstituted for purified condensin in vitro30,31. These find- arms. We refer to this kink as the ‘elbow’, as it connects the ings support the idea that SMC–kleisin complexes are motor pro- upper and lower parts of the arms (Fig. 1d). Fully extended teins that use the ATPase activity of their SMC subunits to track particles were also observed but were less apparent. Using 1MRC Laboratory of Molecular Biology, Cambridge, UK. 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK. 3Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany. 4Department of Biochemistry, University of Oxford, Oxford, UK. 5Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK. 6Present address: MRC Laboratory of Molecular Biology, Cambridge, UK. *e-mail: [email protected] NatURE STRUCTURAL & MOLECULAR BIology | VOL 26 | MARCH 2019 | 227–236 | www.nature.com/nsmb 227 ARTICLES NATURE STRUCTURAL & MOLECULAR BIOLOGY a b c IEX Conductivity SEC 29 81 [mS cm–1] 8 MukBEF kDa MukEF MukB 140 6 MukBEF MukB 115 80 4 66 MukF 50 2 40 30 Normalized absorbance 0 MukE 25 0.8 1.0 1.2 1.4 1.6 1.8 2.0 50 nm 15 Elution volume (ml) d Folded Elbow Hinge 20 nm 20 nm 20 nm 20 nm 20 nm Arm Loose 20 nm 20 nm 20 nm 20 nm 20 nm 30 nm Head ~ MukEF Extended 20 nm 20 nm 20 nm 20 nm 20 nm e f Peak 1 Peak 2 8 Peak 2 6 MukBEF(BS3) MukBEF 4 Peak 1 Normalized absorbance 2 Void 0 50 nm 50 nm 0.8 1.0 1.21.4 1.61.8 Elution volume (ml) g Hinge Elbow Hinge 20 nm 20 nm 20 nm 2 20 nm 20 nm 20 nm 1 49 nm Elbow Peak Peak 24 nm 20 nm 20 nm 20 nm 20 nm 20 nm 20 nm 20 nm 20 nm h Cohesin Elbow Elbow 20 nm 20 nm 24 nm Hinge Hinge Joint 20 nm 20 nm 37 nm Joint ~ 20 nm 50 nm Head 20 nm 20 nm Scc3 Fig. 1 | Folded conformation of MukBEF and cohesin. a, Purification of MukBEF. Elution of the MukBEF complex from a Q ion exchange (IEX) column. Peak fractions were separated by SDS-PAGE and stained with Coomassie blue. An uncropped gel image is shown in Supplementary Dataset 1. b, SEC of the MukBEF complex, MukB and MukEF. Proteins were separated on Superose 6 Increase. c, Negative-stain EM of native MukBEF. A typical field of view is shown. d, Particle instances for observed MukBEF conformations are shown on the left. A cartoon highlighting the position of the elbow is shown on the right. e, SEC profiles for native and MukBEF cross-linked with BS3 are shown. f, Negative-stain EM of BS3-cross-linked MukBEF. Typical fields of view for particles from SEC peak 1 and SEC peak 2 are shown. g, Negative-stain 2D class averages for extended (left) and folded (right) conformations, using circular masks of 948 Å and 640 Å, respectively. Data were collected from samples of peak 1 and peak 2 of the SEC shown in e. h, Negative-stain EM of BS3-cross-linked cohesin. A typical field of view is shown on the left. Class averages using a circular mask of 500 Å are shown in the middle panel. 228 NatURE STRUCTURAL & MOLECULAR BIology | VOL 26 | MARCH 2019 | 227–236 | www.nature.com/nsmb NATURE STRUCTURAL & MOLECULAR BIOLOGY ARTICLES ab cWHD N MukF 351 440 1 Head 290 High N Subunits Cross-links 0.05 MD 235 Neck Probability density (a.u. 275 121 nWHD 115 MukF nWHD 1 N 234 Arm MukE cWHD MukE 96 MukB nWHD 666 1 MD ) False discovery rate 1486 Hinge 779 Low 0 C Head 1233 1193 ArmC NeckC d Subunits Cross-links c Arm MukB Joint Head Hinge High Midpoints Probability density (a.u. 0.020 Smc3 365 Elbow e Scc1 0.015 Coordinat 1 1 1,486 Residue nαHD 0.010 ) 0.005 Probability density Low 0.000 050 100 150 200 250 300 350 Head Normalized arm coordinate (aa) e Smc1 Smc3 Arm Arm Head Hinge Head Hinge Midpoints Midpoints 0.030 323 323 e Elbow 0.025 e Elbow y 0.025 Coordinat 0.020 Coordinat 1 1 0.020 1 1,225 11,230 Residue 0.015 Residue 0.015 0.010 0.010 Probability densit 0.005 Probability density 0.005 0.000 0.000 050 100 150 200 250 300 050100 150200 250300 Normalized arm coordinate (aa) Normalized arm coordinate (aa) Fig.
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