Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF

Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP‐modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB–DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss‐of‐function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss‐of‐function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB–DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them.     

DNA sliding and loop formation by E. coli SMC complex: MukBEF

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.     

MukEF Is required for stable association of MukB with the chromosome

MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.     

Antagonistic interactions of kleisins and DNA with bacterial Condensin MukB

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for faithful chromosome segregation in Escherichia coli. The SMC subunit of the complex, MukB, promotes DNA condensation in vitro and in vivo; however, all three subunits are required for the function of MukBEF. We report here that MukEF disrupts MukB x DNA complex. Preassembled MukBEF was inert in DNA binding or reshaping. Similarly, the association of MukEF with DNA-bound MukB served to displace MukB from DNA. When purified from cells, MukBEF existed as a mixture of MukEF-saturated and unsaturated complexes. The holoenzyme was unstable and could only bind DNA upon dissociation of MukEF. The DNA reshaping properties of unsaturated MukBEF were identical to those of MukB. Furthermore, the unsaturated MukBEF was stable and proficient in DNA binding. These results support the view that kleisins are not directly involved in DNA binding but rather bridge distant DNA-bound MukBs.     

Comparison of MukB homodimer versus MukBEF complex molecular architectures by electron microscopy reveals a higher-order multimerization

The complex of MukF, MukE, and MukB proteins participates in organization of sister chromosomes and partitioning into both daughter cells in Escherichia coli. We purified the MukB homodimer and the MukBEF complex and analyzed them by electron microscopy to compare both structures. A MukB homodimer shows a long rod-hinge-rod v-shape with small globular domains at both ends. The MukBEF complex shows a similar structure having larger globular domains than those of the MukB homodimer. These results suggest that MukF and MukE bind to the globular domains of a MukB homodimer. The globular domains of the MukBEF complex frequently associate with each other in an intramolecular fashion, forming a ring. In addition, MukBEF complex molecules tend to form multimers by the end-to-end joining with other MukBEF molecules in an intermolecular fashion, resulting in fibers and rosette-form structures in the absence of ATP and DNA in vitro.     

Chromosome condensation in the absence of the non-SMC subunits of MukBEF

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for chromosome partitioning in Escherichia coli. We report that overproduction of MukBEF results in marked chromosome condensation. This condensation is rapid and precedes the effects of overproduction on macromolecular synthesis. Condensed nucleoids are often mispositioned; however, cell viability is only mildly affected. The overproduction of MukB leads to a similar chromosome condensation, even in the absence of MukE and MukF. Thus, the non-SMC subunits of MukBEF play only an auxiliary role in chromosome condensation. MukBEF, however, was often a better condensin than MukB. Furthermore, the chromosome condensation by MukB did not rescue the temperature sensitivity of MukEF-deficient cells, nor did it suppress the high frequency of anucleate cell formation. We infer that the role of MukBEF in stabilizing chromatin architecture is more versatile than its role in controlling chromosome size. We further propose that MukBEF could be directly involved in chromosome segregation.     

The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.     

ATP-induced shrinkage of DNA with MukB protein and the MukBEF complex of Escherichia coli

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-gammaS, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.     

Structural basis for the MukB‐topoisomerase IV interaction and its functional implications in vivo

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB–topo IV complex to 2.3 Å resolution. The structure shows that the so‐called ‘hinge’ region of MukB forms a heterotetrameric assembly with a C‐terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.     

The MukB-topoisomerase IV interaction mutually suppresses their catalytic activities

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein–DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.     

DNA reshaping by MukB. Right-handed knotting, left-handed supercoiling

MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations.     

A new family of bacterial condensins

Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled-coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes.     

Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli.

mukF, mukE and mukB genes are essential for the process of chromosome partitioning in Escherichia coli. We have studied protein-protein interactions among MukB, MukE and MukF proteins by co-immunoprecipitation and sucrose gradient sedimentation experiments, using mukFEB null cells harboring plasmids carrying the wild-type or mutant-type mukFEB operon. MukB forms a complex with MukF and MukE. Analysis of mutant MukB proteins suggested that MukF and MukE bind the C-terminal globular domain of MukB. MukF is indispensable for an interaction between MukB and MukE; however, MukF itself is able to associate with MukB even in the absence of MukE. We have also found that MukF has a Ca(2+)-binding activity. Although purified MukF was able to make a complex either with MukE or MukB, a complex consisting of the three Muk proteins was barely detected in vitro. However, increasing the Ca(2+) or Mg(2+) concentration in the reaction partially restored complex formation. This suggests that Ca(2+) or Mg(2+) may be required for the formation of a complex consisting of the three Muk proteins, and thus may participate in a particular step during chromosome partitioning.     

The role of MukE in assembling a functional MukBEF complex

The MukB-MukE-MukF protein complex is essential for chromosome condensation and segregation in Escherichia coli. The central component of this complex, the MukB protein, is related functionally and structurally to the ubiquitous SMC (structural maintenance of chromosomes) proteins. In a manner similar to SMC, MukB requires the association of two accessory proteins (MukE and MukF) for its function. MukF is a constitutive dimer that bridges the interaction between MukB and MukE. While MukB can condense DNA on its own, it requires MukF and MukE to ensure proper chromosome segregation. Here, we present a novel structure of the E. coli MukE-MukF complex, in which the intricate crystal packing interactions reveal an alternative MukE dimerization interface spanning both N- and C-terminal winged-helix domains of the protein. The structure also unveils additional cross-linking interactions between adjacent MukE-MukF complexes mediated by MukE. A variant of MukE encompassing point mutations on one of these surfaces does not affect assembly of the MukB-MukE-MukF complex and yet cannot restore the temperature sensitivity of the mukE∷kan strain, suggesting that this surface may mediate critical protein-protein interactions between MukB-MukE-MukF complexes. Since the dimerization interface of MukE overlaps with the region of the protein that interacts with MukB in the MukB-MukE-MukF complex, we suggest that competing MukB-MukE and MukE-MukE interactions may regulate the formation of higher-order structures of bacterial condensin.     

Crystal structure of the MukB hinge domain with coiled‐coil stretches and its functional implications

The structural maintenance of chromosomes (SMC) family proteins are commonly found in the multiprotein complexes involved in chromosome organization, including chromosome condensation and sister chromatid cohesion. These proteins are characterized by forming a V‐shaped homo‐ or heterodimeric structure with two long coiled‐coil arms having two ATPase head domains at the distal ends. The hinge domain, located in the middle of the coiled coil, forms the dimer interface. In addition to being the dimerization module, SMC hinges appear to play other roles, including the gateway function for DNA entry into the cohesin complex. Herein, we report the homodimeric structure of the hinge domain of Escherichia coli MukB, which forms a prokaryotic condensin complex with two non‐SMC subunits, MukE and MukF. In contrast with SMC hinge of Thermotoga maritima which has a sizable central hole at the dimer interface, MukB hinge forms a constricted dimer interface lacking a hole. Under our assay conditions, MukB hinge does not interact with DNA in accordance with the absence of a notable positively charged surface patch. The function of MukB hinge appears to be limited to dimerization of two copies of MukB molecules. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The MukB-ParC Interaction Affects the Intramolecular,Not Intermolecular,Activities of Topoisomerase IV

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.     

The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication

SMC (structural maintenance of chromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of the Escherichia coli SMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning of ori and other loci in steady-state cells. These observations led to an earlier proposal that MukBEF positions new replicated sister oris. We show here that MukBEF generates and maintains the cellular positioning of chromosome loci independently of DNA replication. Rapid impairment of MukBEF function by depleting a Muk component in the absence of DNA replication leads to loss of MukBEF foci as well as mispositioning of ori and other loci, while rapid Muk synthesis leads to rapid MukBEF focus formation but slow restoration of normal chromosomal locus positioning.     

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.     

Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.

BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.     

FtsZ rings in mukB mutants with or without the Min system

The site of cell division in Escherichia coli is defined by formation of the Z ring between the two segregated daughter nucleoids. Positioning of the Z ring, composed of the highly conserved and tubulin-like FtsZ protein, appears to be negatively regulated by both the nucleoid and the oscillating MinCD inhibitor proteins. MukB protein is probably involved in nucleoid condensation, and in the absence of MukB, the negative effect of the nucleoid on Z rings appears to be partially suppressed. In this study, we examined the localization of Z rings in cells lacking both the Min system and MukB. In the Deltamin DeltamukB double null mutant, essentially all nucleoid-free zones, either at the cell poles or at non-polar sites between nucleoids, contained Z rings. However, a significant proportion of Z rings also formed on top of nucleoids. Interestingly, Z ring clusters often formed at gaps between nucleoids, and some of the rings within the clusters were clearly positioned on top of nucleoids. These results provide further evidence that the negative topological effect of nucleoids in cells lacking MukB is partially but not totally suppressed, and that the absence of the Min system allows more promiscuous Z ring formation.