Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 2;81(23):4891-4906.e8.
doi: 10.1016/j.molcel.2021.10.011. Epub 2021 Nov 4.

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites

Frank Bürmann  1 , Louise F H Funke  2 , Jason W Chin  2 , Jan Löwe  3
Affiliations

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites

Frank Bürmann et al. Mol Cell. .

Abstract

The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.

Keywords: MukBEF; SMC; chromosome organization; cohesin; condensin; cryo-EM; loop extrusion.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Cryo-EM structure of MukBEF-MatP bound to two distinct DNA double helices (A) Reconstitution of MukBEF dimers. Co-purified MukBEF and free MukB were mixed (top) and resolved by SEC (bottom). (B) Composition of the MukBEF-MatP-matS sample used for structure determination. (C) Coomassie stained SDS-PAGE gel of the reconstituted complex used for cryo-EM. (D) Example micrograph of the sample used for structure determination. (E) A 4.6-Å-resolution cryo-EM density map (left, EMDB: EMD-12657) and complete atomic model (middle, right, PDB: 7NYX) of the DNA-bound MukBEF-MatP monomer. (F) Slice through a 3.1-Å-resolution cryo-EM density map of the DNA-binding region of MukBEF-MatP (EMDB: EMD-12656, PDB: 7NYW). (G) κ-MukB and ν-MukB superimposed on the head domain. The arms adopt radically different conformations. (H) DNA binding topology on plectonemic loops inferred from the DNA crossing angle Θ. The crossing angle convention employed by Rawdon et al. (2016) is used. The schematic on the right shows the simplified topology used for clarity throughout, with the in-reality elbow-folded conformation flattened into a ring. See also Figures S1–S3 and Video S1.
Figure 2
Figure 2
DNA binding and subunit interfaces of the MukBEF-MatP complex (A) Model of the DNA-bound head module (PDB: 7NYW). (B) Path of the kleisin MukF and DNA contacts of the MukB larynx. (C) Interface between the MukF linker and the clamped DNA. (D) Interface between the top surface of the ν-MukB head and the clamped DNA. (E) Interface between MukE and MatP. (F) Interface between the MukB joint and MatP. (G) Interfaces between MukB and AcpP and between the κ-MukB joint and the hinge-proximal arm of ν-MukB. See also Figures S2 and S3 and Video S1.
Figure 3
Figure 3
Architecture of apo-MukBEF and the MukBEF dimer (A) Model of the apo-MukBEF monomer (PDB: 7NYY) and 6.8-Å cryo-EM density (EMDB: EMD-12658) at low contour level. (B) Model for the apo-MukBEF dimer (PDB: 7NZ4) and 13-Å cryo-EM density (EMDB: EMD-12664). (C) The 11-Å cryo-EM density (EMDB: EMD-12662) and model (PDB: 7NZ2) for two MukBEF dimers bridged by four MatP-DNA complexes (“MukBEF tetrad”). The apo-MukBEF dimer is shown on the left. (D) The 11-Å cryo-EM density (EMDB: EMD-12663) and model (PDB: 7NZ3) for a MukBEF tetrad with closely apposed dimers. Only one monomer for each MukBEF dimer was modeled due to weak density for their partner monomers. (E) Schematic for variable positioning of the clamp DNA binding site, as shown in (C) and (D). Only a single MukBEF dimer and only two of the four DNAs are shown for clarity. See also Figures S2 and S3 and Video S2.
Figure 4
Figure 4
Conformational changes associated with release of MatP/DNA/ATP (A) Cryo-EM densities for the MukBEF-MatP-DNA complexes with different arm conformations (EMDB: EMD-12660, EMD-12659, EMD-12657, and EMD-12658; PDB: 7NZ0, 7NYZ, 7NYX, and 7NYY). (B) Blocking of MatP and DNA binding sites at the MukB joint and larynx. Structures were superimposed on the ATPase domains. MatP/DNA/ATP-bound conformation is shown in color, and apo conformation is in gray. (C) Conformational change at the MukB neck/hinge interface (left) and at the hinge-proximal arm (right). Structures were superimposed on the ATPase domain (left) or the hinge (right). (D) Cryo-EM density at the neck gate in the MatP/DNA/ATP-bound state. The solvent accessible cleft between MukB and MukF is indicated by a double arrow. (E) Superimposition of the neck gate in apo and MatP/DNA/ATP-bound states (top). Minimum backbone VDW distances of the interface are given (bottom). See also Figures S2 and S3 and Video S3.
Figure 5
Figure 5
Detection of arm folding in vivo (A) Residues employed as sensors for the folded conformation. The folded conformation and a tentative extended conformation based on the structure of the extended elbow (PDB: 6H2X) are shown on the left. A close-up on the P. thracensis structure is shown on the right. Corresponding E. coli residues are in parentheses. (B) BMOE reaction scheme (top) and BMOE mediated in vivo cysteine cross-linking of E. coli strains carrying sensor cysteine mutations (bottom). Reaction products were detected by SDS-PAGE and in-gel fluorescence using a TMR fluorophore bound to MukB-HaloTag. See also Figures S4–S6.
Figure 6
Figure 6
Mapping of DNA binding topology in vivo (A) Principles (left) and workflow (right) of the chromosome entrapment assay in agarose plugs. (B) Combinations of cross-links used for probing DNA entrapment in the ring, clamp, and frame compartments. Hinge cross-link, C730 and R771C in MukB; cap cross-link, Q412C in MukF and R143C in MukB; neck cross-link, D227C in MukF and K1246C in MukB; head cross-link, G67C in MukB (Figures S6A and S7A). (C) Combinatorial cross-linking for identification of reaction species. Combinations: hinge, cap, and neck cross-links (left); cap, neck, and head cross-links (middle); head and hinge (right). C730 was mutated to serine when indicated by a minus sign. Cells were grown to stationary phase. Detection as in Figure 5B. (D) Chromosome entrapment in MukBEF with a covalently closed ring compartment. Input and agarose plug eluate are shown. Detection as in (C). The circular species is retained only in WT ATPase cells. DA, D1406A (blocks ATP binding); EQ, E1407Q (blocks ATP hydrolysis). ATPase is WT if not indicated otherwise. (E) Chromosome entrapment in MukBEF with covalently closed clamp compartment. As in (D). Species that have undergone chemical cross-link reversal during sample preparation are marked with asterisks. (F) Chromosome entrapment in MukB with covalently closed frame compartment. As in (D). Species produced by higher-order oligomers in EQ mutants (trans cross-links) are indicated. (G) Structure-based topological interpretation of the entrapment reactions. Only the frame species can slide off DNA because it does not form a protein/DNA catenane. (H) DNA entrapment in the ring compartment in the absence of MatP. Signal of the plug eluate relative to WT is shown for biological triplicates. Black lines indicate means, purple lines indicate standard deviations, and colored bars indicate 95% credible intervals. See also Figures S6 and S7.
Figure 7
Figure 7
Model for DNA binding and unloading at matS sites (A) Schematic for association of MukBEF with MatP-matS in the Ter macrodomain. MukBEF organizes the chromosome into loops. Upon invasion of Ter, MukBEF encounters MatP-matS and unloads via the double-lock topology in the context of a plectonemic loop. (B) Model for unloading of DNA. A MatP-matS encounter is followed by ATP hydrolysis and opening of the head gate to permit exit of the clamped DNA. matS DNA follows through neck and head gates, facilitated by the bridge between MatP and MukE. Arm zip-up prevents reversal, and the neck gate closes after MatP/DNA dissociation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Afonine P.V., Poon B.K., Read R.J., Sobolev O.V., Terwilliger T.C., Urzhumtsev A., Adams P.D. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 2018;74:531–544. - PMC - PubMed
    1. Badrinarayanan A., Reyes-Lamothe R., Uphoff S., Leake M.C., Sherratt D.J. In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science. 2012;338:528–531. - PMC - PubMed
    1. Banigan E.J., Mirny L.A. Limits of Chromosome Compaction by Loop-Extruding Motors. Phys. Rev. X. 2019;9:031007.
    1. Bürmann F., Shin H.-C., Basquin J., Soh Y.-M., Giménez-Oya V., Kim Y.-G., Oh B.-H., Gruber S. An asymmetric SMC-kleisin bridge in prokaryotic condensin. Nat. Struct. Mol. Biol. 2013;20:371–379. - PubMed
    1. Bürmann F., Lee B.-G., Than T., Sinn L., O’Reilly F.J., Yatskevich S., Rappsilber J., Hu B., Nasmyth K., Löwe J. A folded conformation of MukBEF and cohesin. Nat. Struct. Mol. Biol. 2019;26:227–236. - PMC - PubMed

Publication types

MeSH terms

Supplementary concepts

LinkOut - more resources