Asymmetric MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin

The Escherichia coli SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerization domain interacts functionally with the MukB coiled-coiled ‘neck’ adjacent to the ATPase head, forming an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerization is preserved during this interaction, MukF directs the formation of dimer of dimers MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate ATPase independently and additively, consistent with them each targeting only one of the two MukB ATPase active sites in the asymmetric complex. We demonstrate that MukF interaction with the MukB neck turns over during cycles of ATP binding and hydrolysis in vivo and that impairment of this interaction leads to MukBEF release from chromosomes.

The distinctive architecture of SMC proteins is conserved with the N-and Cterminal globular domains coming together to form an ATPase head and the intervening polypeptide folding upon itself to form ~50 nm long intramolecular coiled-coil arms, with a dimerization hinge distal from the head (Figure 1).
Upon ATP binding, the heads of SMC dimers engage to generate two ATPase active sites (Haering et al., 2002Lemmens et al., 2014. In eukaryotes, SMC complexes are exclusively heterodimeric, whilst those in bacteria are homodimers. Nevertheless, the distinctive SMC architecture is conserved, with a kleisin protein linking the two ATPase heads of an SMC dimer, thereby forming a large tripartite proteinaceous ring (Figure 1 inset).
E. coli and its closest γ-proteobacterial relatives, encode an apparently distant SMC relative, MukBEF, with little primary sequence homology to other SMCs (Nolivos and Sherratt, 2014). Organisms encoding MukBEF have coevolved a number of other distinctive proteins, some of which interact with MukB physically and/or functionally; specifically, topoisomerase IV and MatP both interact with MukB in vitro and in vivo (Brézellec et al., 2006;Hayama and Marians, 2010;Hayama et al., 2013;Li et al., 2010;Nicolas et al., 2016;Nolivos et al., 2016;Vos et al., 2013). MukB forms SMC homodimers, whereas MukF is the kleisin and MukE the kite protein that binds MukF (Palecek and . All three proteins of the MukBEF complex are required for function and their impairment leads to defects in chromosome segregation, manifested by impairment of segregation of newly replicated origins (ori) and mis-orientation of chromosomes with respect to their genetic map within cells (Danilova et al., 2007). In rich media, this leads to inviability at higher temperatures and formation of anucleate cells during permissive low-temperature growth Yamanaka et al., 1996). Here, we reveal that MukF, like other characterized kleisins, interacts functionally with the SMC MukB neck, through a 4-helix bundle in its Nterminal domain, while its C-terminal domain interacts with the MukB head at the cap. We show that this interaction with the MukB neck is required for MukBEF function in vivo, and infer that this interaction is established and broken during cycles of ATP binding and hydrolysis. Impairment of this interaction in vivo leads to release of MukBEF clusters from chromosomes. Inset centre shows a cartoon of a typical SMC-kleisin tripartite ring.

The MukF N-terminal domain interacts with the MukB neck
Because of the intriguing distinction between dimeric MukF and monomeric kleisins (Figure 1) (Figure 2B bottom right panel).
We conclude that helices 8 and 9 of the 4-helix bundle are sufficient for interaction with the MukB neck. Helix 9 is essential either because it interacts with the neck, or because it is required for proper folding of the helix 8-9 polypeptide.  In agreement with the Flag-MukF-MukB interaction assays, FC2, but not FN2, formed complexes with MukB H (Figure 3A middle and right panels).
FN6, which lacks the two C-terminal helices, 8 and 9, of the 4-helix bundle, failed to form complexes with MukB HN (Figure 3B). Addition of ATP did not significantly alter the nature or abundance of complexes containing MukB HN and FN2 or FC2 (Figure 3-figure supplement 2). This is consistent with MukB HN , which is a monomer in solution, being unable to form stable headsengaged dimers with either FN2 or FC2 in the presence of ATP.

Characterization of the interactions between the MukB neck and MukF
To  Whether such complex is generated at any stage of MukBEF activity cycle remains to be determined.

DNA binding to MukB relieves MukE-mediated ATPase inhibition
Previous reports have shown no effect of DNA on the ATPase of MukBEF  (Hirano and Hirano, 2004). We confirmed that MukB ATPase is independent of the presence of DNA ( Figure 6B)  doesn't bind to FN2, which lacks the middle region ( Fig. 7 supplementary figure 1) (Nolivos et al., 2016). Equivalent interfaces between the kleisin and SMC3 neck in the yeast, drosophila and human cohesin complexes have also been proposed to act as DNA exit gates and it has been proposed that this interaction, which is not required for loading onto chromosomes, turns-over in response to ATP binding and hydrolysis (

Two independently regulated ATPases in asymmetric MukBF complexes
The work reported here provides new insight into how kleisin interaction with the SMC heads leads to an asymmetric complex that has two putative independently controlled ATPases, one activated by the kleisin C-terminal which loads slowly onto DNA in the ter region but is unable to undergo the multiple cycles of ATP binding and hydrolysis required to re-locate to ori (Badrinarayanan et al., 2012b;Nolivos et al., 2016). Uhlmann, 2016), they have evolved the ability to held newly replicated sisters stably together (cohesion), until controlled release is triggered. The latter activity requires that ATPase-hydrolysis-dependent turnover of cohesion complexes on chromosomes, mediated by Wapl, is inhibited by acetylation of the SMC3 subunit, which presumably inhibits SMC3 ATPase (Ben-Shahar et al., Chan et al., 2012;Kueng et al., 2006;Unal et al., 2008). In addition to this inhibition of turnover, cohesion requires entrapping of the two sister chromatids within the SMC complexes, an activity separable from that involved into loading onto single chromosomes, and a process that must involve an altered association of DNA segments within the SMC complex.

Analysis of steady state
The detailed mechanism by which SMC complexes transport

Protein Purification
MukB, MukB H, MukB HN , MukE, were 6xHis-tagged at the C-terminus, while MukF and its C-and N-terminal truncations were 6xHis-tagged at the Nterminus and were expressed from plasmid pET21 and pET28, respectively in C3013I cells (NEB). 2L cultures were grown in LB with appropriate antibiotics at 37°C to A 600~0 .6 and induced by adding IPTG at final concentration of 0.4 mM. After 2 hours at 30°C, cells were harvested by centrifugation, resuspended in 30ml lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5%glycerol, 10 mM imidazole) supplemented with 1 tablet of protease

MALS)
Purified proteins were fractionated on a Superose 6 10/300 GL or a Superose 12 10/300 column equilibrated with 50 mM HEPES, pH 7.5 buffer containing 100 mM NaCl, 1 mMDTT, 1 mM EDTA, at flow rate of 0.5 ml/min. 500 µl samples containing analysed proteins were injected on the column and run at a flow rate of 0.5 ml/min. SEC-MALS analysis was performed at 20°C using a Shimadzu (Kyoto, Japan) chromatography system, connected in-line to a Heleos8+multi-angle light scattering detector and an Optilab T-rEX refractive index (RI) detector (Wyatt Technologies, Goleta, CA). Protein samples in 50 mM HEPES pH 7.5, 100 mM NaCl, 1mM DTT, 1mM EDTA, 10% glycerol, were injected in this system, and the resulting MALS, RI and UV traces processed in ASTRA 6 (Wyatt Technologies).

Pull-down Assays
MukF FLAG-tagged fragments were expressed from pET DUET plasmids in

Mutagenesis
Point mutations in plasmid-encoded genes were made using Q5 Site-Directed Mutagenesis Kit (NEB). Primers were designed with NEBase Changer.
10 ng of the template was taken to the reaction. Plasmids were isolated and mutations confirmed by sequencing.

Complementation assays
The ability of leaky plasmid-encoded MukB expression from pET21, in the absence of IPTG, to complement the temperature-sensitive growth defect of ∆mukB AB1157 cells at 37 0 C in LB was assayed. Cells were transformed with pET21 carrying MukB or MukB variants and allowed to recover for 8 hr post transformation at permissive temperature then plated in duplicates on LB plates containing carbenicillin (100µg/ml). One plate was incubated at nonpermissive (37 0 C) and the other one at permissive (20 0 C) temperature. Six colonies from plates incubated at permissive temperature were streaked in duplicate and grown at permissive and non-permissive temperature along with positive and negative controls.