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

2005; Springer Nature; Volume: 24; Issue: 11 Linguagem: Inglês

10.1038/sj.emboj.7600680

1460-2075

Rachel Fennell-Fezzie, Scott Gradia, David L. Akey, James M. Berger,

Bacterial Genetics and Biotechnology

Article19 May 2005free access The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins Rachel Fennell-Fezzie Rachel Fennell-Fezzie Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Scott D Gradia Scott D Gradia Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author David Akey David Akey Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author James M Berger Corresponding Author James M Berger Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Rachel Fennell-Fezzie Rachel Fennell-Fezzie Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Scott D Gradia Scott D Gradia Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author David Akey David Akey Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author James M Berger Corresponding Author James M Berger Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Author Information Rachel Fennell-Fezzie1, Scott D Gradia1, David Akey1 and James M Berger 1 1Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA *Corresponding author. Department of Molecular and Cell Biology, University of California Berkeley, 327B Hildebrand Hall #3206, Berkeley, CA 94720-3206, USA. Tel.: +1 510 643 9483; Fax: +1 510 643 9290; E-mail: [email protected] The EMBO Journal (2005)24:1921-1930https://doi.org/10.1038/sj.emboj.7600680 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 Å 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. Introduction The appropriate organization and condensation of chromosomes is crucial to all cells. The systematic folding of extended DNA molecules into compact structures protects chromosomes from entanglements that can lead to DNA breakage during segregation and helps cells partition their genomes efficiently and evenly into two daughter cells (Koshland and Strunnikov, 1996; Hirano, 1999, 2000, 2002). Throughout the cell cycle, chromosomes are maintained in a highly organized state, although the degree of compaction varies from organism to organism. For instance, bacteria compress their DNA to approximately 1/1000th of its theoretical free, random-coil size, while eukaryotes compact their chromosomes nearly 100 000-fold (Trun and Marko, 1998; Holmes and Cozzarelli, 2000). Although chromosome complexity and organization vary enormously among different domains of life, virtually all cells rely in part on a multiprotein complex known as condensin to achieve chromosome compaction (Koshland and Strunnikov, 1996; Hirano, 2002). The ability to modulate DNA supercoiling appears to be one facet of condensin function. Direct evidence for supercoiling by condensin was first noted in Xenopus extracts (Kimura and Hirano, 1997; Kimura et al, 1999). Researchers have since observed that the net superhelical densities of plasmid and chromosomal DNAs also change in condensin-deficient strains of Bacillus subtilis and Escherichia coli (Weitao et al, 2000; Lindow et al, 2002). In E. coli, genetic studies have demonstrated that certain condensin mutants can suppress the phenotypes of other mutations that increase DNA supercoiling levels inside cells (Weitao et al, 1999; Sawitzke and Austin, 2000). In eukaryotes, the condensin complex consists of an SMC (structural maintenance of chromosomes) heterodimer (XCAP-C/Smc2 and XCAP-E/Smc4) and three non-SMC subunits (XCAP-D2, XCAP-G, and XCAP-H/barren) (Hirano et al, 1997; Sutani et al, 1999; Lavoie et al, 2000; Hirano, 2002). A striking structural characteristic of SMC monomers is that they possess two globular domains connected by an extended coiled-coil segment (Niki et al, 1991; Hirano and Mitchison, 1994; Melby et al, 1998; Anderson et al, 2002; Yoshimura et al, 2002). A globular 'hinge' disrupts the central helical region and reverses the orientation of the polypeptide chain (Haering et al, 2002), creating an antiparallel coiled coil and juxtaposing the N- and C-terminal regions to form a bipartite domain of the ABC superfamily of ATPases at one end of the protein. Although the ATPase domains can dimerize in the presence of nucleotide, SMC proteins principally form dimers through the association of hinge regions (Niki et al, 1992; Melby et al, 1998; Hopfner et al, 2000; Hirano et al, 2001; Haering et al, 2002; Hirano, 2002; Hopfner and Tainer, 2003). In contrast to eukaryotes, bacteria and archaea rely on a three-subunit condensin complex (Yamanaka et al, 1996; Mascarenhas et al, 2002; Soppa et al, 2002). Most prokaryotes employ an SMC homodimer along with two non-SMC proteins, ScpA and ScpB, although clear ScpB homologs do not appear to be present in all species (Mascarenhas et al, 2002). By contrast, E. coli and other members of the γ-proteobacter family use the MukB, MukE, and MukF and proteins for chromosome condensation (Niki et al, 1991, 1992; Yamanaka et al, 1996). Although MukB is a distant relative of the SMC protein family, the architecture and evolutionary lineage of the accessory MukE and MukF subunits have not been determined (Niki et al, 1992; Melby et al, 1998; van den Ent et al, 1999; Cobbe and Heck, 2004). Deletion of accessory condensin subunits causes defects similar to those of SMC knockouts in all systems studied to date (Bhat et al, 1996; Yamanaka et al, 1996; Sutani et al, 1999; Lavoie et al, 2000; Mascarenhas et al, 2002). In bacteria, the phenotype of null mutations includes diffuse nucleoids, a high incidence of anucleate cells, elongated or filamentous cells, and an inability to form colonies at temperatures above 30°C (Niki et al, 1991; Yamanaka et al, 1996; Britton et al, 1998; Mascarenhas et al, 2002; Jensen and Shapiro, 2003; Volkov et al, 2003). Nonetheless, the precise biochemical functions of the non-SMC components of condensins are still poorly understood. In general, these subunits bear little or no homology to other proteins, although HEAT repeats have been identified in eukaryotic XCAP-D2 and XCAP-G (Neuwald and Hirano, 2000). More recently, phylogenetic analyses have indicated that at least one non-SMC subunit is conserved across many diverse complexes that employ SMC proteins. These factors, known as 'kleisins', include bacterial ScpA, eukaryotic condensin XCAP-H (barren), Rec8, and Scc1/Mcd1/Rad21 (Schleiffer et al, 2003). Of these, the functional role of Scc1 has been best delineated; in the cohesin complex, it anchors together the ATPase domains of the Smc1/Smc3 heterodimer and is selectively cleaved by the separin protease to initiate anaphase (Uhlmann et al, 1999, 2000). Although no Muk subunits have been categorized as kleisin members, Schleiffer et al have suggested that E. coli MukF might function as a kleisin, since it associates with MukB (Yamanaka et al, 1996; Schleiffer et al, 2003). To better understand the structure and function of the MukBEF condensin, as well as the relationship of its non-SMC subunits to those of other condensins, we have carried out a series of structural, informatic, and biochemical studies on the Muk system. We report here the crystal structure of a 33 kDa N-terminal fragment of MukF(1–287), and show that this region forms a doubly domain-swapped dimer of a novel overall architecture. Protein interaction assays reveal that a domain of MukF C-terminal to this dimerization module binds specifically to MukB and MukE, while sequence analyses show that two domains at the extreme N- and C-termini of MukF are helix–turn–helix (HTH) folds homologous to the extreme termini of kleisin proteins. Together, these data indicate that MukF serves as a kleisin, and highlight the organizational basis by which it links together MukB subunits. Results MukF(1–287) monomer structure Using limited proteolysis and deletion mapping, we identified a stable 33 kDa N-terminal fragment of MukF comprising residues 1–287 of the 440-amino-acid protein. When purified, this fragment crystallized in the space group P61, with unit cell dimensions a=b=58.7 Å and c=307.5 Å. Preliminary crystals suffered from perfect merohedral twinning (Yeates and Fam, 1999); however, the addition of divalent metals to crystallization drops, notably Ca2+ ions, reduced the fraction of twinned crystals. We solved the structure of MukF(1–287) by multiwavelength anomalous dispersion (MAD) methods and refined it to a resolution of 2.9 Å with a free R-value of 27.3% and a working R-factor of 23.3% (Figure 1 and Table I). Despite the presence of Ca2+ in the crystallization conditions, and reports that MukF may be a Ca2+-binding protein (Yamazoe et al, 1999), we observed no electron density for divalent metals. Figure 1.MukF(1–287) structure. (A) Superposition of MukF(1–287) amino-acid sequence and secondary structure. Helices (cylinders) and strands (arrows) are labeled and colored as follows: N-terminal extension, gold; domain I WHD, red; domain II coiled coil, blue. (B) Ribbon diagram of the MukF(1–287) monomer. Secondary structural labels correspond to those in panel A. (C) Experimentally phased electron density (Fobs, αflattened, 1.5σ contour level) for strand β1 looking down the noncrystallographic two-fold axis and showing the unambiguous separation between protomers. (D) Superposition of WHDs from MukF(1–287) (red) and the C-terminal domain of Scc1 (green) (Haering et al, 2004). HTH and wing (W) motifs are labeled. Molecular figures in the paper are rendered with PYMOL (DeLano, 2002). Download figure Download PowerPoint Table 1. Structure determination statistics Remote Peak Data collection Radiation (Å) 1.1272 0.9797 Resolution (Å) (last shell) 20–2.9 (3.1–2.9) 20–2.9 (3.1–2.9) Completeness (%) (last shell) 95.5 (93.0) 94.7 (95.5) Rsyma (%) (last shell) 7.7 (29.2) 8.2 (39.0) I/σ (last shell) 12.2 (4.2) 11.3 (4.2) Multiplicity (last shell) 5.3 (4.2) 5.5 (5.4) Phasing No. of sites (identified/total) 8/12 Mean FOM (SOLVE) 0.47 Structure refinementb Resolution range (Å) 20–2.9 No. of protein atoms per dimer 4228 No. of reflections (overall) 13152 No. of reflections (test set) 625 Rwork/Rfreec (%) 23.3/27.3 R.m.s.d.bonds/angles 0.009 Å/1.14° Ramachandran analysis Most favored 92.5 Allowed 6.6 Generously allowed(%) 0.9 a Rsym=∑∑j∣Ij−〈I〉∣/∑Ij, where Ij is the intensity measurement for reflection j and 〈I〉 is the mean intensity for multiply recorded reflections. b Coordinates for MukF(1–287) have been deposited in the RCSB database under accession number 1T98. c Rwork,free=∑∣∣Fobs∣−∣Fcalc∣∣/∑∣Fobs∣, where the working and free R-factors are calculated using the working and free reflection sets, respectively. The free reflections (5.1% of the total) were held aside throughout refinement. Within the crystal, there are two copies of the MukF(1–287) protomer per asymmetric unit. Each adopts a highly helical and extended structure (Figure 1B). The fragment contains nine α-helices and three short β-strands partitioned between two domains. Helix α1 and strand β1 form an N-terminal extension that stands apart from the other regions of the protomer and makes no intra-subunit contacts. A six-amino-acid linker region between helices α5 and α6 connects domains I and II. Domain I spans residues 26–115 and is roughly globular in shape. This region is composed of helices α2–α5 and strands β2–β3. Residues 92–99, which fall between β2 and β3, were disordered in our electron density maps and are absent from the model. A search of the PDB using DALI and SSM (Holm and Sander, 1996; Krissinel and Henrick, 2004) found an extensive number of proteins similar to domain I. Of these, all top hits corresponded to winged helix domains (WHDs) within nucleic acid-binding proteins or DNA-associated assemblies. Interestingly, the closest spatial match to the MukF N-terminal WHD was the C-terminal domain of Scc1 (top Q-score match from SSM, r.m.s.d.=1.6 Å over 59 Cα residues; Figure 1D) (Haering et al, 2004), a kleisin that binds to the Smc1/Smc3 heterodimer of cohesin. In WHDs, the second and third helices (α3 and α4 in MukF) form an HTH motif. These elements are immediately followed by two β-strands that comprise the signature wing region (β2–β3 in MukF). Typically, the second helix of the HTH motif (often called the recognition helix) and the wing interact with DNA, although other binding modes have also been observed, both to nucleic acids and to other proteins (Gajiwala and Burley, 2000; Mer et al, 2000; Haering et al, 2004). Despite extensive screens using gel mobility shift, filter-binding, and fluorescence polarization assays, we have detected no association between linear or supercoiled duplex DNA and either full-length MukF or MukF(1–287) (data not shown), suggesting that the primary purpose of this region may not be to bind nucleic acids. Domain II is formed by amino acids 122–287, which fold into an extended and slightly bent helical bundle that includes helices α6–α9. Three of these helices (α6, α8, and α9) contain several glycines that allow them to curve around α7, which is essentially straight except for a slight bend near its N-terminus induced by Pro159. The tertiary organization of secondary structural elements within domain II is such that it forms a three-helix coiled coil at one end and transitions into a four-helix coiled coil at the other. Comparisons of MukF(1–287) to the structural database reveal that this domain is globally similar in structure to numerous highly helical proteins of diverse function. None of these relationships appear to bear significantly on function. The architecture of domain II provides a structural rationale for the null phenotype of the mukF233 mutation, which changes Leu233 to proline (Yamanaka et al, 1996). Leu233 lies on helix α8 and is buried in the core of the four-helix region of the coiled coil. Substitution of this amino acid by proline would most likely distort α8 and compromise the packing and stability of the domain, either impairing the ability of MukF to accommodate a specific aspect of condensin function or promoting protein misfolding and degradation. MukF(1–287) dimer structure In the asymmetric unit of the crystal, two protomers of MukF(1–287) associate with each other to form a dimer of dimensions 70 Å × 85 Å × 55 Å (Figure 2A and B). The shape of the dimer is reminiscent of a 'skull-and-crossbones' in which the coiled coils of domain II form an 'X' that cradles the globular WHDs. Remarkably, inspection of experimental electron density maps unambiguously revealed that two sets of secondary structural elements are exchanged between the two protomers to form a doubly domain-swapped quaternary arrangement (Figure 2). Helix α1 of each protomer nestles against domain I of its partner subunit, burying a portion of α2 on the opposite monomer and sealing off this region's hydrophobic core. C-terminal to α1, strand β1 extends across the molecular two-fold axis and forms a short, antiparallel sheet with β1 of the neighboring protomer. Following domain I, a second swapping event occurs as the polypeptide chain between α5 and α6 winds back around the molecular two-fold axis. Conserved residues Leu24 from β1 and Tyr113/Tyr114 from α5 form the hydrophobic domain I•domain I interface. As a consequence, domain I of one protomer abuts domains I and II of its partner, but does not contact its own domain II region. In contrast to the domain I•domain I interface, contacts between domains I and II are largely polar and are mediated between α7 of one subunit and the undersides of strands β2 and β3 of the other. Figure 2.MukF(1–287) forms a stable, domain-swapped dimer. (A) Stereo diagram of the MukF(1–287) dimer. Protomer A is colored as per Figure 1, while the other subunit is colored gray. (B) The MukF(1–287) dimer viewed orthogonally to panel A and looking down on the WHDs. The two protomers are colored as in panel A, with one shown as a surface representation and the other shown as a ribbon to highlight the entwined structure of the oligomer. (C) Analytical ultracentrifugation data of MukF(1–287). Data (circles) fitting to a single species model calculated a mass of 69.1 kDa, consistent with a dimeric species in solution (see Materials and methods). Theoretical models for the monomeric, dimeric, and trimeric species of the MukF(1–287) protein are shown as lines indicated in the legend. Residuals to the fitted data are shown above. Download figure Download PowerPoint The domain-swapped structure we observe for the MukF(1–287) dimer is likely to be the physiological state of the molecule. Previous co-immunoprecipitation and sucrose gradient sedimentation experiments with MukF (Yamazoe et al, 1999), as well as our own findings from size-exclusion chromatography and light scattering (data not shown), indicate that MukF forms stable dimers in solution. To further validate this finding for MukF(1–287), we performed analytical ultracentrifugation studies of the purified protein used for crystallization. As can be seen in Figure 2C, data obtained for the particle fit cleanly to a stable dimer model, but not to models predicted for monomeric or trimeric oligomeric states. An inspection of the contacts between protomers reveals that inter-subunit interactions arising from domain swapping account for more than three-quarters of the total surface area buried in the dimerization interface (∼3700 Å2 per protomer). Moreover, the linker region between helices α5 and α6 is too short to permit domains I and II within one monomer to associate with each other using the interdomain interactions we observe in the MukF(1–287) dimer. The extensive, intertwined character of its oligomerization interface suggests that this interaction is obligate (Nooren and Thornton, 2003) and probably imparts a high degree of stability to the MukF dimer. The N- and C-termini of MukF are homologous to kleisin domains Kleisins are a recently identified superfamily of proteins that are distributed throughout many SMC-based complexes (Schleiffer et al, 2003). Kleisins include bacterial ScpA, XCAP-H/Brn1 of eukaryotic condensin, Scc1/Mcd1/Rad21 of cohesin, and Rec8 of the Smc5/Smc6 DNA repair system. These proteins all appear to associate with the ATPase domains of SMC proteins. In the case of bacterial condensin, ScpA specifically associates with Smc subunits (Volkov et al, 2003; Dervyn et al, 2004; Hirano and Hirano, 2004), while for eukaryotic cohesin, the N- and C-termini of Rad21/Mcd1/Scc1 have been demonstrated to bind directly to Smc3 and Smc1, respectively (Haering et al, 2002, 2004). Given that MukB is part of the SMC superfamily (van den Ent et al, 1999; Lowe et al, 2001; Cobbe and Heck, 2004), it has been suggested that the functions and perhaps structures of its accessory subunits may overlap with those of the non-SMC components found in other SMC complexes. MukF has been put forth as a potential kleisin in this regard (Schleiffer et al, 2003). Given the close structural similarity between the MukF N-terminal WHD and the C-terminal Scc1 WHD, coupled with the fact that MukF forms a stable complex with MukB and MukE, we decided to investigate more closely whether MukF might be related to kleisin proteins at the amino-acid sequence level. To accomplish this, we aligned sequences from multiple members of the MukF and kleisin families (Figure 3). Our analysis shows that the N- and C-termini of MukF align well with the equivalent N- and C-terminal regions of ScpA, as well as with those of eukaryotic kleisins. An examination of our alignments in light of the structural data for the MukF N-terminal region and the Scc1 C-terminus (Haering et al, 2004) shows that the two regions of homology correspond to WHDs that form the extreme termini of both proteins. There is particularly good overlap between secondary structural elements predicted to form HTH motifs from primary sequence analysis and the regions now known to contain such architectures from structural studies (Figures 1D and 3) (Haering et al, 2004). Significantly, our alignment of MukF with other kleisin proteins independently recapitulated the homology between members of the kleisin family (Schleiffer et al, 2003), even though we did not use this information to guide or bias our comparisons. Taken together, this analysis supports the idea that MukF is a member of the kleisin superfamily. Figure 3.Conservation between MukF and kleisins. Sequence alignment of several kleisin N- (upper panel) and C-terminal (lower panel) domains with MukF using MAFFT (Katoh et al, 2002) and Jalview (Clamp et al, 2004). Secondary structural elements for the N-terminal domain of MukF (red) and the C-terminal domain of Scc1 (green) are highlighted as seen in their respective crystal structures (Figure 1) (Haering et al, 2004). Download figure Download PowerPoint Identification of MukF regions responsible for binding MukE and MukB To further ascertain the extent to which the behavior of MukF reflects that of kleisins, we set out to define more narrowly the regions of MukF that associate with MukB and MukE. Previous findings from Hiraga and co-workers (Yamazoe et al, 1999) had already shown that MukF binds both MukB and MukE, and that MukE interacts only weakly, if at all, with MukB in the absence of MukF. Some of these associations were proposed to be mediated by the acidic linker that lies beyond MukF(1–287), as well as by a predicted coiled-coil region in MukF that our structure shows to comprise a portion of domain II (Yamanaka et al, 1996; Yamazoe et al, 1999). However, the boundaries and relative relationship of MukB- and MukE-binding sites on MukF have remained unclear. To determine whether MukF(1–287) could bind either MukB or MukE, we first performed pull-down assays with lysates containing N-terminally hexahistidine-tagged MukF(1–287) (bait) and either untagged MukB or MukE(1–209) (prey). MukE(1–209) is a construct of the 243-amino-acid MukE protein that recapitulates the functions of the full-length protein in vitro but that lacks several poorly conserved and hydrophilic residues from its C-terminus to improve solubility (Materials and methods). Bait and prey proteins were expressed independently of one another in different cell cultures and, after mixing the lysates together for a short incubation time, binding was assayed batch-wise using Ni-NTA resin (Materials and methods). Control experiments using untagged MukF(1–287), MukE(1–209), or MukB showed that these proteins do not associate independently with the Ni-NTA resin (Supplementary Figure 1). Analysis of the bound and unbound pools in assays using histidine-tagged MukF(1–287) demonstrated binding of the tagged protein to the Ni-NTA resin, and also showed that this construct is unable to pull down either MukE(1–209) or MukB (Figure 4A and B). Figure 4.MukF, MukB, and MukE domain interactions. Results of filter-binding assays between different Muk constructs are shown. The Muk fragments and subunits assayed are identified by cartoons: the MukF N-terminal domains are shown as a gray and crosshatched dimer; MukF C-terminal domain fragments are dark gray; MukB (170 kDa) and the MukB ATPase domain (72 kDa) are light gray with coiled squiggles; MukE(1–209) (24 kDa) is white. Representations for the two MukF C-terminal domain constructs can be distinguished by the presence of a small 'tail' that corresponds to the acidic linker region (residues 302–330) between MukF(1–287) and MukF(331–440). Gel lanes are labeled as follows: 'L', lysate; 'U', unbound; 'B', bound. Hexahistidine-tagged fragments are preceded by 'H−'. (A) MukF(1–287) does not bind MukE(1–209). (B) MukF(1–287) does not bind MukB. (C) His-tagged MukF(302–440) binds MukE(1–209). (D) H-F(331–440) does not associate with MukE(1–209). (E) H-F(302–440) pulls down both E(1–209) and MukB. (F) Tagged MukF(331–440) binds MukB. (G) Tagged MukF(331–440) binds the ATPase domain of MukB. (H) Histidine-tagged MukF(302–440) pulls down MukE(1–209) but does not associate with MukF(1–287). Download figure Download PowerPoint We next assayed the ability of two different MukF C-terminal fragments to bind MukE(1–209). The larger fragment, MukF(302–440), was obtained by deletion mapping using amino-acid homology and contains a conserved acidic region that has been predicted to bind Ca2+ ions (Yamazoe et al, 1999). The other, MukF(331–440), was identified from limited proteolysis of the full-length protein by N-terminal sequencing and mass spectrometry. As with MukF(1–287), both tagged C-terminal domain constructs of MukF bound the Ni-NTA resin on their own. MukF(302–440) additionally proved capable of pulling down coexpressed MukE(1–209) (Figure 4C). By contrast, tagged MukF(331–440) showed no stable interaction with untagged MukE(1–209) (Figure 4D). When assayed for MukB binding, both MukF C-terminal fragments pulled down untagged MukB from crude lysates (Figure 4E and F). The MukF C-terminus also proved capable of associating with a construct of MukB that comprises only the ATPase domains (residues 1–302 and 1180–1486) of the protein (Figure 4G). However, the MukF C-terminal region could not pull down MukF(1–287) (Figure 4H). Since neither untagged MukE(1–209) nor MukB bound appreciably to the nickel resin (Supplementary Figure 1), their interactions with the C-terminal region of MukF appear specific. This observation explains the null phenotype of the mukF303 mutation (Yamanaka et al, 1996), which truncates the MukF protein at residue 302: since the regions responsible for MukB and MukE binding are missing, the truncated protein is not competent for assembling with a MukBEF complex. Taken together, these data demonstrate that the last 110 amino acids of MukF are responsible for binding the ATPase domain of MukB, while the acidic linker region between the N- and C-terminal domains of MukF (residues 302–330) is essential for associating with MukE. By contrast, the N-terminal two-thirds of MukF does not interact tightly with MukB, MukE, or the C-terminal domain of MukF. This latter finding is consistent with our structural work showing that the N-terminus of MukF forms a homodimerization module, and that the predicted leucine-zipper region of the protein is actually an integral part of the four-helix coiled-coil segment. Overall, the MukB-binding behavior of the MukF C-terminus is reminiscent of the association of the Scc1 C-terminus with Smc1 (Haering et al, 2002, 2004), further supporting a functional role for MukF as a kleisin. Discussion MukF function within the MukBEF complex The relationship of MukF to kleisins provides an important clue to its function. Several studies have shown that SMC subunits can bring together distant DNA segments (Hirano and Hirano, 1998, 2002, 2004; Yoshimura et al, 2002; Volkov et al, 2003). The subunits that associate with SMCs appear to regulate and direct this function to help different SMC complexes manifest distinct biochemical activities. For example, in the Muk system, MukB can bind DNA on its own, but is converted into an active condensin only when functional MukF and MukE subunits are associated (Niki et al, 1992; Saleh et al, 1996; Yamanaka et al, 1996; Yamazoe et al, 1999). In eukaryotes, distinct regulatory subunits associate with different SMC dimers to create complexes that act specifically in chromosome segregation and condensation, DNA repair, and gene regulation (Kimura and Hirano, 2000; Hirano, 2002; Hagstrom and Meyer, 2003; Ono et al, 2003; Haering et al, 2004). What physical purpose might MukF serve? Our structural studies demonstrate that the N-terminal two-thirds of this protein forms a stable, domain-swapped dimerization element. Our work further shows that it is the extreme C-terminus of MukF that is responsible for binding the ATPase domains of MukB, and that this domain likely forms an HTH or WH fold related to the C-terminal, SMC-binding domain of kleisins (Figures 3 and 4). Given that the C-terminus of Scc1 binds specifically to the ATP-binding domains of Smc1 (Haering et al, 2002, 2004; Gruber et al, 2003), we anticipate that the C-terminus of MukF interacts in a similar manner with the ABC cassette of MukB. Observations from Yamazoe et al (1999) support this idea, showing that deletion of the extreme MukB C-terminus (residues 1372–1486) de