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Structural biology of plasmid segregation proteins
Structural biology of plasmid segregation proteins Maria A Schumacher DNA segregation, or partition, ensures stable genome transmission during cell division. In prokaryotes, partition is best understood for plasmids, which serve as tractable model systems to decipher the molecular underpinnings of this process. Plasmid partition is mediated by par systems, composed of three essential elements: a centromere-like site and the proteins ParA and ParB. In the first step, ParB binds the centromere to form a large segrosome. Subsequently, ParA, an ATPase, binds the segrosome and mediates plasmid separation. Recently determined ParB–centromere structures have revealed key insights into segrosome assembly, whereas ParA structures have shed light on the mechanism of plasmid separation. These structures represent important steps in elucidating the molecular details of plasmid segregation. Addresses Department of Biochemistry and Molecular Biology, University of Texas, MD Anderson Cancer Center, Unit 1000, Houston, TX 77030, USA Corresponding author: Schumacher, Maria A ([email protected])
Current Opinion in Structural Biology 2007, 17:103–109 This review comes from a themed issue on Protein–nucleic acid interactions Edited by James M Berger and Christoph W Mu¨ller Available online 11th December 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.11.005
Introduction Partition, or segregation, is the process whereby genetic material is accurately moved and positioned to daughter cells during cell division. Most of our understanding of prokaryotic partition has derived from cellular, genetic and biochemical studies on the segregation of plasmid DNA. Plasmid partition ( par) systems represent simplified model systems with which to address the molecular and structural details of partition — they are composed of only three required elements: a cis-acting centromere-like (partition) DNA site(s) and two proteins typically called ParA and ParB [1–5]. Studies have shown that the first step of plasmid partition involves binding of the centromere-like partition site by the ParB protein [1–5]. Subsequently, multiple ParB molecules spread onto and around the site, leading to the creation of a large nucleoprotein structure called the partition complex or segrosome. ParB then mediates the next critical step in partition, which is plasmid pairing [6,7]. Once the plaswww.sciencedirect.com
mids are paired, the partition complexes are recognized by ParA, an ATPase, which acts as a molecular switch, as governed by its nucleotide-bound state, to drive plasmid separation (Figure 1). Plasmid-encoded par systems can be divided into two main types based on the kind of ParA protein present. Type I par systems, which are the most numerous, contain ParA proteins with putative deviant Walker-A type ATPase folds, whereas type II systems employ ParA proteins with actin-like folds [1,2]. The type I systems can be further divided into type Ia and type Ib based on the homology and size of the Par proteins. Type Ia systems contain ParA and ParB proteins of 321–420 and 312–342 residues, respectively, whereas the type Ib proteins are smaller, with ParA homologues containing 192–308 residues and ParB proteins of 46–131 residues. In the type Ia systems, ParA proteins also serve as transcriptional repressors in the autoregulation of its par operon, whereas in the type Ib and II systems, this function is carried out by ParB. Surprisingly, the ParB proteins show little to no homology, which is why the par system classification is based on ParA. Moreover, the centromere-like sites bound by ParB are also diverse. Indeed, although they typically consist of multiple direct or inverted repeats, the number of repeats, their sequence and length differ dramatically. Despite the unexpected diversity exhibited by the functionally homologous ParB proteins and centromere-like components, it appears that the partition complexes they form have a similar overall structural organization, which is used as a recognition and recruitment site by the ParA protein. Although a general outline of the plasmid partition process has been obtained, critical questions remain, largely due to a lack of structural information. Such questions include: what are the DNA-binding properties of the ParB proteins that enable them to recognize their elaborate centromere-like sites and form large nucleoprotein complexes; how do ParA proteins use adenine nucleotides to ‘switch’ conformational states; and how do the ParA proteins function to move the plasmids apart? This review provides an overview of structures of ParB and ParA, and ParB–DNA and ParA–nucleotide complexes that have become available in the past few years and that have started to shed light on these key questions.
ParB structural diversity Recently determined structures of three ParB proteins have confirmed what previous sequence analyses suggested, which is that these functional homologues display a surprising degree of structural diversity. The recent Current Opinion in Structural Biology 2007, 17:103–109
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Figure 1
Schematic diagram showing the general steps of plasmid partition. The plasmids are represented by large circles and the ParB molecules by small circles. The first overall step is segrosome formation by the ParB protein. This is followed by plasmid pairing (interacting circles). Once the plasmids are paired, the ParA molecule enters the segrosome and drives the plasmids apart. The exact mechanism(s) used by all ParA proteins are not yet resolved and thus the ParA molecule is not shown, although its function is implied.
structures include representatives from the two subgroups of ParB-like proteins: the type Ia proteins P1 ParB and RP4 KorB, and the type Ib protein ParG [8,9,10,11]. ParG is the ParB homologue from the Escherichia coli multidrug resistance plasmid TP228. Like all characterized ParB and ParB-like proteins, ParG is a functional dimer. The NMR structure of the full-length, 76-residue protein revealed a bipartite structure, with an N-terminal disordered region (residues 1–32) and a C-terminal region (residues 33–76) with a ribbon-helixhelix (RHH) fold similar to that of members of the Arc/ MetJ superfamily of DNA-binding proteins (Figure 2a). The dimeric RHH fold of ParG indicates that it probably binds its centromere-like DNA site by forming a dimer of dimers and inserting its double-stranded b sheet into the major groove of the DNA [10]. The ParG structure underscores what appears to be a recurrent theme among ParB homologues — the multidomain, flexible nature of these proteins. Structures of RP4 KorB and P1 ParB confirm this supposition. In fact, the structures of the RP4 KorB C-terminal dimerization domain and N-terminal DNA-binding domain were solved separately. The KorB C-terminal dimerization domain (residues 297–358) was found to consist of a five-stranded, antiparallel all-b structure with striking Current Opinion in Structural Biology 2007, 17:103–109
homology to the Src homology 3 (SH3) domain found in eukaryotic signaling proteins (Figure 2b) [8]. Unlike the SH3 domain, however, the KorB C-terminal domain is missing an extended loop between b1 and b2 that is required for recognition of proline-rich motifs by SH3 proteins, while it contains a significantly elongated b5 strand that plays an essential role in KorB dimerization. The KorB C-terminal domain is not involved in centromere binding. This role is fulfilled by the N-terminal region of KorB. Insight into how KorB binds its individual centromere-like palindromic repeats was recently provided by the structure determination of the KorB DNA-binding domain (residues 101–294) bound to one of its 12 operator/centromere-like sites, Ob [9]. The KorB DNA-binding domain consists of eight helices, two of which belong to the DNA-binding helix-turn-helix (HTH) motif. Each half-site of the palindromic operator site is bound by one KorB subunit in its major groove (Figure 2b). Although the KorB HTH has a canonical HTH structure, it utilizes residues outside the recognition helix for base-specific contacts.
P1 ParB–centromere complexes and segrosome assembly The structures of ParG and KorB have provided key insight into the structural diversity of ParB proteins; however, they leave in question how these proteins mediate the assembly of large segrosomes. Recent crystal structures of the archetypical ParB protein, P1 ParB [11], have shed light on this issue (Figures 2 and 3 Figures 2c and 3a,b) [11]. The E. coli P1 par system has served as a paradigm for studies on plasmid partition. The P1 centromere-like site, parS, is an 74 bp DNA element and one of the most complex partition sites. It can be divided into three regions: a central integration host factor (IHF)-binding site; a right-hand region with two A-boxes and one B-box; and a left-hand site with one A-box and one B-box (Figure 3a) [12–18]. Interestingly, the A- and B-boxes are somehow both recognized by ParB. Although not required for partition, the E. coli IHF ab heterodimer increases partition efficiency by bending the central region of parS to juxtapose the ‘arms’ containing the A- and B-boxes, facilitating ParB binding across the bend (Figure 3c). Full-length parS confers maximal partition efficiency; however, the right-hand parS site, also called parS small, is sufficient for partition. P1 ParB residues 142–333 have all the determinants required for centromere-like binding, whereas residues 1–141 comprise the ParA-binding site. Recent structures of ParB(142–333)– parS small complexes showed that ParB(142–333) is composed of two separate domains. The first, an N-terminal (residues 147–270) HTH domain, consists of seven a helices and contains an HTH motif. This domain is connected by a flexible linker (residues 271–274) to the C-terminal domain (residues 275–333). The C-terminal dimerization domain has a novel fold consisting of three antiparallel b strands and a C-terminal a helix, which lock www.sciencedirect.com
Plasmid partition structural biology Schumacher 105
Figure 2
ParB structural diversity. (a) NMR structure of the TP228 ParB homologue ParG. (b) Crystal structure of the RP4 ParB homologue KorB. The C-terminal dimerization domain (residues 297–358) and N-terminal DNA-binding domain (residues 101–294) were solved as separate domains (not all residues were visible). The KorB dimer was modeled by linking the two domains using a flexible region (indicated by dashed lines). (c) Crystal structure of the P1 ParB(142–333)–parS small complex. The first and last observed residues of the ParB homologues are numbered. If these residues correspond to the N and C termini, they are also labeled N and C. In all ParB structures, one subunit of each dimer is colored blue and the other pink. The DNA bound by the KorB DNA-binding domain and ParB DNA-binding regions is shown as a yellow surface representation. This figure serves to underscore the surprising and dramatic structural variety present among the ParB proteins, which are functional homologues. Figures 2, 3b and 4a made using PyMOL [32].
together in the dimer to form a continuous antiparallel b sheet and coiled coil (Figures 2c and 3b). Unique structural features evident in the ParB–DNA structures explain how ParB can interact with multiple arrays of box elements in the looped parS site [11]. First, each box element is bound by a separate DNA-binding module; the A-boxes are bound by the N-terminal HTH motif, whereas the B-boxes are recognized by the C-terminal dimerization domain. Importantly, the two DNA-binding modules do not interact with each other and freely rotate, enabling them to contact multiple arrangements of A- and B-boxes, as found in parS (Figure 3b). Residues from the recognition helix of the HTH contact the major groove of the A-box elements. Interestingly, only one of these base contacts appears to be specific, suggesting the possibility that the HTH domain may play a role in non-specific DNA spreading, a property that appears to be conserved among ParB proteins [19,20]. By contrast, the novel dimerization domain, which represents a completely new type of DNA-binding motif, makes highly specific interactions with the B-box. It is notable that, although the HTH of P1 www.sciencedirect.com
ParB is structurally similar to that of KorB, the two motifs recognize their DNA sites differently. Moreover, although both proteins contain a similarly localized dimerization domain, the structures of these domains are completely different. This domain is essential for centromere binding by P1 ParB, but not by KorB. The most striking aspect of the P1 ParB–DNA structures, however, is the finding that each DNA-binding module in a given ParB dimer binds a box element located on an adjacent DNA duplex. This unique DNA-binding feature reveals how ParB can act as a bridging factor between the right-hand and left-hand arms of the bent parS site. This unusual bridging capability would also permit P1 ParB to bind between the two arms of one looped parS site while simultaneously contacting a parS site on a second plasmid, thus providing a very attractive model for plasmid pairing.
ParA proteins and mechanisms of plasmid separation Once the segrosome is assembled, the final step in partition is plasmid separation by the ParA protein, which is Current Opinion in Structural Biology 2007, 17:103–109
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Figure 3
Assembling the P1 segrosome: structures of P1 ParB–parS small. (a) Sequence of the full-length P1 parS centromere-like site. The site is divided into right and left ParB-binding sites, with an IHF-binding site positioned in the center. The A- and B-boxes are labeled. The left-hand B1 and A1 boxes are colored blue and green, whereas the right-hand A2, A3 and B2 boxes are colored yellow, green and blue, respectively. The right-hand parS site, called parS small, encompasses A2–B2 and is the minimal site required for partition. (b) P1 ParB is a DNA-bridging factor. Two structures (from two crystal forms) of P1 ParB(142–333)–parS small show four distinct bridging interactions. One ParB(142–333) subunit is colored magenta and the other cyan. The DNA is shown as transparent surfaces with box elements colored according to (a). The ParB dimerization domains are shown in the same orientation to underscore the ability of DNA-binding modules to undergo free rotation, which would enable them to contact multiple orientations of box elements in distinct or looped DNA duplexes. (c) Model of the P1 ParB–IHF–full-length parS complex. The a and b subunits of IHF are colored pink and green, and the ParB subunits are colored blue and red. The DNA is shown as sticks and colored according to atom type. The bent IHF–DNA structure is based on the IHF–DNA crystal structure (PDB code1IHF; [33]). Shown is just one possible arrangement of the ParB bridging interaction, between A1 and B2, with the looped parS site. After the initial loading of one ParB dimer, it is thought that additional ParB dimers bind the remaining box elements as well as non-specific DNA around the parS site.
either an actin-like protein (type II) or, more commonly, a Walker-A type protein (type I). Currently, structural information is only available for the type II ParA protein ParM from the R1 plasmid. Electron microscopy (EM) data for ParM showed that it polymerizes in the presence of Mg2+ATP, Mg2+ATPgS and Mg2+AMP-PNP to form double-helical protofilaments with longitudinal repeats similar to those of F-actin [21–23] (Figure 4a,b). Subsequent crystal structures of ParM confirmed that it has the characteristic actin fold [23]. Based on these data, an elegant model for plasmid separation was formulated. According to this model, ParM–Mg2+ATP contact with plasmid-paired ParR (the R1 ParB homologue) triggers nucleation of ParM filaments [22]. Filament growth would be promoted by the addition of ParM–Mg2+ATP Current Opinion in Structural Biology 2007, 17:103–109
molecules inserting into the extending polymer. This insertional polymerization of ParM–Mg2+ATP at the ParM–ParR interface would cause the ParM filaments to extend bidirectionally, pushing or pulling the two plasmids apart [23]. Subsequently, ATP hydrolysis within the ParM filaments would lead to disassembly of the filaments, as observed for actin (Figure 4c). Can a similar model explain partition by the more abundant type I par systems? Although early studies revealed no evidence of filamentation by the Walker-type ParA ATPases, more recent data have shown otherwise [24– 26]. For example, ParA homologues ParF and SopA were recently shown to form extended filaments in the presence of Mg2+ATP and ParB homologue [25,26]. www.sciencedirect.com
Plasmid partition structural biology Schumacher 107
Figure 4
Structures of the actin-like ParA homologue ParM and models of plasmid separation. (a) Crystal structures of R1 ParM in its apo and ADP-bound state. Comparison of the ParM apo and ADP-bound states reveals that ADP binding induces a 258 relative closure of the two domains. The structure of ParM is homologous to eukaryotic actin (shown at far right in its ADP-bound state). Helices are colored red, b strands are yellow and loop regions are green. The ADP molecules are shown as spheres, with carbon, nitrogen, oxygen and phosphorous atoms colored magenta, blue, red and orange, respectively. (b) EM studies on R1 ParM. In the left panel is a typical ParM filament. Filtered images are shown to its right, with arrows indicating protofilament cross-overs. The averaged diffraction from six of the straightened filaments is shown in a separate panel (bottom box), with the layer lines 45 A˚, 53 A˚ and 300 A˚ labeled. At the extreme right, colored yellow, is the final three-dimensional reconstruction, showing the calculated 49 A˚ distance between subunits observed in the diffraction studies and the cross-over distance of 300 A˚ for paired ParM protofilaments. These images reveal a ‘two-start’ helix with a somewhat shorter longitudinal spacing but very similar filament structure to that of F-actin. Figure reproduced from [21]. (c) Plasmid segregation model. The speculative steps in active segregation are as follows. (i) The plasmids align at mid-cell and are bound by the centromere-like-binding ParB proteins (yellow) via the partition sites (red). (ii) ParA–ATP (blue) is recruited to the partition complex and (iii–iv) polymerizes bidirectionally, pushing the plasmids towards the opposite cell poles. (v) Following (and perhaps during) cell division, the polymers disassemble via ATP hydrolysis. This model is consistent with data examining plasmid separation by ParM and may prove generally applicable to all par systems, including those with Walker-A type ParA proteins. Figure obtained from Hayes and Barilla [25].
In addition, the ParA protein of pB171 oscillates to form spiral-like structures over the cell nucleoid [24]. Although there are no structures available for a type I plasmid ParA protein, structures were recently obtained for the bacterial chromosomal ParA protein from Thermus thermophilus, Soj [27]. These structures show that Soj, which possesses a clear Walker-A motif, is monomeric in www.sciencedirect.com
its apo form and bound to Mg2+ADP, but dimeric when complexed with Mg2+ATP [27]. Unfortunately, how Soj might form extended filaments was not apparent from these structures. However, complementary biochemical studies demonstrated that Soj does indeed form filaments, but only in the presence of ATP and DNA. These recent data, combined with data from ParF and SopA, led Current Opinion in Structural Biology 2007, 17:103–109
108 Protein–nucleic acid interactions
to the intriguing possibility that filamentation by all ParA proteins may, in fact, be of general importance in plasmid segregation. Although the accumulating data seem to indicate that this is the case, other possibilities cannot yet be excluded. For example, the oscillation behavior of pB171 ParA may indicate a more passive role for some ParA proteins in plasmid localization, similar to how oscillation by the MinCDE system dictates the location of FtsZ [28–31].
Conclusions The past few years have resulted in the first structure determinations for ParB and ParA proteins and their complexes. These structures have started to fill in the missing pieces of the partition puzzle left after decades of cell biology and genetic studies. These pieces include how ParB proteins can bind their elaborate centromerelike sites to form segrosomes and how ParA may function as a switch to direct the movement of plasmids to their correct intracellular addresses. However, key questions remain. ParB structures have started to enumerate the surprisingly diverse structures adopted by these functional analogs. A critical challenge for the future is to elucidate how these structurally distinct proteins can bind different DNA sites and still create similar large nucleoprotein handles that are used to recruit their cognate ParA molecules. Ultimately, a complete understanding of segrosome assembly will require structures of higher order ParB– centromere complexes, including a complex with ParA. Finally, the mechanism or mechanisms employed by the various ParA proteins to drive plasmid separation are not yet resolved. Future structural and biochemical analyses will be needed to elucidate this vital step in partition.
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Jensen RB, Lurz R, Gerdes K: Mechanism of DNA segregation in prokaryotes: replicon pairing by ParC of plasmid R1. Proc Natl Acad Sci USA 1998, 95:8550-8555.
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Delbruck H, Ziegelin G, Lanka E, Heinemann U: An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4. J Biol Chem 2002, 277:4191-4198.
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Khare D, Ziegelin G, Lanka E, Heinemann U: Sequence-specific DNA binding determined by contacts outside the helix-turnhelix motif of the ParB homolog KorB. Nat Struct Mol Biol 2004, 11:656-663. The structure of the DNA-binding domain of KorB (residues 101–294) shows how it identifies its DNA site by using residues outside its HTH. The authors constructed a model showing how the C-terminal dimerization domain might connect with the DNA-binding domains. 10. Golovanov AP, Barilla D, Golovanova M, Hayes F, Lian LY: ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon-helix-helix structure. Mol Microbiol 2003, 50:1141-1153.
11. Schumacher MA, Funnell BE: Structures of ParB bound to DNA reveal mechanism of partition complex formation. Nature 2005, 438:516-519. The crystal structure of P1 ParB(142–333) bound to its minimal centromere-like site reveals several novel DNA-binding capabilities that illuminate how this protein can bind multiple box elements in different orientations, and bridge between looped or adjacent DNA sites to create condensed higher order nucleoprotein complexes. In addition, the observed bridging capabilities suggest a mechanism for plasmid pairing. 12. Funnell BE: Participation of Escherichia coli integration host factor in P1 plasmid partition system. Proc Natl Acad Sci USA 1988, 85:6657-6661. 13. Funnell BE, Gagnier L: The P1 plasmid partition complex at parS: II. Analysis of ParB protein binding activity and specificity. J Biol Chem 1993, 268:3616-3624. 14. Bouet JY, Surtees JA, Funnell BE: Stoichiometry of P1 plasmid partition complexes. J Biol Chem 2000, 275:8213-8219. 15. Surtees JA, Funnell BE: The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex. J Biol Chem 2001, 276:12385-12394. 16. Martin KA, Friedman SA, Austin SJ: Partition site of the P1 plasmid. Proc Natl Acad Sci USA 1987, 84:8544-8547.
Acknowledgements
17. Martin KA, Davis MA, Austin SJ: Fine structure analysis of the P1 plasmid partition site. J Bacteriol 1991, 173:3630-3634.
The author would like to acknowledge support from Burroughs Wellcome (Burroughs Wellcome Career Development Award 992863) and the University of Texas MD Anderson Trust Fellowship.
18. Treptow N, Rosenfeld R, Yarmolinsky M: Partition of nonreplicating DNA by the par system of bacteriophage P1. J Bacteriol 1994, 176:1782-1786.
References and recommended reading
19. Rodionov O, Yarmolinsky M: Plasmid partitioning and the spreading of P1 partition protein ParB. Mol Microbiol 2004, 283:1215-1223.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
20. Rodionov O, Lobocka M, Yarmolinsky M: Silencing of genes flanking the P1 plasmid centromere. Science 1999, 283:546-549.
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Ebersbach G, Gerdes K: Plasmid segregation mechanism. Annu Rev Genet 2005, 39:453-479.
21. van den Ent F, Møller-Jensen J, Amos LA, Gerdes K, Lo¨we J: F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J 2002, 21:6935-6943.
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Surtees JA, Funnell BE: Plasmid and chromosome traffic control: how ParA and ParB drive partition. Curr Top Dev Biol 2003, 56:145-180.
22. Møller-Jensen J, Jensen RB, Lo¨we J, Gerdes K: Prokaryotic DNA segregation by an actin-like filament. EMBO J 2002, 17:3119-3127.
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Hayes F, Barilla D: Assembling the bacterial segrosome. Trends Biochem Sci 2006, 31:247-250.
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Hayes F, Barilla D: The bacterial segrosome: a dynamic nucleoprotein machine for DNA trafficking and segregation. Nat Rev Microbiol 2006, 4:133-143.
23. Møller-Jensen J, Borch J, Dam M, Jensen RB, Roepstorff P, Gerdes K: Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism. Mol Cell 2003, 12:1477-1487.
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Gerdes K, Møller-Jensen J, Bugge Jensen R: Plasmid and chromosome partitioning: surprises from phylogeny. Mol Microbiol 2000, 37:455-466.
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Funnell BE: Partition-mediated plasmid pairing. Plasmid 2005, 53:119-125.
Current Opinion in Structural Biology 2007, 17:103–109
24. Ebersbach G, Gerdes K: Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell. Mol Microbiol 2004, 52:385-398. The first case of type I ParA oscillation is described in this study, which demonstrates that pB171 ParA forms stationary helices in the absence of ParB–centromere, but oscillates in spiral-shaped structures in the presence of the partition complex. Based on these data, passive and active models of plasmid separation are proposed. www.sciencedirect.com
Plasmid partition structural biology Schumacher 109
25. Barilla D, Rosenberg MF, Nobbmann U, Hayes F: Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF. EMBO J 2005, 24:1453-1464. Clear filamentation by the ParA homologue ParF was revealed in this study, which utilized microscopy and biophysical techniques to reveal detailed images of the filaments and their properties. 26. Lim GE, Derman AI, Poliano J: Bacterial DNA segregation by dynamic SopA polymers. Proc Natl Acad Sci USA 2005, 102:17658-17663. In addition to providing more evidence of Walker-A ParA protein filamentation, this paper shows that SopA filaments emanate from plasmid DNA in radial asters. Based on their findings, the authors propose a mechanism in which plasmid separation is driven by ParA polymerization, much like the mechanism proposed for R1 ParM. However, the authors go on to propose that the radial asters function in plasmid positioning as well. 27. Leonard TA, Butler PJ, Lo¨we J: Bacterial chromosome segregation: structure and DNA binding of the Soj dimer-a conserved biological switch. EMBO J 2005, 24:270-283. This paper describes the first structure of a ParA Walker-A protein, solved in its apo form, as well as ADP- and ATP-bound forms. The structure shows that ATP binding leads to dimerization, whereas the apo and ADPbound forms are monomers. Critically, biochemical studies show that Soj forms filaments in the presence of ATP and DNA, suggesting the pos-
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sibility that specific conditions may be required for filamentation by each Walker-A type ParA protein. 28. Lo¨we J, Amos LA: Crystal structure of the bacterial cell-division protein FtsZ. Nature 1998, 391:203-206. 29. Hayashi I, Oyama T, Morikawa K: Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus. EMBO J 2001, 20:1819-1828. 30. Cordell SC, Anderson RE, Lo¨we J: Crystal structure of the bacterial cell division inhibitor MinC. EMBO J 2001, 20:2454-2461. 31. Addinall SG, Holland B: The tubulin ancestor, FtsZ, draughtsman, designer and driving force for bacterial cytokinesis. J Mol Biol 2002, 318:219-236. 32. Delano WL: The PyMOL molecular graphics system. (DeLano Scientific, San Carlos, California, 2002). 33. Rice PA, Yang S, Mizuuchi K, Nash HA: Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 1996, 87:1295-1306.
Current Opinion in Structural Biology 2007, 17:103–109
Current Opinion in Structural Biology 2007, 17:103–109 This review comes from a themed issue on Protein–nucleic acid interactions Edited by James M Berger and Christoph W Mu¨ller Available online 11th December 2006 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.11.005
Introduction Partition, or segregation, is the process whereby genetic material is accurately moved and positioned to daughter cells during cell division. Most of our understanding of prokaryotic partition has derived from cellular, genetic and biochemical studies on the segregation of plasmid DNA. Plasmid partition ( par) systems represent simplified model systems with which to address the molecular and structural details of partition — they are composed of only three required elements: a cis-acting centromere-like (partition) DNA site(s) and two proteins typically called ParA and ParB [1–5]. Studies have shown that the first step of plasmid partition involves binding of the centromere-like partition site by the ParB protein [1–5]. Subsequently, multiple ParB molecules spread onto and around the site, leading to the creation of a large nucleoprotein structure called the partition complex or segrosome. ParB then mediates the next critical step in partition, which is plasmid pairing [6,7]. Once the plaswww.sciencedirect.com
mids are paired, the partition complexes are recognized by ParA, an ATPase, which acts as a molecular switch, as governed by its nucleotide-bound state, to drive plasmid separation (Figure 1). Plasmid-encoded par systems can be divided into two main types based on the kind of ParA protein present. Type I par systems, which are the most numerous, contain ParA proteins with putative deviant Walker-A type ATPase folds, whereas type II systems employ ParA proteins with actin-like folds [1,2]. The type I systems can be further divided into type Ia and type Ib based on the homology and size of the Par proteins. Type Ia systems contain ParA and ParB proteins of 321–420 and 312–342 residues, respectively, whereas the type Ib proteins are smaller, with ParA homologues containing 192–308 residues and ParB proteins of 46–131 residues. In the type Ia systems, ParA proteins also serve as transcriptional repressors in the autoregulation of its par operon, whereas in the type Ib and II systems, this function is carried out by ParB. Surprisingly, the ParB proteins show little to no homology, which is why the par system classification is based on ParA. Moreover, the centromere-like sites bound by ParB are also diverse. Indeed, although they typically consist of multiple direct or inverted repeats, the number of repeats, their sequence and length differ dramatically. Despite the unexpected diversity exhibited by the functionally homologous ParB proteins and centromere-like components, it appears that the partition complexes they form have a similar overall structural organization, which is used as a recognition and recruitment site by the ParA protein. Although a general outline of the plasmid partition process has been obtained, critical questions remain, largely due to a lack of structural information. Such questions include: what are the DNA-binding properties of the ParB proteins that enable them to recognize their elaborate centromere-like sites and form large nucleoprotein complexes; how do ParA proteins use adenine nucleotides to ‘switch’ conformational states; and how do the ParA proteins function to move the plasmids apart? This review provides an overview of structures of ParB and ParA, and ParB–DNA and ParA–nucleotide complexes that have become available in the past few years and that have started to shed light on these key questions.
ParB structural diversity Recently determined structures of three ParB proteins have confirmed what previous sequence analyses suggested, which is that these functional homologues display a surprising degree of structural diversity. The recent Current Opinion in Structural Biology 2007, 17:103–109
104 Protein–nucleic acid interactions
Figure 1
Schematic diagram showing the general steps of plasmid partition. The plasmids are represented by large circles and the ParB molecules by small circles. The first overall step is segrosome formation by the ParB protein. This is followed by plasmid pairing (interacting circles). Once the plasmids are paired, the ParA molecule enters the segrosome and drives the plasmids apart. The exact mechanism(s) used by all ParA proteins are not yet resolved and thus the ParA molecule is not shown, although its function is implied.
structures include representatives from the two subgroups of ParB-like proteins: the type Ia proteins P1 ParB and RP4 KorB, and the type Ib protein ParG [8,9,10,11]. ParG is the ParB homologue from the Escherichia coli multidrug resistance plasmid TP228. Like all characterized ParB and ParB-like proteins, ParG is a functional dimer. The NMR structure of the full-length, 76-residue protein revealed a bipartite structure, with an N-terminal disordered region (residues 1–32) and a C-terminal region (residues 33–76) with a ribbon-helixhelix (RHH) fold similar to that of members of the Arc/ MetJ superfamily of DNA-binding proteins (Figure 2a). The dimeric RHH fold of ParG indicates that it probably binds its centromere-like DNA site by forming a dimer of dimers and inserting its double-stranded b sheet into the major groove of the DNA [10]. The ParG structure underscores what appears to be a recurrent theme among ParB homologues — the multidomain, flexible nature of these proteins. Structures of RP4 KorB and P1 ParB confirm this supposition. In fact, the structures of the RP4 KorB C-terminal dimerization domain and N-terminal DNA-binding domain were solved separately. The KorB C-terminal dimerization domain (residues 297–358) was found to consist of a five-stranded, antiparallel all-b structure with striking Current Opinion in Structural Biology 2007, 17:103–109
homology to the Src homology 3 (SH3) domain found in eukaryotic signaling proteins (Figure 2b) [8]. Unlike the SH3 domain, however, the KorB C-terminal domain is missing an extended loop between b1 and b2 that is required for recognition of proline-rich motifs by SH3 proteins, while it contains a significantly elongated b5 strand that plays an essential role in KorB dimerization. The KorB C-terminal domain is not involved in centromere binding. This role is fulfilled by the N-terminal region of KorB. Insight into how KorB binds its individual centromere-like palindromic repeats was recently provided by the structure determination of the KorB DNA-binding domain (residues 101–294) bound to one of its 12 operator/centromere-like sites, Ob [9]. The KorB DNA-binding domain consists of eight helices, two of which belong to the DNA-binding helix-turn-helix (HTH) motif. Each half-site of the palindromic operator site is bound by one KorB subunit in its major groove (Figure 2b). Although the KorB HTH has a canonical HTH structure, it utilizes residues outside the recognition helix for base-specific contacts.
P1 ParB–centromere complexes and segrosome assembly The structures of ParG and KorB have provided key insight into the structural diversity of ParB proteins; however, they leave in question how these proteins mediate the assembly of large segrosomes. Recent crystal structures of the archetypical ParB protein, P1 ParB [11], have shed light on this issue (Figures 2 and 3 Figures 2c and 3a,b) [11]. The E. coli P1 par system has served as a paradigm for studies on plasmid partition. The P1 centromere-like site, parS, is an 74 bp DNA element and one of the most complex partition sites. It can be divided into three regions: a central integration host factor (IHF)-binding site; a right-hand region with two A-boxes and one B-box; and a left-hand site with one A-box and one B-box (Figure 3a) [12–18]. Interestingly, the A- and B-boxes are somehow both recognized by ParB. Although not required for partition, the E. coli IHF ab heterodimer increases partition efficiency by bending the central region of parS to juxtapose the ‘arms’ containing the A- and B-boxes, facilitating ParB binding across the bend (Figure 3c). Full-length parS confers maximal partition efficiency; however, the right-hand parS site, also called parS small, is sufficient for partition. P1 ParB residues 142–333 have all the determinants required for centromere-like binding, whereas residues 1–141 comprise the ParA-binding site. Recent structures of ParB(142–333)– parS small complexes showed that ParB(142–333) is composed of two separate domains. The first, an N-terminal (residues 147–270) HTH domain, consists of seven a helices and contains an HTH motif. This domain is connected by a flexible linker (residues 271–274) to the C-terminal domain (residues 275–333). The C-terminal dimerization domain has a novel fold consisting of three antiparallel b strands and a C-terminal a helix, which lock www.sciencedirect.com
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Figure 2
ParB structural diversity. (a) NMR structure of the TP228 ParB homologue ParG. (b) Crystal structure of the RP4 ParB homologue KorB. The C-terminal dimerization domain (residues 297–358) and N-terminal DNA-binding domain (residues 101–294) were solved as separate domains (not all residues were visible). The KorB dimer was modeled by linking the two domains using a flexible region (indicated by dashed lines). (c) Crystal structure of the P1 ParB(142–333)–parS small complex. The first and last observed residues of the ParB homologues are numbered. If these residues correspond to the N and C termini, they are also labeled N and C. In all ParB structures, one subunit of each dimer is colored blue and the other pink. The DNA bound by the KorB DNA-binding domain and ParB DNA-binding regions is shown as a yellow surface representation. This figure serves to underscore the surprising and dramatic structural variety present among the ParB proteins, which are functional homologues. Figures 2, 3b and 4a made using PyMOL [32].
together in the dimer to form a continuous antiparallel b sheet and coiled coil (Figures 2c and 3b). Unique structural features evident in the ParB–DNA structures explain how ParB can interact with multiple arrays of box elements in the looped parS site [11]. First, each box element is bound by a separate DNA-binding module; the A-boxes are bound by the N-terminal HTH motif, whereas the B-boxes are recognized by the C-terminal dimerization domain. Importantly, the two DNA-binding modules do not interact with each other and freely rotate, enabling them to contact multiple arrangements of A- and B-boxes, as found in parS (Figure 3b). Residues from the recognition helix of the HTH contact the major groove of the A-box elements. Interestingly, only one of these base contacts appears to be specific, suggesting the possibility that the HTH domain may play a role in non-specific DNA spreading, a property that appears to be conserved among ParB proteins [19,20]. By contrast, the novel dimerization domain, which represents a completely new type of DNA-binding motif, makes highly specific interactions with the B-box. It is notable that, although the HTH of P1 www.sciencedirect.com
ParB is structurally similar to that of KorB, the two motifs recognize their DNA sites differently. Moreover, although both proteins contain a similarly localized dimerization domain, the structures of these domains are completely different. This domain is essential for centromere binding by P1 ParB, but not by KorB. The most striking aspect of the P1 ParB–DNA structures, however, is the finding that each DNA-binding module in a given ParB dimer binds a box element located on an adjacent DNA duplex. This unique DNA-binding feature reveals how ParB can act as a bridging factor between the right-hand and left-hand arms of the bent parS site. This unusual bridging capability would also permit P1 ParB to bind between the two arms of one looped parS site while simultaneously contacting a parS site on a second plasmid, thus providing a very attractive model for plasmid pairing.
ParA proteins and mechanisms of plasmid separation Once the segrosome is assembled, the final step in partition is plasmid separation by the ParA protein, which is Current Opinion in Structural Biology 2007, 17:103–109
106 Protein–nucleic acid interactions
Figure 3
Assembling the P1 segrosome: structures of P1 ParB–parS small. (a) Sequence of the full-length P1 parS centromere-like site. The site is divided into right and left ParB-binding sites, with an IHF-binding site positioned in the center. The A- and B-boxes are labeled. The left-hand B1 and A1 boxes are colored blue and green, whereas the right-hand A2, A3 and B2 boxes are colored yellow, green and blue, respectively. The right-hand parS site, called parS small, encompasses A2–B2 and is the minimal site required for partition. (b) P1 ParB is a DNA-bridging factor. Two structures (from two crystal forms) of P1 ParB(142–333)–parS small show four distinct bridging interactions. One ParB(142–333) subunit is colored magenta and the other cyan. The DNA is shown as transparent surfaces with box elements colored according to (a). The ParB dimerization domains are shown in the same orientation to underscore the ability of DNA-binding modules to undergo free rotation, which would enable them to contact multiple orientations of box elements in distinct or looped DNA duplexes. (c) Model of the P1 ParB–IHF–full-length parS complex. The a and b subunits of IHF are colored pink and green, and the ParB subunits are colored blue and red. The DNA is shown as sticks and colored according to atom type. The bent IHF–DNA structure is based on the IHF–DNA crystal structure (PDB code1IHF; [33]). Shown is just one possible arrangement of the ParB bridging interaction, between A1 and B2, with the looped parS site. After the initial loading of one ParB dimer, it is thought that additional ParB dimers bind the remaining box elements as well as non-specific DNA around the parS site.
either an actin-like protein (type II) or, more commonly, a Walker-A type protein (type I). Currently, structural information is only available for the type II ParA protein ParM from the R1 plasmid. Electron microscopy (EM) data for ParM showed that it polymerizes in the presence of Mg2+ATP, Mg2+ATPgS and Mg2+AMP-PNP to form double-helical protofilaments with longitudinal repeats similar to those of F-actin [21–23] (Figure 4a,b). Subsequent crystal structures of ParM confirmed that it has the characteristic actin fold [23]. Based on these data, an elegant model for plasmid separation was formulated. According to this model, ParM–Mg2+ATP contact with plasmid-paired ParR (the R1 ParB homologue) triggers nucleation of ParM filaments [22]. Filament growth would be promoted by the addition of ParM–Mg2+ATP Current Opinion in Structural Biology 2007, 17:103–109
molecules inserting into the extending polymer. This insertional polymerization of ParM–Mg2+ATP at the ParM–ParR interface would cause the ParM filaments to extend bidirectionally, pushing or pulling the two plasmids apart [23]. Subsequently, ATP hydrolysis within the ParM filaments would lead to disassembly of the filaments, as observed for actin (Figure 4c). Can a similar model explain partition by the more abundant type I par systems? Although early studies revealed no evidence of filamentation by the Walker-type ParA ATPases, more recent data have shown otherwise [24– 26]. For example, ParA homologues ParF and SopA were recently shown to form extended filaments in the presence of Mg2+ATP and ParB homologue [25,26]. www.sciencedirect.com
Plasmid partition structural biology Schumacher 107
Figure 4
Structures of the actin-like ParA homologue ParM and models of plasmid separation. (a) Crystal structures of R1 ParM in its apo and ADP-bound state. Comparison of the ParM apo and ADP-bound states reveals that ADP binding induces a 258 relative closure of the two domains. The structure of ParM is homologous to eukaryotic actin (shown at far right in its ADP-bound state). Helices are colored red, b strands are yellow and loop regions are green. The ADP molecules are shown as spheres, with carbon, nitrogen, oxygen and phosphorous atoms colored magenta, blue, red and orange, respectively. (b) EM studies on R1 ParM. In the left panel is a typical ParM filament. Filtered images are shown to its right, with arrows indicating protofilament cross-overs. The averaged diffraction from six of the straightened filaments is shown in a separate panel (bottom box), with the layer lines 45 A˚, 53 A˚ and 300 A˚ labeled. At the extreme right, colored yellow, is the final three-dimensional reconstruction, showing the calculated 49 A˚ distance between subunits observed in the diffraction studies and the cross-over distance of 300 A˚ for paired ParM protofilaments. These images reveal a ‘two-start’ helix with a somewhat shorter longitudinal spacing but very similar filament structure to that of F-actin. Figure reproduced from [21]. (c) Plasmid segregation model. The speculative steps in active segregation are as follows. (i) The plasmids align at mid-cell and are bound by the centromere-like-binding ParB proteins (yellow) via the partition sites (red). (ii) ParA–ATP (blue) is recruited to the partition complex and (iii–iv) polymerizes bidirectionally, pushing the plasmids towards the opposite cell poles. (v) Following (and perhaps during) cell division, the polymers disassemble via ATP hydrolysis. This model is consistent with data examining plasmid separation by ParM and may prove generally applicable to all par systems, including those with Walker-A type ParA proteins. Figure obtained from Hayes and Barilla [25].
In addition, the ParA protein of pB171 oscillates to form spiral-like structures over the cell nucleoid [24]. Although there are no structures available for a type I plasmid ParA protein, structures were recently obtained for the bacterial chromosomal ParA protein from Thermus thermophilus, Soj [27]. These structures show that Soj, which possesses a clear Walker-A motif, is monomeric in www.sciencedirect.com
its apo form and bound to Mg2+ADP, but dimeric when complexed with Mg2+ATP [27]. Unfortunately, how Soj might form extended filaments was not apparent from these structures. However, complementary biochemical studies demonstrated that Soj does indeed form filaments, but only in the presence of ATP and DNA. These recent data, combined with data from ParF and SopA, led Current Opinion in Structural Biology 2007, 17:103–109
108 Protein–nucleic acid interactions
to the intriguing possibility that filamentation by all ParA proteins may, in fact, be of general importance in plasmid segregation. Although the accumulating data seem to indicate that this is the case, other possibilities cannot yet be excluded. For example, the oscillation behavior of pB171 ParA may indicate a more passive role for some ParA proteins in plasmid localization, similar to how oscillation by the MinCDE system dictates the location of FtsZ [28–31].
Conclusions The past few years have resulted in the first structure determinations for ParB and ParA proteins and their complexes. These structures have started to fill in the missing pieces of the partition puzzle left after decades of cell biology and genetic studies. These pieces include how ParB proteins can bind their elaborate centromerelike sites to form segrosomes and how ParA may function as a switch to direct the movement of plasmids to their correct intracellular addresses. However, key questions remain. ParB structures have started to enumerate the surprisingly diverse structures adopted by these functional analogs. A critical challenge for the future is to elucidate how these structurally distinct proteins can bind different DNA sites and still create similar large nucleoprotein handles that are used to recruit their cognate ParA molecules. Ultimately, a complete understanding of segrosome assembly will require structures of higher order ParB– centromere complexes, including a complex with ParA. Finally, the mechanism or mechanisms employed by the various ParA proteins to drive plasmid separation are not yet resolved. Future structural and biochemical analyses will be needed to elucidate this vital step in partition.
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Khare D, Ziegelin G, Lanka E, Heinemann U: Sequence-specific DNA binding determined by contacts outside the helix-turnhelix motif of the ParB homolog KorB. Nat Struct Mol Biol 2004, 11:656-663. The structure of the DNA-binding domain of KorB (residues 101–294) shows how it identifies its DNA site by using residues outside its HTH. The authors constructed a model showing how the C-terminal dimerization domain might connect with the DNA-binding domains. 10. Golovanov AP, Barilla D, Golovanova M, Hayes F, Lian LY: ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon-helix-helix structure. Mol Microbiol 2003, 50:1141-1153.
11. Schumacher MA, Funnell BE: Structures of ParB bound to DNA reveal mechanism of partition complex formation. Nature 2005, 438:516-519. The crystal structure of P1 ParB(142–333) bound to its minimal centromere-like site reveals several novel DNA-binding capabilities that illuminate how this protein can bind multiple box elements in different orientations, and bridge between looped or adjacent DNA sites to create condensed higher order nucleoprotein complexes. In addition, the observed bridging capabilities suggest a mechanism for plasmid pairing. 12. Funnell BE: Participation of Escherichia coli integration host factor in P1 plasmid partition system. Proc Natl Acad Sci USA 1988, 85:6657-6661. 13. Funnell BE, Gagnier L: The P1 plasmid partition complex at parS: II. Analysis of ParB protein binding activity and specificity. J Biol Chem 1993, 268:3616-3624. 14. Bouet JY, Surtees JA, Funnell BE: Stoichiometry of P1 plasmid partition complexes. J Biol Chem 2000, 275:8213-8219. 15. Surtees JA, Funnell BE: The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex. J Biol Chem 2001, 276:12385-12394. 16. Martin KA, Friedman SA, Austin SJ: Partition site of the P1 plasmid. Proc Natl Acad Sci USA 1987, 84:8544-8547.
Acknowledgements
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The author would like to acknowledge support from Burroughs Wellcome (Burroughs Wellcome Career Development Award 992863) and the University of Texas MD Anderson Trust Fellowship.
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24. Ebersbach G, Gerdes K: Bacterial mitosis: partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell. Mol Microbiol 2004, 52:385-398. The first case of type I ParA oscillation is described in this study, which demonstrates that pB171 ParA forms stationary helices in the absence of ParB–centromere, but oscillates in spiral-shaped structures in the presence of the partition complex. Based on these data, passive and active models of plasmid separation are proposed. www.sciencedirect.com
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25. Barilla D, Rosenberg MF, Nobbmann U, Hayes F: Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF. EMBO J 2005, 24:1453-1464. Clear filamentation by the ParA homologue ParF was revealed in this study, which utilized microscopy and biophysical techniques to reveal detailed images of the filaments and their properties. 26. Lim GE, Derman AI, Poliano J: Bacterial DNA segregation by dynamic SopA polymers. Proc Natl Acad Sci USA 2005, 102:17658-17663. In addition to providing more evidence of Walker-A ParA protein filamentation, this paper shows that SopA filaments emanate from plasmid DNA in radial asters. Based on their findings, the authors propose a mechanism in which plasmid separation is driven by ParA polymerization, much like the mechanism proposed for R1 ParM. However, the authors go on to propose that the radial asters function in plasmid positioning as well. 27. Leonard TA, Butler PJ, Lo¨we J: Bacterial chromosome segregation: structure and DNA binding of the Soj dimer-a conserved biological switch. EMBO J 2005, 24:270-283. This paper describes the first structure of a ParA Walker-A protein, solved in its apo form, as well as ADP- and ATP-bound forms. The structure shows that ATP binding leads to dimerization, whereas the apo and ADPbound forms are monomers. Critically, biochemical studies show that Soj forms filaments in the presence of ATP and DNA, suggesting the pos-
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