Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF

Abstract

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk−, with the complexes failing to associate with chromosomes.

Acyl Carrier Protein is essential for MukBEF action inEscherichia colichromosome organization-segregation

“…Analysis of samples containing AcpP and MukB N or MukB HN using native Mass Spectrometry (nMS) revealed AcpP interacts with MukB with a 1:1 monomer-monomer stoichiometry (Fig. 1d), supporting data previously reported for WT MukB 14 . In addition, complexes with a mass corresponding to MukB N 2-AcpP2 were also identified, likely arising through interactions between the coiled-coils.…”

“…The nature and function of the interaction between AcpP and MukB has been unclear, despite numerous reports describing an interaction 14,17,19,24,27,28 . We therefore set out to determine whether the interaction between AcpP and MukB is specific and to identify any interaction site on MukB.…”

“…Unusually among kleisins, MukF dimerizes through an additional N-terminal dimeric winged-helix domain (WHD). This enables the joining of two dimeric MukBEF complexes into dimer of dimer (DoD) complexes that are essential for in vivo MukBEF function 3,13,14 .…”

“…MukE dimers interact with MukF; thus the complete MukBEF complex has a 4:4:2 B:E:F stoichiometry 3,14 . MukB ATP hydrolysis results from the engagement of two head domains that create two shared ATP binding sites.…”

SMC complexes organize the bacterial chromosome by lengthwise compaction

“…The mechanistic and functional differences between MukBEF and SMC–ScpAB complexes remain elusive, but in our opinion, it is likely that they both act through ATP hydrolysis-driven loop extrusion. The requirement of MukBEF dimers of dimers for function (Badrinarayanan et al 2012 ; Rajasekar et al 2019 ) provides a conceptually straightforward way of having a symmetrical loop extrusion mechanism, which in our model is essential for efficient lengthwise compaction (Mäkelä and Sherratt 2020 ). Whether other SMC complexes form dimers of dimers, or other-higher order cooperative structures, is hotly debated, although the apparent coordination of putative loop extrusion on the two B. subtilis arms could be explained by higher order SMC action.…”