Biochem. J. (2008) 412, 1–18 (Printed in Great Britain)
1
doi:10.1042/BJ20080359
REVIEW ARTICLE
Structural biology of plasmid partition: uncovering the molecular
mechanisms of DNA segregation
Maria A. SCHUMACHER1
Department of Biochemistry and Molecular Biology, University of Texas, M.D. Anderson Cancer Center, Unit 1000, Houston, TX 77030, U.S.A.
DNA segregation or partition is an essential process that ensures
stable genome transmission. In prokaryotes, partition is best
understood for plasmids, which serve as tractable model systems
to study the mechanistic underpinnings of DNA segregation at
a detailed atomic level owing to their simplicity. Specifically,
plasmid partition requires only three elements: a centromerelike DNA site and two proteins: a motor protein, generally an
ATPase, and a centromere-binding protein. In the first step of the
partition process, multiple centromere-binding proteins bind cooperatively to the centromere, which typically consists of several
tandem repeats, to form a higher-order nucleoprotein complex
called the partition complex. The partition complex recruits the
ATPase to form the segrosome and somehow activates the ATPase
for DNA separation. Two major families of plasmid par systems
have been delineated based on whether they utilize ATPase
proteins with deviant Walker-type motifs or actin-like folds. In
contrast, the centromere-binding proteins show little sequence
homology even within a given family. Recent structural studies,
however, have revealed that these centromere-binding proteins
appear to belong to one of two major structural groups: those
that employ helix–turn–helix DNA-binding motifs or those with
ribbon–helix–helix DNA-binding domains. The first structure of
a higher-order partition complex was recently revealed by the
structure of pSK41 centromere-binding protein, ParR, bound to its
centromere site. This structure showed that multiple ParR ribbon–
helix–helix motifs bind symmetrically to the tandem centromere
repeats to form a large superhelical structure with dimensions
suitable for capture of the filaments formed by the actinlike ATPases. Surprisingly, recent data indicate that the deviant
Walker ATPase proteins also form polymer-like structures, suggesting that, although the par families harbour what initially
appeared to be structurally and functionally divergent proteins,
they actually utilize similar mechanisms of DNA segregation.
Thus, in the present review, the known Par protein and Par–
protein complex structures are discussed with regard to their
functions in DNA segregation in an attempt to begin to define,
at a detailed atomic level, the molecular mechanisms involved in
plasmid segregation.
INTRODUCTION
partition reaction (Figure 1A). However, the amounts of each of
the Par proteins are carefully regulated, as deficiency or excess
of either protein can completely or seriously impede the partition
process [14 –17]. Fluorescence microscopy studies have provided
a visual picture of the highly dynamic partition reaction. For
the most common type of par system, immunofluorescence and
FISH (fluorescent in situ hybridization) studies have shown that
newborn cells usually have one central plasmid focus, whereas
larger older cells have two foci located at the one-fourth and
three-fourth positions of the cell, indicative of plasmid segregation
[18–23]. Studies using GFP (green fluorescent protein)-tagged
plasmids gave visualized plasmid movement in live cells and have
revealed that plasmid movement and separation are relatively
rapid events in the entire cell cycle. The challenge has been to
relate these visual observations with the molecular steps involved
in plasmid partition.
A general understanding of the steps involved in DNA partition
has been obtained from biochemical, cellular and genetic studies.
Specifically, the first step involves binding of the centromerelike partition site by the centromere-binding protein, ultimately
leading to the creation of a large nucleoprotein complex [3–11]
(Figure 1A). Next, interactions between the centromere-binding
proteins and the DNA lead to pairing between partition complexes
on two different plasmids. Once the plasmids are paired, the
DNA partition or segregation, is the process whereby the genetic
material is accurately moved and positioned to daughter cells
during cell division. The partition of newly replicated eukaryotic
chromosomes is carried out by the formation of microtubulebased spindles, which pull chromosomes to opposite cell poles
[1]. In contrast, the molecular machinery that mediates the
segregation of prokaryotic DNA has been less clear. Much of our
understanding of prokaryotic partition has resulted from cellular,
genetic and biochemical analyses on the segregation of low-copynumber plasmids. Such plasmids play a significant role in the
spread of multidrug resistance and also play a central role in DNA
manipulation technology. Thus understanding the mechanism
behind their maintenance has important consequences. Whereas
high-copy-number plasmids primarily rely on passive diffusion
for plasmid maintenance, low-copy-number plasmids utilize socalled partition (par) systems, which are carried on the plasmid
DNA [2–13]. The majority of par operons or cassettes contain
two genes: one encoding a motor protein, which is typically
an ATPase, and the second encoding a centromere-binding
protein [2–11]. In addition, the centromere-like site bound by
the centromere-binding protein is located near the par cassette.
These three components are all that is required to direct the
Key words: deviant Walker box motif, DNA segregation,
insertional polymerization model, partition, partition complex,
plasmid.
Abbreviations used: EM, electron microscopy; FP, fluorescence polarization; HTH, helix–turn–helix; IHF, integration host factor; p[NH]ppG, guanosine
5 -[β,γ-imido]triphosphate; RHH, ribbon–helix–helix; SH3, Src homology 3.
1
email [email protected]
c The Authors Journal compilation c 2008 Biochemical Society
2
Figure 1
M. A. Schumacher
Genetic organization of par operons
(A) Schematic diagram of the general steps of plasmid partition. Plasmids are represented as large circles, and the partition complexes formed by centromere-binding proteins binding to the
centromere are shown as small circles. The first step is partition complex formation. Subsequently, plasmid pairing (interacting ovals) occurs. Once the plasmids are paired, the motor protein is
recruited to the partition complex and drives plasmid separation. (B) Genetic organizations for the two main families, type I and II, of par loci. Shown are representatives from each class (type Ia, F
and P1; type Ib, pTP228; double loci, pB171; and type II, R1). Orange arrows represent genes that encode motor proteins (ATPases) and blue arrows represent genes encoding centromere-binding
proteins. Centromere sites are indicated by black bars and are labelled (e.g. parS , sopC , parC1, parC2 and parC ). Arcs represent DNA-binding properties of the indicated gene products, continuous
arcs show regulation of promoter activity by the indicated gene products and broken arcs represent formation of partition complexes.
partition complexes are recognized by the motor protein leading
to the assembly of the segrosome, which then actively mediates
the separation of the plasmids (Figure 1A). The simplicity of the
plasmid par systems marks them as extremely attractive model
systems to address the fundamental biological question of how
DNA is segregated to daughter cells at the atomic level. As
described in the present review, structural work carried out in
the last 5 years has started to elucidate the detailed molecular
mechanisms utilized by these proteins in each step of the DNA
segregation process.
PLASMID PARTITION CASSETTES AND FAMILIES
The first identified plasmid partition systems and their associated
genetic loci are those of the Escherichia coli P1 and F plasmid
par systems. These discoveries were made over 25 years ago by
the Hiraga and Austin groups [11,24,25]. Since then, multiple par
cassettes have been found experimentally and by bioinformatics
approaches in plasmids from very diverse bacteria [7]. Most of
these cassettes have very similar genetic organizations whereby
they consist of three elements: a cis-acting centromere-like site
and two trans-acting proteins: a centromere-binding protein and
a motor or force-generating protein (Figure 1B). Although the
force-generating proteins were all originally found to be ATPases,
recent work shows that tubulin-like GTPases and, possibly, coiledcoil proteins can also function as the force-generating protein
to drive partition. To reflect this, these proteins are referred to
as motor proteins. The upstream gene in the operon encodes
the motor protein, whereas the downstream gene encodes the
centromere-binding protein (Figure 1B). The centromere-like site
itself is typically located either upstream or downstream of the
par operon [3–11]. An important property of par cassettes is their
ability to stabilize heterologous replicons, which means that they
can act independently of the replication system and still mediate
plasmid separation, ensuring plasmid maintenance.
c The Authors Journal compilation c 2008 Biochemical Society
An analysis of all the par systems that had been identified to
date by Gerdes et al. in 2000 [7] led to their classification into
two main types. This categorization was based on the kind of
motor protein that is present, as the centromere-binding proteins
exhibit significant sequence diversity. Accordingly, the par motor
proteins can readily be broken into two main groups. In these
two groups, the motor protein belongs to one of two families
of ATPases: one with a deviant Walker box motif (also called
P-loop) and the second containing an actin-like fold. In the present
review, this categorization of par systems as suggested by Gerdes
et al. [7] has been used because it provides a useful framework
for describing and comparing Par protein structure. More recently,
additional putative par systems that are unrelated to the two major
par families have been identified. However, structures of these
proteins have yet to be determined. We shall begin by defining
and describing the two par major families as well as two more
recently discovered putative par systems, which we delineate as
the type III and type IV systems, as this sets the stage for the
ensuing discussion on partition biology and Par protein structure–
function relationships.
Type I and II par systems
The type I are the most numerous of the par systems and contain motor proteins with Walker-type or P-loop ATPase motifs
[5]. These ATPase proteins are typically called ParA, and their
centromere-binding protein counterpart is called ParB. The type I
par systems can be divided further into type Ia and type Ib on
the basis of the genetic organization of the par operon and the
limited sequence and size homologies of the Par proteins encoded
by these systems (Figure 1B). In both type Ia and Ib systems,
the gene for the Walker-type ATPase is located upstream of that
for the centromere-binding protein. However, the location of the
centromere site differs: for the type Ia systems, the centromerelike site is located downstream of the par operon, whereas, in the
type Ib systems, the centromere is found upstream (Figure 1B).
Structure and function of plasmid partition proteins
The type Ia Par proteins are characteristically larger than type Ib
proteins. Specifically, the type Ia ParA and ParB proteins typically
consist of 251– 420 residues and 182–336 residues respectively.
In contrast, type Ib ParA and ParB proteins generally contain
208–227 residues and 46–113 residues respectively. Notably, the
type Ia ParA proteins have a dual role in partition; they function in
both partition and transcription autoregulation of their respective
par operons [3–11,26–29]. Consistent with this additional role,
type Ia ParA proteins contain an N-terminal region consisting
of 108–130 residues in addition to their C-terminal Walker box
domain. This N-terminal region contains a putative HTH (helix–
turn–helix) DNA-binding motif, which is not found in the smaller
type Ib Walker-box ATPase proteins. Thus, in the type Ib system,
the motor proteins do not function as transcriptional regulators.
However, maintenance of the correct cellular concentrations
of Par proteins is essential for proper partitioning by all par
systems. In the type Ib and type II par systems, the centromerebinding proteins carry out this important autoregulatory function
(Figure 1B). There is no significant sequence identity or similarity
between the type Ia and Ib centromere-binding proteins. In
addition, type I centromere-binding proteins are sequentially
diverse compared with the type II centromere-binding proteins.
The most conserved region among the type Ia ParB proteins
is a predicted HTH motif. The type Ib centromere-binding
proteins display even more distinct sequences than the type Ia
ParB proteins and contain no identifiable DNA-binding motif(s).
Indeed, it was not until recent structures were solved that the
DNA-binding motifs of these proteins were revealed.
The type Ia par loci from the E. coli P1 and F plasmids are
among the best characterized par systems [10,11,24,25]. The
P1 par system contains the prototypical ParA ATPase and
the centromere-binding protein ParB, whereas the F par system
encodes an ATPase called SopA and a centromere-binding protein
named SopB. Studies on these systems revealed that the ParB and
SopB proteins bind to specific centromere sites and form higherorder complexes called partition complexes, which recruit the
ParA/SopA proteins for completion of the partition reaction [30–
32]. More recently, Hayes and co-workers have carried out an
extensive analysis of the type Ib par system from the Salmonella
Newport TP228 plasmid [33–35]. These studies revealed that the
TP228 par system contains functional analogues of ParA and
ParB, called ParF and ParG respectively [33–35].
The second type of par system, termed type II, contains
ATPase proteins called ParM (for motor) that belong to the
actin/hsp70 (heat-shock protein 70) superfamily. The centromerebinding proteins in type II systems are called ParR. The genetic
organization of type II systems is very similar to those of the
type Ib systems, wherein the parM gene is located upstream
of the parR gene and the centromere site is generally located
upstream of the parMR genes (Figure 1B). The type II ParM
proteins contain 236 –336 residues, whereas the ParR proteins,
similarly to the type Ib ParB proteins, are small, generally 46 –
120 residues in length [7,36 – 44]. Because the type II centromerebinding proteins also function in transcription autoregulation, they
were named ParR, for Par repressor. In contrast, the ParM proteins
show no DNA-binding activity. Similarly to the type I centromerebinding proteins, the ParR proteins show little to no sequence
similarity (or identity). Again, until structures were solved, the
DNA-binding motif and manner of DNA recognition employed
by these proteins was unknown.
The best investigated type II par system is that harboured on the
E. coli R1 plasmid [36 – 44]. Like all type II systems, R1 contains
a ParM ATPase and a ParR centromere-binding protein. The R1
centromere, parC, is somewhat unusual in that it is discontinuous;
it consists of two clusters of five 11 bp repeats, which are located
Figure 2
3
Representative par centromere sites
Well-characterized centromere-like sites are shown for two of each class of par systems (type Ia,
type Ib and type II). The arrows represent repeat elements within each centromere. The arrows
are schematic; the lengths of the arrows do not represent nucleotide length. The number of
nucleotides in each repeat are indicated. For example, each sopC repeat is 43 bp, the P1 parS
B-box elements are 6 bp (orange), and the A-box elements are 7 bp (blue). The central IHF site
is 29 bp (grey). The TP228 parH repeats contain 19 bp repeats, whereas the pTAR parS
site contains consensus repeats that are 7 bp. The type II pSK41 parC repeats are 20 bp,
whereas the R1 repeats within parC are 11 bp in length.
on either side of the − 10 and − 35 DNA promoter motifs [13,40]
(Figure 2). The R1 par system has been thoroughly dissected
from a cellular, biological and biochemical standpoint. On the
basis of these detailed analyses, the insertional polymerization
model was formulated [45]. The foundation of this model is
based on the important discovery that the R1 ParM protein
forms filaments in an ATP-dependent manner [45]. According
to the model, ParM filaments are first captured between the
paired partition complexes, one end of each filament attaching
to each partition complex. Continued filament growth between
the plasmids then forces the plasmids to the cell poles, hence
leading to DNA segregation. Recent studies have demonstrated
that this process can be accomplished in vitro using only the three
components ParM, ParR and parC [46]. Thus this latter study
firmly established that cellular factors are not essential for the
general mechanism of type II plasmid separation in vitro, although
a role for such factors in vivo cannot yet be excluded [46].
Double par loci on a single plasmid
The E. coli pB171 and Salmonella enterica R27 plasmids are
unique in the plasmid par systems studied thus far in that both
contain a type I and a type II par system [47–53] (Figure 1B). To
date, however, plasmids carrying two par loci of the same type
(type I or II) have not been identified. Koonin [54] and Mushegian
and Koonin [55] have speculated that all biological functions
have evolved independently at least twice and that analogous
genes can often replace each other in a process called non-orthologous gene displacement. Given the fact that type I systems are
more abundant and also more efficient than type II par systems in
plasmid retention, Gerdes and co-workers have proposed that the
type I par systems, may in fact be replacing the type II systems
[51]. The increased abundance of type I loci compared with
type II systems appears to support this idea. They cite the pB171
plasmid as a possible evolutionary snapshot in non-orthologous
gene displacement whereby the type I par locus is in the process
of replacing the less efficient type II locus [51].
Type III and IV par systems
The majority of par systems can be categorized as either type I or
II. However, recent data indicate that there are other types of par
systems. A possible addition to the family of plasmid par systems,
which we shall term type III, was identified on the Bacillus
thuringiensis virulence plasmid pBtoxis [56 –58]. This putative
new par system encodes an upstream gene called tubR and a
downstream gene, tubZ. Thus this genetic organization, which
c The Authors Journal compilation c 2008 Biochemical Society
4
M. A. Schumacher
places the gene encoding the centromere-binding protein before
the motor protein, is distinct from the type I and II par systems
[56]. A possible centromere site for the B. thuringiensis israelensis
subspecies has been identified and consists of four 12 bp direct
repeats [57]. The TubR protein, which contains a putative HTH
domain, was shown to bind specifically to these repeats. Perhaps
the most interesting feature of this system, however, was the
finding that the TubZ protein contains neither a deviant Walker box
motif nor an actin-like fold and instead appears to be a member of
the tubulin/FtsZ GTPase superfamily of motor proteins. In fact,
it was clearly demonstrated that TubZ assembles into dynamic
polymers that exhibit directional polymerization with plus and
minus ends, indicating that plasmid partition of pBtoxis may be
mediated by tubulin-like polymers [56]. How TubZ might attach to
plasmids to drive segregation remains unclear. However, it appears
that TubR can recruit TubZ polymers to the partition site, similarly
to how the centromere-binding proteins of the type I and II par
systems engage their requisite ATPase motor proteins. The recent
finding that the Bacillus anthracis pXO1 plasmid RepX protein
(now called TubZ-Ba) forms two stranded filaments identical with
those formed by the B. thuringiensis TubZ protein (TubZ-Bt)
supports the contention that there may be several type III par
systems [58–60]. Indeed, at least four TubZ-like sequences have
been found on different Bacillus plasmids [56].
Finally, a potential type IV partition system that utilizes only
one protein has been identified on the Staphylococcus aureus
plasmid pSK1 [61]. This protein, called Par, contains 245 residues
and was shown to be critical for extending the segregational
stability of the pSK1 plasmid, supporting the idea that it may
function in partition. It seems unlikely that a second protein is
present in the pSK1 par operon as only one significant open
reading frame, belonging to par, has been identified in the coding
region. How this protein may play both centromere-binding
and motor functions is unknown. However, structural prediction
studies suggest that Par contains an N-terminal HTH domain,
which could function in centromere-binding, and a central coiledcoil domain, which might form polymeric structures similar to
those formed by coiled-coil-containing proteins in the cytoskeleton of eukaryotic cells [62–64]. Therefore the pSK1 Par protein
contains domains that could conceivably perform both roles in partition. Clearly, more studies are needed to elucidate the mechanism of pSK1 segregation and the involvement of the pSK1 Par
protein in this process. Interestingly, Par homologues have been
found on plasmids in a range of Gram-positive bacteria, including Staphylococcus, Streptococcus, Lactococcus, Lactobacillus,
Clostridium and Tetragenococcus, suggesting that such systems
are not uncommon [65].
these examples of bacterial chromosomal DNA segregation by
plasmid-like Par proteins.
Bacterial chromosomal par systems appear to be hybrids of
plasmid Type Ia and Ib systems in that the chromosomal ParA
proteins contain deviant Walker box motifs of the small Type Ib
category, whereas the corresponding chromosomal centromerebinding proteins contain HTH motifs and are very similar to
plasmid type Ia centromere-binding proteins [7]. Initial indications that Par-like proteins might be involved in chromosome
segregation in bacteria came from genetic studies showing that
mutation of the chromosomally encoded B. subtilis ParB homologue, spo0J, resulted in a 100-fold increase in the production of
anucleate cells [71]. Found on the same operon as spo0J is a
gene, soj, which encodes a deviant Walker box-containing protein.
Initial studies on Soj mutants suggested that it is not involved
in DNA segregation [71,72]. However, more recent data show
that Spo0J and Soj together play an important role in bringing
together centromere sites that are far apart on the B. subtilis
chromosome and organizing the origin region into a compact
structure that facilitates separation of replicated origins [73]. To
do this, Spo0J, like its plasmid counterparts, binds a conserved
DNA site, numerous copies of which are clustered about the soj–
spo0J operon and the replicated chromosomal origins.
There are only a few cases in which chromosomal Par proteins
have been shown to be essential for bacterial DNA partition under
normal cellular conditions [74 –76]. In C. crescentus, the ParB and
ParA homologues are required for chromosome segregation,
and inactivation of either one is lethal. Another example
is chromosome II of Vibrio cholerae. V. cholerae contains two
chromosomes and each has a parAB system. Recent studies have
found that segregation of chromosome II is absolutely dependent
upon its parAB system [77,78]. It is interesting to speculate that
this chromosome may actually represent a co-opted plasmid
that has taken on a role as a host chromosome. In contrast with
these examples in which par systems are essential for DNA
segregation, some bacterial par systems are important only under
specialized conditions. For example, deletion of the Streptomyces
coelicolor par system causes serious chromosome defects only
during the formation of chain spores [79]. Consistent with this,
parAB transcription is greatly stimulated during S. coelicolor
sporulation [79]. In P. putida, the parAB locus plays a vital role
in DNA segregation during the transition from exponential to
stationary phase in cells grown in minimal medium [80,81]. Thus
these combined data suggest that plasmid Par homologues play
important roles in DNA segregation in some bacteria, whereas
they may be important only under certain conditions in others.
Consistent with this supposition, more than 50 homologues of
ParA and ParB have been identified in bacteria thus far [82].
Bacterial chromosomal par systems
It is interesting that, although the mechanism(s) of DNA segregation utilized by eukaryotes and plasmids have been delineated
in general terms, the means by which most bacteria partition
chromosomes is still largely a mystery. This is notably and
surprisingly true for the model Gram-negative organism, E. coli.
Indeed, no partition proteins with homology with plasmid Par
proteins have been identified in E. coli genome searches, nor have
any Par-like proteins been isolated from genetic screens [66].
However, DNA segregation of some bacterial chromosomes do
involve Par protein homologues and these proteins are surprisingly
similar to their plasmid counterparts. The best-studied examples
of chromosomal par loci are from Bacillus subtilis, Caulobacter
crescentus, Pseudomonas putida and Pseudomonas aeruginosa
[67–70]. Although the present review focuses on studies on
plasmid partition machinery, we shall briefly mention some of
c The Authors Journal compilation c 2008 Biochemical Society
CELLULAR AND BIOCHEMICAL STUDIES REVEAL PLASMID
SEGREGATION STEPS
Cellular and biochemical studies have provided a general outline
of the steps involved in the plasmid partition process. The
important findings from these studies will be summarized next
to set the stage for the discussion of structural studies, which, in
turn, have begun to elucidate the detailed molecular mechanisms
involved in DNA segregation.
First step: formation of the partition complex
The first step of the partition reaction involves the co-operative
binding of the centromere-binding proteins to their cognate centromere site. Just as eukaryotic centromeres serve as attachment
Structure and function of plasmid partition proteins
sites for spindle microtubules, plasmid segregation depends on
the creation of a defined nucleoprotein complex, called the
partition complex, which is formed by the accurate assembly of
centromere-binding proteins on to the centromere site. As noted,
little homology exists between centromere-binding proteins,
and the centromere-like sites that they bind are also diverse.
However, most plasmid centromere-like sites share an important
characteristic, which is that they consist of multiple DNA repeat
elements (Figure 2). The repeats can be extensive, as exemplified
by the F plasmid centromere, sopC, which contains 12 tandem
repeats of a 43 bp element [30,83]. Also, the parS centromere of
the Agrobacterium tumefaciens pTAR plasmid contains 13 repeats
that are separated by an integral number of turns of the DNA
double helix [84]. The type II R1 parC centromere has a complex
arrangement in that it consists of two sets of 11 bp repeats that
are separated by a 39 bp region that includes the parMR promoter
[13]. But the most complex centromere is the parS centromere
of the P1 plasmid. Similar, albeit less well studied, centromeres
are found on related plasmids such as the E. coli P7 plasmid
[85–88]. These centromeres contain a central binding site for the
E. coli protein IHF (integration host factor) (Figure 2). IHF is an
architectural protein that bends DNA by ∼ 180◦ [89]. On either
side of the IHF site in the P1 centromere are asymmetrically
arranged DNA-binding motifs called A-boxes and B-boxes that
are recognized by the P1 ParB protein. Not only are the boxes
asymmetrically arranged, they also differ in number on each arm
whereby the ‘left’ arm (left of the IHF-binding site) contains
one B-box followed by a single A-box, whereas the ‘right’ arm
contains one B-box sandwiched between two A-boxes on the
left and one A-box on the right (Figure 2). Less complicated
centromeres include the plasmid TP228 parH centromere, which
consists of four direct repeats and the type II pSK41 centromere,
parC, which also contains four tandem DNA repeats [35,90]
(Figure 2).
Despite the apparent diversity among centromeres and
centromere-binding proteins, in all cases it appears that the
protein–centromere interaction leads to the formation of higherorder partition complexes. The higher-order structure of the
partition complex is postulated to be important in the capture
and activation of the motor protein for DNA segregation [90].
The strongest evidence for higher-order protein–DNA structure,
including DNA wrapping, in formation of the partition complexes
comes from studies on the interactions between the centromerebinding proteins SopB and R1 ParR with their centromere sites.
The dramatic effect on DNA topology caused by formation
of the SopB–sopC partition complex is consistent with a structure
in which the centromere DNA is wrapped in a right-handed
manner around a multimeric SopB protein core [91]. The R1
parC centromere itself appears to be intrinsically bent, and ParR
binding leads to further distortion of the site [92]. Similarly, the
centromere of the Enterococcus faecium gentamicin-resistance
plasmid pGENT is also intrinsically curved [93]. In this regard,
it is notable that analogous structural features are associated with
eukaryotic centromeres. Specifically, studies indicate that there
is inherent curvature in Saccharomyces cerevisiae centromere
DNA sites and binding of yeast centromere-binding proteins with
these DNA elements promotes DNA wrapping into higher-order
complexes [92–94]. These combined findings indicate that the
specific architecture of the higher-order partition complex plays
a critical role in its function. In the case of partition systems, the
function would probably be assembly of the motor protein to form
a fully active segrosome. Indeed, as will be described, the recent
structure of the pSK41 ParR–centromere complex shows a highly
wrapped protein–DNA superstructure, with overall dimensions
ideal for ParM filament binding [90].
5
Recruitment of the motor protein is thought to take place after
the two plasmids have paired. The pairing event is apparently
mediated by interactions between centromere-binding proteins
and/or centromere sites located on the partition complexes of the
replicated plasmids. Pairing was initially suggested by the finding
that plasmid centromeres act as incompatibility determinants.
This means that plasmids that replicate or segregate using similar
mechanisms cannot coexist in the same cell [95]. In contrast,
plasmids with distinct partition systems are compatible, can
coexist and localize to alternative subcellular positions. Although
pairing has been somewhat controversial, it has been demonstrated
in vitro for the E. coli plasmids R1 and pB171 and in vivo for the
P1 plasmid [8,96 –98].
Second step: DNA separation as mediated by motor proteins
After plasmid pairing, the motor protein is recruited to create the
active segrosome and mediates plasmid separation. The mechanism(s) by which motor proteins carry out this feat was initially
unclear, especially for the Walker-type ParA ATPase motor proteins. To be activated for partition, both type II ParM proteins
and type I Walker-type proteins must bind ATP. As noted, for the
type II ParM proteins, ATP binding was found to stimulate filamentation, thus providing an explanation for the ATP requirement
and also providing a model for plasmid separation. According
to this model, growth of ParM filaments anchored between two
ParR/parC-containing plasmids pushes or pulls the plasmids
apart. Interestingly, a recent study reported that GTP binding to
ParM stimulates the formation of filamentous structures identical
with those formed in the presence of ATP [99]. Whether ATP
or GTP, or a mixture of these nucleotides, functions in type II
partition in vivo remains to be resolved.
The Walker-type ATPase proteins also require ATP binding
for partition [26 –28]. How ATP binding stimulates segregation
by the type I motor proteins, however, has been less clear than
for the type II motor proteins. Initial models, based primarily
on knowledge regarding other Walker-type proteins, suggested
that type I motor proteins are monomers in the absence of ATP
and form nucleotide sandwich dimers upon ATP binding [26].
Dimerization is mediated via cross-contacts from one subunit to
the ATP molecule bound in the second subunit. However, it is
difficult to speculate how such a simple monomer to dimer switch
could cause the large-scale propulsion of paired plasmid DNA
from the cell centre to opposite cell poles. More recent structural
and biochemical data, which have revealed that these Walkertype proteins can form polymers upon ATP binding, suggest
that they actually function in a manner similar to the ParM
proteins in the type II par systems [3,6]. The ability of Walkertype proteins to form polymers is not unprecedented. Indeed, the
Walker-type protein MinD forms polymers upon binding ATP.
The MinD protein is part of the MinCDE system that is involved
in cell division control, and ATP binding to MinD causes the
release of its C-terminal region, which forms an amphipathic
helix when localized to the cell membrane. Once attached to
the membrane, multiple MinD proteins coalesce and form long
filaments [100,101]. Along with MinC, the role of MinD is to
prevent formation of the cell division septum at random locations
in E. coli, particularly near the cell poles. The inhibition of septum formation by MinCD is caused by a rapid oscillation of
this complex between the cell poles. This remarkable oscillation
correlates with the polymerization of the MinD protein [101].
The theory that Par Walker-type proteins utilize filaments
formation for segregation has been somewhat controversial.
However, data showing that several type Ia and Ib Walker-type
proteins can form polymers provide strong support for the idea that
c The Authors Journal compilation c 2008 Biochemical Society
6
M. A. Schumacher
this capability is important functionally. The pB171 ParA, which
is a member of the type Ib ParA family, was shown to both oscillate
over the nucleoid and form polymers [52,102]. This led to a model
for pB171 partition in which polymerization by pB171 ParA
pushes the plasmids apart by the insertion of ParA–ATP molecules
between the ParA–ParB/parC interface, similar to the insertional
polymerization model proposed for type II segregation of the
R1 plasmid [102]. However, an important feature of this model,
called the ‘sweeping par model’ is that the force exerted by pB171
ParA is assumed to be proportional to the number of filaments
involved in forming the polymer at that point. This model differs
from the type II insertional polymerization model in some ways.
Perhaps the major difference is that, unlike ParM, which forms
non-oscillating filaments that separate plasmids paired by ParR
and pushes them to cell poles, oscillating pB171 ParA positions
plasmids near quarter cell locations. As a result, whereas the
type II R1 reaction is binary because the mitotic machinery can
only accommodate one pair of plasmids at a time, each attached
at the ends of the growing ParM filament, the pB171 ParA can
position many plasmids at a time because of the oscillating nature
of its polymer, and so all plasmids present at a given moment
can participate in the segregation process. Notably, this model
is consistent with the observed higher efficiency of type I loci
compared with type II. Another type Ib motor protein that has been
shown to form polymers is the TP228 ParF protein [34]. Similarly
to pB171 ParA, the polymerization of ParF is dependent on ATP
and is stimulated by the presence of its centromere-binding protein
[34].
The E. coli type Ia ParA protein SopA has also been shown to
form filament-like structures [18,103,104]. Several detailed mechanisms have been posited for SopA-mediated segregation based
on its ability to form polymers [103,104]. One model proposes
that SopA forms asters that project from the partition complex
[103]. It is hypothesized that the asters not only function to push
the plasmids apart, but also to position the plasmids at midcell
before replication and maintain the position of daughter plasmids
after separation. Thus, according to this model, the pushing
and/or pulling forces that are imparted by the projecting asters
within the cell would allow for the correct positioning of the
plasmids in the cell. A second model, based on studies in which
there was no evidence for aster formation, proposes that nucleoid
DNA inhibits SopA polymerization. In this model, increased concentrations of SopB bound to sopC alleviate the DNA inhibition
of SopA polymerization, allowing polymers to attach to SopB–
sopC partition complexes [104]. Interestingly, in the studies used
to formulate the aster hypothesis, there was no evidence for DNA
inhibition of SopA polymerization. A third study, which shows
evidence for SopA oscillation between cell poles, suggests that
SopA filaments function as a railway track for the movement of
the plasmids [18]. Despite the differences in detail, all of these
models propose that F plasmid segregation is powered by SopA
polymers, which are formed in a manner stimulated by ATP and
SopB/sopC.
STRUCTURAL STUDIES ON PARTITION PROTEINS
Cellular and biochemical studies have provided a general outline
of the plasmid partition process. However, multiple questions
regarding the structure–function relationship of Par molecules
remain and must be addressed in order to provide a complete and
detailed understanding of the mechanisms employed by the Par
proteins to segregate DNA. Outstanding questions regarding the
first step include: what types of DNA-binding motifs do centromere-binding proteins utilize; how do these motifs recognize
c The Authors Journal compilation c 2008 Biochemical Society
centromere repeats; how do multiple centromere-binding proteins
bind co-operatively to multiple repeats to form a higher-order
partition complexes; and what kind of structures are adopted by
these higher-order complexes? In terms of the second step of
plasmid partition, important questions include: what structures
are adopted by the putative Walker-type proteins (do they really
contain canonical Walker-type folds?); how does ATP binding
lead to a ‘switch’ in conformational states that ultimately results
in plasmid separation; how does interaction with the partition
complex stabilize and possibly stimulate this process; and what
role does ATP hydrolysis play in plasmid separation? As discussed
below, recent structural information has started to shed light on
some of these questions.
Centromere-binding proteins: sequence diversity belies
structural homology
Within the last year, new structures have been obtained for several
centromere-binding proteins. As a result, structures are now
available for each type of centromere-binding protein: type Ia,
type Ib and type II. Remarkably, these combined structures indicate that, despite the significant lack of sequence homology
among these proteins, they share common DNA-binding folds.
In fact, it appears that there are two primary structural classes of
centromere-binding proteins: those that contain HTH folds, which
are of the type Ia family, and those that contain RHH (ribbon–
helix–helix) DNA-binding motifs, which unexpectedly include
both the type Ib and type II centromere-binding proteins.
Structures of type Ib centromere-binding proteins
The type Ib centromere-binding proteins are typically small,
consisting of 46 –113 residues. The sequences of these proteins
provided no hint as to how centromere binding is mediated.
However, recent structures of these proteins and their complexes
with DNA have revealed the DNA-binding motif they utilize and
how they bind DNA (Figure 3).
Structure of TP228 ParG protein
One of the best-characterized type Ib par systems is that of
the E. coli multidrug-resistance plasmid, TP228. The TP228
centromere-binding protein, called ParG, is a 76 residue, 8.6 kDa
dimer that is essential for TP228 plasmid retention. The structure
of apo ParG was solved by NMR and revealed that it contains a
bipartite structure with an N-terminal disordered region (residues
1–32) and a C-terminal region (residues 33–76) that has a RHH
DNA-binding motif [105] (Figure 3B). The RHH topology is
β1, residues 34 – 41; α1, residues 43–55; α2, residues 60–75.
The RHH is a very common DNA-binding motif present in the
Arc/MetJ superfamily of DNA-binding proteins [106]. In fact, it
is predicted that there are over 2000 RHH-containing proteins
in various bacterial sources, 55 in bacteriophages and 300 such
proteins in archaea [106]. Thus far, the RHH fold appears to
be absent from eukaryotes. However, RHH folds are difficult
to predict without experimental data as sequence conservation
is strikingly low among these proteins and there is a general
lack of strictly conserved residues within the fold. As was noted,
this was the case for the centromere-binding proteins. Further
impeding the identification of RHH proteins is the fact they
regulate multiple diverse cell processes such as transcription, the
lytic cycle of bacteriophages and cell division [106]. However, all
RHH proteins whose structures have been solved to date form
dimers-of-dimers upon DNA binding, whereby each β-strand
inserts into consecutive major grooves. The defining characteristic
of the RHH motif is the utilization of β-strand residues for making
Structure and function of plasmid partition proteins
Figure 3
7
Structures of type Ib centromere-binding proteins
(A) Schematic diagram showing the domain structure of the type Ib centromere-binding proteins. The N-terminal domain (grey) is a flexible region that functions in ATPase stimulation, ATPase
recruitment and pairing. In addition, a role in DNA binding has been suggested. The C-terminal domain (magenta) contains the DNA-binding RHH motif. In some cases, there is a short region
following the RHH (shown in grey). The RHH motifs of the structures in (B) and (C) are shown in the same orientations for comparison purposes. (B) Structure of the ParG protein from the E. coli
TP228 plasmid shown colour-coded as in (A) [105]. The protein is a dimer. The N-terminal flexible region is grey, and the RHH, which mediates all the dimer contacts, is magenta. (C) Structure of
Streptococcus pyogenes plasmid pSM19035 ω protein colour-coded as in (A) [110,111]. On the left is the apo structure, and on the right is the ω–DNA structure. The DNA is shown as sticks and
is coloured blue. N- and C-termini regions are labelled for one subunit in each apo structure. (B) and (C) were modelled using PyMOL (DeLano Scientific).
base-specific DNA contacts. Therefore it is likely that the RHH
fold of ParG binds its DNA centromere-like site by forming a
dimer-of-dimers and inserting its double-stranded β-sheet into
the DNA major groove.
In addition to binding their centromere sites to form higherorder partition complexes that recruit and stabilize motor proteins,
centromere-binding proteins also stimulate the ATPase activity of
the motor protein. This presents a conundrum as ATP hydrolysis
leads to depolymerization of ATPase motor proteins. To date, it
is not understood at a molecular level how these two opposing
functions are mediated and co-ordinated to achieve accurate
plasmid separation and this remains one of the important questions
in partition biology. In studies aimed to address this issue, Hayes
and co-workers have shown that there are two separate regions
within the flexible N-terminal region of ParG that mediate these
activities: one acts to promote polymerization of the Walkerbox protein ParF and the other to enhance its ATPase activity
30-fold [107]. Specifically, they identified residue Arg19 as part
of a putative arginine finger motif within the N-terminal region of
ParG that is responsible for stimulation of the ParF ATPase
activity, but which is dispensable for the ParG-mediated affects
on ParF polymerization. Arginine fingers are common motifs
utilized by P-loop NTPases, either in cis, where it is from the same
protein, or trans, where, similar to the case of ParG and ParF, it is
located on another protein [108]. These motifs, which are typically
found on extended loop regions on a protein, stimulate nucleotide
hydrolysis by inserting into the active site and stabilizing the
transition state. Because ATPase-interacting regions located on
type Ia and Ib centromere-binding proteins are usually located
in extended regions and contain arginine residues, an attractive
hypothesis, proposed by Hayes and co-workers, is that all these
proteins contain arginine finger motifs that are used in trans
to enhance the ATPase activity of their requisite ATPase motor
proteins [107].
Remarkably, two additional functions have been posited for
the N-terminal domains of type Ib proteins: DNA binding and
pairing. Specifically, studies on ParG indicate that residues in
its N-terminal region can affect how it organizes the formation
of higher-order ParG–DNA structures [109]. Moreover, the continued deletion of this N-terminal tail greatly perturbs its DNAbinding kinetics [109]. Additionally, studies on the ω protein
from the Streptococcus pyogenes plasmid pSM1903, another
type Ib centromere-binding protein with a RHH fold, revealed
that removal of its entire N-terminal tail causes a 2-fold reduction
in its DNA-binding affinity [110]. Indications that the N-terminal
region of type Ib centromere-binding proteins impact pairing
come from studies on the pB171 ParB protein, which showed that
removal of its N-terminal tail abrogated ParB–ParB interactions
between plasmids [53]. Combined, these data demonstrate that the
flexible N-terminal regions of type Ib centromere-binding proteins
play several pivotal roles in partition (Figure 3A). However, the
precise locations of these functional regions within the N-terminal
domains of these proteins have yet to be mapped, and their specific
molecular mechanisms have yet to be resolved.
Structure of inc18 ω protein in the presence and absence of DNA
In addition to ParG, the structure of the ω protein from the
Streptococcus pyogenes plasmid pSM19035 found in the Grampositive broad host range inc18 family of plasmids has also been
c The Authors Journal compilation c 2008 Biochemical Society
8
M. A. Schumacher
determined [110,111]. Like most type Ib centromere-binding
proteins, ω functions in transcriptional autoregulation. On the
other hand, ω is distinct from other type Ib centromere-binding
proteins in that it also acts as a global regulator of transcription
[111]. To carry out these functions, ω binds co-operatively to DNA
sites containing seven to ten consecutive non-palindromic
DNA heptad repeats (5 -A /T ATCACA /T -3 ). Interestingly, these
sites can be arranged as direct or inverted repeats. ω binds with
low affinity (K d of >500 nM) to one heptad, but it binds strongly
to DNA sites containing two or more consecutive heptad repeats
[110,111]. The heptad arrangement also affects the affinity of ω
for the DNA site, with direct or converging repeats providing the
strongest binding interaction.
Structural and biochemical studies on the Streptococcus
pyogenes ω protein revealed that, similarly to ParG, it has a
bipartite structure with an N-terminal flexible region and a Cterminal domain containing a RHH motif (Figure 3C). The
flexibility of the N-terminal region is reflected in the structure
of apo ω, whereby the N-terminal 20 amino acids were lost to
proteolysis during crystallization [111]. The C-terminal RHH has
the topology of β, residues 28–32; α1, residues 34 – 47; and α2,
residues 51–66. To gain insight into the unusual DNA-binding
preferences exhibited by ω, structures were solved in the presence
of direct and converging 17 bp DNA repeats using a deletion
mutant of the ω protein, 19ω, in which the first 19 residues were
removed. As noted, the N-terminal region has been implicated to
be important in DNA binding, and this deletion results in a 2fold reduction in DNA binding compared with wild-type protein
[110,111]. However, the role(s) played by the N-terminal tails
of type Ib centromere-binding proteins in DNA binding has yet
to be determined as, so far, the 19ω–DNA complexes are the
only structures available for a type Ib centromere-binding protein
bound to cognate DNA.
The ω–DNA structures show that ω binds its DNA as a
dimer-of-dimers, wherein each dimer binds a consecutive DNA
major groove. Unexpectedly, the crystallographic asymmetric
unit contains a 19ω dimer-of-dimers bound to a 17 bp DNA
site, which in turn is arranged pseudocontinuously with a second
uncomplexed 17 bp DNA site. As a result, there is an unbound
17 bp DNA site on either side of each ω–DNA complex in the
crystal lattice (Figure 3C). The DNA bound by ω is essentially
straight and B-DNA in character. The presence of the uncomplexed operator in the structure permitted the direct comparison of
protein-bound and protein-free DNA conformations. This analysis
showed that the unbound DNA adopts the same conformation
as that bound by 19ω. Therefore any deviations from ideal
B-DNA observed in the structure of the 19ω-bound DNA
are the likely result of nucleotide sequence-induced alterations.
Comparison of the structures of ω bound to direct and converging
repeats showed that the protein can bind these different sites
because the contacts of ω to the two DNA sites are related by a
pseudo-2-fold rotation axis, and, as a result, the specific protein–
DNA interactions are comparable, despite the heptad orientation
[110]. Presumably, the formation of the higher-order ω–DNA
partition complex involves the co-operative interaction of multiple
ω dimers-of-dimers binding to consecutive DNA heptad repeats.
However, confirmation of this awaits structural studies on ω and
other type Ib centomere-binding proteins bound to full-length
centromere sites.
Structures of type II ParR proteins
Structures for type II ParR centromere-binding proteins have
only become available within the last year. Similarly to the
c The Authors Journal compilation c 2008 Biochemical Society
type Ib centromere-binding proteins, the amino acid sequences
of the type II centromere-binding proteins revealed no clues about
the structures these proteins adopt. However, these proteins are
similar in size to the type Ib centromere-binding proteins. Thus it
was intriguing when the structures of the pB171 and the pSK41
type II ParR proteins were solved and shown to belong to the
same family of DNA-binding proteins as the type Ib centromerebinding proteins, namely the RHH family (Figure 4).
Structure of pB171 ParR protein
The E. coli pB171 ParR protein functions as the centromerebinding protein for the type II locus on the pB171 plasmid, which
contain both type I and II par systems [48–53]. The structure
of the full-length pB171 ParR protein was recently solved in
the absence of DNA [112]. The C-terminal 35 residues were not
resolved in this structure, neither were residues 1–5. The structure,
consisting of residues 6 –95, revealed a RHH motif and a Cterminal domain composed of a three helix ‘cap’ (Figure 4B).
The topology is β1, residues 7–14; α1, residues 20 –30; α2,
residues 36 –52; α3, residues 56 –65; α4, residues 71–81; and
α5, residues 88–94. The three-helix cap at the C-terminal region
not only reinforces the tight dimerization of the RHH, but also
stabilizes interactions between dimers-of-dimers. Several RHH
proteins contain domains outside the RHH motif that similarly
act to bolster the dimer and dimer-of-dimer; however, none of
these structures is similar to the pB171 three-helix cap. A notable
finding in the crystal structure was the creation in the lattice of a
continuous helical structure created by the assembly of 12 ParR
dimers per turn. In this arrangement, the N-terminal RHH motifs
face one direction, whereas the C-termini point in the opposite
direction. This was interesting because EM (electron microscopy)
images of the R1 ParR protein bound to its centromere site parC,
revealed that the ParR–parC partition complex forms ring-like
structures on pre-linearized DNA that are between 15 and 20 nm
in diameter [112]. Because R1 and pB171 ParR proteins probably
form similar higher-order partition complexes, it was postulated
that the EM projections of R1 ParR–parC were possibly hiding
the helical nature of the arcs observed in the pB171 ParR apo
structure. However, it was not possible to distinguish the exact
nature of the superstructure formed by the R1 or pB171 ParR–
DNA partition complexes by these data. The resolution to the
issue was obtained by the crystal structure of the type II pSK41
ParR–centromere complex, which revealed the first structure of a
higher-order partition complex [90].
pSK41–ParR–centromere complex reveals partition complex structure
pSK41 is a prototypical multiresistance plasmid from Staphylococcus aureus and is stably maintained by the presence of a type II
partition system encoding ParR and ParM proteins [113]. The
pSK41 Par proteins show little sequence homology with R1
ParR and ParM. Nonetheless, the pSK41 ParM protein is readily
identifiable as an actin-like protein, thus establishing the system
as a type II par system. The pSK41 centromere site was identified by a combination of footprinting and FP (fluorescence
polarization)-based DNA-binding studies [90]. These analyses
revealed that the centromere consists of four 20 bp elements,
in which the 20 bp repeat constitutes the minimal element for
binding of a ParR molecule (Figure 2). The simplicity of this
centromere site made it an ideal candidate for attempts to obtain
the structure of a higher-order partition complex. As data-quality
crystals of the pSK41-full-length ParR–centromere complex
could not be obtained, the protein was subjected to limited
Structure and function of plasmid partition proteins
Figure 4
9
Structures of type II centromere-binding proteins
(A) Schematic diagram showing the domain structure of the type II centromere-binding proteins. The N-terminal domain (magenta) forms a RHH domain responsible for centromere binding. The
C-terminal region is a partially flexible domain that functions in ParM binding and also contributes to the stabilization of the RHH dimer and dimer-of-dimers. The RHH motifs of the structures in
(B) and (C) are shown in the same orientations for comparison purposes. (B) Structure of the E. coli pB171 ParR colour-coded as in (A) [112]. The N-terminal RHH domain is coloured magenta,
and the C-terminal region, which consists of a three-helix cap and additional disordered residues, is grey. N- and C-terminal regions are labelled. (C) Structure of the Staphylococcus aureus pSK41
ParR protein coloured as in (A) [90]. On the left is the protein in the absence of DNA, and on the right is the ParR–centromere complex. N- and C-termini regions of one subunit are labelled in the
left-hand panel. The DNA is shown as sticks and coloured blue. (B) and (C) were modelled using PyMOL (DeLano Scientific). An interactive three-dimensional version of the structure shown in
(C) can be seen at http://www.BiochemJ.org/412/0001/bj4120001add.htm.
proteolysis, and the minimal DNA-binding domain was found
to reside in the first 53 residues of the 109 residue protein. This
domain, termed ParRN, contains all of the determinants required
for centromere binding as ascertained by FP-binding studies
[90]. The structure of the ParRN-centromere was determined by
utilizing a 20 bp repeat, which stacked pseudocontinuously in the
crystal to form the full-length centromere (Figure 4C).
The structure revealed that, like pB171 ParR, pSK41 ParR
contains a RHH motif in its N-terminal DNA-binding domain
with topology of β1, residues 5–12; α1, residues 16 –25; and α2,
residues 33– 47. Like all RHH proteins, the β-strands combine
in an antiparallel fashion and helices interdigitate to form the
extensive RHH dimer. The DNA elements bound by each ParR
dimer-of-dimer stack pseudocontinuously to generate the fulllength centromere. Thus the most notable feature of the structure
is the creation of the higher-order partition complex that mediates
full segrosome assembly (Figure 4C). Consistent with the cooperative nature of ParR binding, the protein dimer-of-dimers that
bind the DNA form intimate protein interactions that lead to the
generation of a continuous protein superstructure that has distinct
positive and negative electrostatic surfaces. As would be expected,
the positive face wraps the centrosomal DNA about itself to
create the unique superstructure that is best described as a large
superhelix. Characteristic of the superstructure is its superhelical
parameters: it has a pitch of ∼ 24 nm with six ParR dimers-ofdimers in one turn of the superhelix and a measured diameter of
∼ 18 nm. Within this structure, the C-terminal regions of the ParR
protein would face inwards, towards the pore, which suggests that
it plays a role in ParM recruitment. This was confirmed by binding
experiments, which showed that ParM bound only to complexes
containing full-length ParR and not ParRN [90].
DNase I-protection studies carried out on the pSK41 ParR
binding to the centromere revealed striking nucleosome-like
periodicity in the protection pattern consistent with the crystal
structure. As would be expected from binding a continuous protein
superstructure, the footprint showed almost complete protection.
Indeed, only eight symmetrically disposed adenine nucleotides
on each strand are susceptible to DNase I attack. The basis for
the susceptibility of these nucleotides is revealed by the crystal
structure, which shows that these adenines reside in the only
minor groove positions that are exposed outside the partition
complex superhelix and between ParR molecules that bind each
repeat (Figure 4C). Thus the multiple features of the DNase I
footprint are readily explained by the crystal structure. In this
same study, cryo-EM was also carried out whereby the partition
complex formed by binding of ParR to the centromere site with
no surrounding DNA regions was examined. The image revealed
a circular superhelical structure with dimensions essentially
identical with those observed in the crystal structure.
The structural studies on the pSK41 ParR–centromere complex
resolved the issue of whether R1 and pB171 ParR–centomere
complexes formed rings or helices as, clearly, the structure is a
superhelix [90,112]. Indeed, the measured diameter of the pSK41
superhelical partition complex crystal structure was ∼ 18 nm,
which closely matches the 15 and 20 nm diameter obtained for
the superhelical rings of the R1 ParR–centromere complex by
EM [112]. These data, which indicate a conserved structure
of the type II segrosome assembly, seem to support the notion
that the higher-order structure of the partition complex is likely
to be crucial to its function of recruitment of ParM filaments and
their stabilization. Indeed, as discussed below, recent EM data on
ParM filaments combined with the structure of the pSK41 partition
c The Authors Journal compilation c 2008 Biochemical Society
10
Figure 5
M. A. Schumacher
Structures of type Ia centromere-binding proteins
(A) Schematic diagram showing the domain structure of the type Ia centromere-binding proteins. The N-terminal domain (grey) is a flexible region involved in ATPase activation and recruitment.
Motif B (cyan) functions as a higher-order oligomerization domain. The HTH unit (magenta) is a three-helix unit containing an HTH motif. This unit is highly conserved in the type Ia structures
solved to date. The linker domain (blue) is a helical domain that links the HTH unit to the dimer domain. The dimer domain (light grey) mediates dimerization and is not conserved among the type Ia
centromere-binding proteins. The HTH units of each structure are shown in the same orientation with its three helices labelled H1–H3 (magenta). (B) Structure of the T. thermophilus Spo0J protein
colour-coded as in (A) [114]. Although included in crystallization, the N-terminal domain was not visible. Motif B (cyan) mediates dimerization between Spo0J subunits at high concentrations. The
HTH unit is magenta and the linker domain is blue. N- and C-termini regions are labelled. (C) Structure of the P1 ParB-(142–333)–parS small complex [123]. The HTH unit is shown as the same
orientation as that of Spo0J and is coloured magenta. The linker domain is blue, the dimer domain is grey, and the N- and C-termini regions of one subunit are labelled. The DNA is shown as a
surface and is coloured yellow. (D) Structure of the RP4 ParB homologue, KorB. The C-terminal dimer-domain (residues 297–358) and DNA-binding domains (residues 101–294) were solved as
separate domains (not all residues were visible) [118,119]. The KorB dimer was modelled by linking the two domains by a flexible region (indicated by broken lines). Part of motif B present in the
structure is coloured cyan. The HTH unit is magenta and the linker domain is blue. The dimer domain, solved separately, is grey and is shown as linked by flexible residues of the linker domain.
The DNA is shown as a yellow surface. (B)–(D) were modelled using PyMOL (DeLano Scientific).
complex provide insight into the role played by the partition complex in ParM filament recruitment and final segrosome assembly.
Structure of type Ia ParB centromere-binding proteins
It had been predicted that the type Ia ParB proteins contain
HTH motifs. Studies on the type Ia centromere-binding protein,
SopB, also clearly showed that these proteins form higher-order
segrosomal structures. The DNA-binding properties of these
proteins that enable such higher-order structure formation are
starting to be resolved with recent structure determinations of
type Ia centromere-binding proteins.
All plasmid centromere-binding proteins share in common
the fact that they are multidomain proteins that contain flexible
extended regions, usually at their N- and/or C-termini. Yet, the
type Ia centromere-binding proteins clearly differ from those in
the type Ib and II families. The type Ia proteins are larger than the
type Ib and II proteins and typically contain between 182 and
336 residues. Sequence comparisons indicate that the type Ia
centromere-binding proteins have two conserved regions, which
correspond to a domain called motif B and a HTH unit. Motif B is
near the N-terminal region and the HTH unit is centrally located
(Figure 5A). The linker domains of type Ia proteins show limited
sequence and, as it turns out, structural homology (Figure 5A).
Like the type Ib and II centromere-binding proteins, all the type Ia
ParB proteins that have been characterized form dimers whereby
c The Authors Journal compilation c 2008 Biochemical Society
the dimer domain is located in the C-terminal ∼ 70 residues. The
C-terminal dimer domain is one of the least conserved domains
among type Ia members. As will be described, this is reflected in
the finding that this domain can adopt very different structures.
In fact, in one case, this domain functions in DNA binding as well
as dimerization.
Structure of Thermus thermophilus chromosomal protein Spo0J
The first described structure of a type Ia centromere-binding
protein was that of the N-terminal domain of the T. thermophilus
Spo0J, which is a chromosomally encoded type Ia protein [114].
Attempts to crystallize the Spo0J protein from B. subtilis and
eight other bacterial genomes all failed. Only a truncated form
consisting of residues 1–222 of the 269 residue T. thermophilus
Spo0J was successfully crystallized. This fragment contains the
regions of highest homology among ParB proteins, motif B and
the HTH unit. Spo0J homologues typically bind DNA sites
containing multiple 16 bp repeats located in the origin proximal
region of the requisite bacterial chromosome [69,115]. Gelretardation assays revealed that the Spo0J-(1–222) fragment is
greatly weakened for DNA binding, despite the fact that it contains
the HTH unit [114]. This is consistent with data on other type Ia
ParB proteins, which indicate that the C-terminal dimer domain
is essential for high-affinity DNA binding. In most cases, this
domain is not directly involved in DNA binding by type Ia
centromere-binding proteins and thus it appears that dimerization
Structure and function of plasmid partition proteins
11
is important in mediating the proper docking of the HTH units on
to successive grooves of the DNA.
The structure of Spo0J-(1–222) is predominantly helical
(Figure 5B). Residues 1–22 and 210 –222 were not visible,
consistent with data indicating that the N-terminal domains of
type Ia proteins are flexible. The structure can be broken into three
main regions based on homology with other type Ia proteins. The
region containing residues 23–118 corresponds to the motif B region and consists of four α-helices and a two-stranded β-sheet,
with topology α1–α2–α3–β1–β2–α4 (Figure 5B). The HTH unit
includes residues 119–160. The linker domain, which in the fulllength protein connects to the dimer domain, includes residues
161–209. Although Spo0J-(1–222) does not contain the dimer
domain, a clear dimer is observed in the crystal structure, which
is formed by contacts primarily involving residues from motif B.
There are two main interaction interfaces within the dimer: one
between residues from α1 of one subunit and residues from α3,
β1 and β2 of the other subunit and the second between α2 of one
subunit and helices 4, 5, 8 and 10 of the other subunit. Despite
the large surface area buried in this dimer interface, biochemical
data show that Spo0J-(1–222) only dimerizes when the protein
is at high concentrations. This is consistent with studies on other
type Ia ParB proteins, such as P1 ParB, which suggest that motif
B is involved in secondary oligomerization when bound to DNA
[86].
Structures of RP4 KorB
The structures described above underscore a conserved feature of
centromere-binding proteins, their flexible multidomain nature.
Following this general theme, the type Ia RP4 KorB protein had
to be broken into domains in order for structures to be obtained.
KorB is carried on the RP4 plasmid and is a member of the
E. coli incompatibility group P (IncP-1α) [116,117]. This plasmid
displays a broad host range and thus can be transferred to a
variety of Gram-negative bacteria [117]. Structures have been
solved for the N-terminal HTH-containing domain of KorB as
well as the C-terminal dimer domain [118,119]. The KorB Cterminal dimer domain (residues 297–358) consists of a fivestranded antiparallel all-β structure with striking homology with
the SH3 (Src homology 3) domain found in eukaryotic signalling
proteins (Figure 5D) [120,121]. However, unlike the SH3 domain,
the KorB C-domain is missing a long loop between β1 and β2
that is essential for the SH3 recognition of proline-rich motifs. In
contrast, the KorB C-domain contains a significantly elongated
β5 compared with SH3 domain structures and is essential for
KorB dimerization.
The KorB C-terminal domain is not directly involved in
centromere binding. Instead, this function is fulfilled by the
N-terminal region of KorB [119]. However, similarly to Spo0J,
the C-terminal domain of KorB is critical for high-affinity DNA
binding by KorB again probably by correctly positioning and
anchoring the DNA-binding domains on cognate DNA. Insight
into how KorB binds its individual centromere-like palindromic
repeats was 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 [120]. The structure of the
KorB DNA-binding domain consists of eight helices, including the C-terminal portion of motif B (coloured cyan in
Figure 5D), the DNA-binding HTH unit (magenta) and part of
the linker region (blue) connecting the HTH unit to the dimer
domain (Figure 5D). Each half site of the palindromic operator
site is bound by one KorB subunit in its major groove (Figure 5D).
Although the KorB HTH has a canonical HTH structure, Thr211
and Arg240 , which are outside the recognition helix and located
Figure 6 Structure of the P1 ParB-(142–333)–parS double B-box
interaction: P1 ParB is a bridging factor
(A) Sequence of the P1 parS centromere site. The site is divided into right and left ParB-binding
sites, with an IHF site located in the centre. The A- and B-boxes are labelled. The leftside B1
and A1 boxes are coloured blue and green, whereas the rightside A2, A3 and B2 boxes are
coloured yellow, green and blue respectively. Note that the third A-box, A4, which is located to
the left of B2, is not necessary for partition and is not shown. The rightside parS site, called parS
small , encompasses A2–A3–B2 and is the minimal site required for partition. (B) Structure of
P1 ParB-(142–333) bound to an A3–B2-containing DNA site in which the protein dimer–DNA
duplex stoichiometry was 1:2 [127]. One ParB-(142–333) subunit is coloured magenta and the
other is yellow. The DNA is shown as surfaces with box elements coloured as in (A). Modelled
using PyMOL (DeLano Scientific). An interactive three-dimensional version of the structure
shown in (B) can be seen at http://www.BiochemJ.org/412/0001/bj4120001add.htm.
on the second and fourth helices in the linker domain, are utilized
for base-specific contacts.
Structures of P1 ParB–centromere complexes
As noted, the E. coli P1 and F par loci were the first partition
systems to be discovered and have since served as paradigms
for studies on plasmid partition [4,10,12,16 –17,24 –29,85–88].
The P1 par system is of particular interest owing to its unusual
centromere-like site. Studies carried out largely in the Funnell and
Austin laboratories have provided a detailed description of the P1
centromere site and how it is recognized by the P1 ParB protein
[24,25,85–88]. These studies showed that the P1 centromere site,
parS, is an unusual ∼ 74 bp DNA centromere element that can
be divided into three main regions: a centralized ∼ 29 bp binding
site for IHF, a so-called ‘rightside’ region with two heptameric
A-boxes, A2 and A3 (the third A-box, A4, to the right of B2
is not essential for partition) and one hexameric B-box, and a
‘leftside’ element with one A-box and one B-box (Figures 2 and
6A). Although not required for partition, the E. coli IHF αβ
heterodimer increases partition efficiency by bending the central
region of parS to juxtapose the A-box- and B-box-containing
c The Authors Journal compilation c 2008 Biochemical Society
12
M. A. Schumacher
‘arms’, which appears to facilitate ParB binding across the bend
(Figure 5C) [122].
Interestingly, data revealed that both the A-boxes and Bboxes are recognized and bound by P1 ParB [86]. This finding
distinguishes ParB from other centromere-binding proteins,
which only bind one cognate DNA element. Although fulllength parS provides maximal partition efficiency, the rightside
parS site composed of A2–A3–B2 is sufficient for partition in
an IHF-independent manner. P1 ParB residues 142–333, which
contains the HTH unit, linker domain and dimer domain, contain
all the determinants required for centromere binding. Similarly
to other type Ia centromere-binding proteins, motif B functions
as a secondary oligomerization domain in P1 ParB when the
protein is present at high local concentrations. The extreme Nterminal region of P1 ParB and nearly all other type Ia centomerebinding proteins are used to bind to their requisite motor proteins
(Figure 5A). An exception is KorB, which binds its motor protein
using residues within its centromere-binding domain [86,116].
Insight into how ParB can recognize two DNA elements
and bind the unusual parS centromere site were provided by
recent structures of ParB-(142–333)–rightside parS complexes
(Figure 5C) [123]. These structures show that ParB-(142–333)
comprises two separate domains. The first corresponds to residues
147–270 and includes the HTH unit and linker region. This
domain consists of seven α-helices and is connected by a short
flexible linker (residues 271–274) to the C-terminal domain
(residues 275–333). As in other type Ia centromere-binding
proteins, the C-terminal domain mediates dimerization. Unlike
KorB, which contains a C-terminal SH3-like domain, the Cterminal domain of P1 ParB contains a novel fold consisting of
three antiparallel β strands and a C-terminal α helix [118,123].
These elements lock together in the dimer to form a continuous
antiparallel β-sheet, flanked by a coiled-coil (Figure 5C). Unique
structural features evident in the ParB–DNA structures explain
how ParB can interact with multiple arrays of box elements on
the looped parS site [123]. Importantly, each box element is
bound by a separate DNA-binding module: the A-boxes are bound
by the HTH motif, whereas the B-boxes are recognized by the
dimer domain. In addition, the structure shows that ParB acts as a
bridging factor between DNA boxes, explaining how it can bind
sites on looped DNA or box elements on adjacent plasmids.
Importantly, the two DNA-binding modules of ParB 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 5C). 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 nonspecific DNA spreading. Indeed, non-specific binding of P1
ParB to DNA enables it to bind several kilobases upstream and
downstream of the parS site [124 –126]. Studies on P1 ParB and
SopB, which both show spreading, suggest that DNA spreading
may be a shared feature of at least some of the centromerebinding proteins [104,115,124 –126]. Initial data on spreadingdefective ParB mutants led to the speculation that it was important
for partition. However, more recent studies in which plasmid
stability was determined after spreading was restricted by the
introduction of roadblocks on either side of parS revealed that
extensive spreading was, in fact, not required for plasmid partition
[124]. One result of spreading is the silencing of genes several
or many kilobases away from the ParB–DNA interaction site
[125]. The exact role(s) that spreading plays in DNA partition
and transcription regulation has yet to be clarified.
Unlike the HTH domain–A-box interaction, the contacts made
by the novel dimer domain to the B-box are highly sequence
c The Authors Journal compilation c 2008 Biochemical Society
specific. Initial structures of ParB-(142–333)–parS rightside
showed only one side of the dimer domain bound to a B-box.
However, this was due to the stoichiometry of box elements to
ParB dimer used in the initial structural studies. Indeed, modelling
suggested that each side of the dimer domain should be capable of
B-box binding. The more recent structure of ParB-(142–333)
bound to a 16 bp A-box/B-box-containing site confirmed this prediction and not only revealed a completely novel type of protein–
DNA interaction, but also indicated that one P1 ParB is able
to interact simultaneously with four separate DNA duplexes or
DNA sites on bent DNA or a mixture of these permutations
[127] (Figure 6). This multi-bridging capability has important
implications for the ability of P1 ParB to form wrapped nucleoprotein structures as well as mediate plasmid pairing. Indeed,
the unique DNA-bridging function of ParB reveals how it can
bind across the rightside and leftside 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. Moreover,
the multi-bridging capability, especially that provided by the
double B-box interaction, which juxtaposes DNA close in space,
suggests multiple strategies for forming higher-order partition
complex superstructures by P1 ParB.
Comparison of the Spo0J, KorB and ParB structures suggest
that there are larger differences among the type Ia proteins as
compared with the type Ib and II centromere-binding proteins.
Although a mechanism for partition complex formation similar
to that demonstrated by pSK41 ParR can be envisioned for all
centromere-binding RHH proteins, a clear structural model for the
partition complex by the type Ia proteins is not yet forthcoming.
Structures of motor proteins: mechanisms of plasmid separation
Once the higher-order partition complex is formed, the final step
in partition is formation of the final segrosome via recruitment
of the motor protein, which is typically either an actin-like
protein (type II) or, more commonly, a Walker-type protein
(type I). Filament growth then propels the plasmids to opposite
cell poles. Currently, structural information is available for one
type II plasmid motor protein, ParM from the E. coli R1 plasmid
[99,128–130]. Although a structure has yet to be reported for a
plasmid type I Walker-type motor protein, the structure of the
chromosomal Walker-type protein, T. thermophilus Soj, which is
related to the type Ib motor proteins, has been described [131].
Structures of R1 ParM and T. thermophilus Soj combined with
EM studies on several type II and I motor proteins have revealed
important insight into how these proteins mediate separation of
replicated plasmids.
Structures of the T. thermophilus Soj protein
The T. thermophilus Soj protein collaborates with the T. thermophilus Spo0J protein to play an important, yet incompletely
characterized, role in chromosome segregation. Soj is also
required together with Spo0J for stable maintenance of a plasmid
bearing the parS centromere site, suggesting that they mediate
plasmid segregation [69]. Localization studies of Soj have revealed dynamic oscillations of the protein similarly to oscillations
observed for the type Ib Walker-type protein pB171 ParA
[101,102,132]. Soj has also been shown to form nucleoprotein filaments, but in a DNA-dependent manner [131]. The structure
of T. thermophilus Soj was solved in its apo and ADP-bound
forms. In addition, the structure of the ATPase hydrolysisdeficient mutant, Soj(D44A), was solved bound to ATP [131].
Structure and function of plasmid partition proteins
Figure 7
Structure of T. thermophilus Soj
On the left (red) is the structure of apo Soj. On the right is the structure of Soj(D44A), which
forms a nucleotide sandwich dimer (one subunit is red and the other green) upon binding ATP.
The ATP molecules are shown as CPK (Corey–Pauling–Koltun) with carbon, nitrogen, oxygen
and phosphorous atoms coloured cyan, blue, red and magenta respectively [131]. Modelled
using PyMOL (DeLano Scientific).
The structure confirmed previous predictions, that the protein
contains a canonical Walker-type fold (Figure 7).
The Soj structure shows the highest homology with the cell
division regulator MinD [133]. Accordingly, Soj consists of a
core of twisted β-strands surrounded by α-helices. The α-helices
cluster on either side of the β-sheet formed by one antiparallel βstrand and seven parallel β-strands. The Soj apo and ADP-bound
forms are monomers, whereas ATP binding to Soj(D44A) leads
to formation of an ATP ‘nucleotide sandwich dimer’. Notably,
the formation of such a dimer had been predicted previously, but
not observed until the structure of the Soj(D44A)–ATP complex
[131]. Comparison of apo and ADP-bound Soj, reveals that ADP
binding is accompanied by minor structural changes in the Ploop region wherein residues GGVG (Gly-Gly-Val-Gly) become
less extended as they make multiple interactions with the ADP
phosphate moieties. Upon ATP binding, dimerization is induced
by numerous hydrogen bonds between residues on adjacent
subunits (Figure 7). However, the key interaction in mediating
the formation of dimer is provided by the Walker box signature
lysine residue, Lys15 , which contacts the α- and γ -phosphates of
the ATP bound in the other subunit.
The formation of Soj polymers requires not only ATP, but also
non-specific DNA as well [131,134]. A recent study aimed at
elucidating what role Soj DNA binding might play in segregation
identified several surface arginine residues, conserved among
chromosomal Soj proteins, that were essential for DNA binding,
but appeared to have no significant role in nucleotide binding
[134]. Remarkably, creation of a DNA-binding-deficient Soj
protein by mutation of these residues led to a partition-deficient
phenotype in a model plasmid partitioning system in E. coli. This
finding clearly demonstrated that DNA binding by Soj is essential
for its segregation function and also suggests a connection
between the DNA-binding and -polymerization functions of Soj in
segregation [134]. However, the arginine residues that are critical
for DNA binding by Soj proteins are not conserved among the
type Ib ParA proteins. Moreover, previous studies on the type Ib
pB171 ParA and ParF motor proteins and the type Ia SopA protein
indicated that DNA binding is not required for polymer formation
by these proteins. Rather, ATP binding seems sufficient to induce
the formation of polymers by the type Ib and Ia motor proteins
and the addition of the requisite centromere-binding protein–centromere complex stimulates the process [34,102].
Structures and EM studies on the R1 ParM protein
Before high-resolution structures were available, the type II ParM
protein had been predicted to belong to the actin superfamily.
Figure 8
13
Structures of actin-like ParA homologue, E. coli R1 ParM
(A) Crystal structure of R1 ParM in its apo state [129]. (B) Structure of the R1 ParM ADP-bound
state. Comparison with the apo structure reveals that ADP binding induces a 25◦ relative closure
of the two domains [129]. (C) Structure of eukaryotic actin bound to ADP [143]. In all structures,
helices are coloured cyan, β-strands are coloured magenta and loop regions are coloured
light pink. Also labelled are subdomains Ia, Ib, IIa and IIb. Modelled using PyMOL (DeLano
Scientific).
Other bacterial proteins that are actin superfamily members
include DnaK, FtsA and MreB [135,136]. Crystal structures of
ParM in its apo and ADP-bound form confirmed that it has
an actin-like fold and, accordingly, consists of a two-domain
architecture [129]. These two domains, called I and II, are
delineated further into Ia and Ib and IIa and IIb (Figure 8). The
larger subdomains, Ia and IIa, share a common fold consisting of
a five-stranded β-sheet surrounded by three α-helices. In contrast,
the two smaller subdomains are much more diverse structurally.
Comparison of the apo ParM and ParM–ADP structures revealed
that domains I and II act as rigid bodies and undergo a 25◦ rotation
upon binding ADP [129] (Figures 8A and 8B). Interestingly,
analysis of the ADP-binding pocket of ParM revealed no
specificity-determining interactions between ParM residues and
the adenine base. Indeed, a recent study showed that, in addition
to ATP, ParM can bind GTP. In this study, the structure of the R1
ParM was solved bound to GDP and the R1 ParM–p[NH]ppG
(guanosine 5 -[β,γ -imido]triphosphate) complex structure was
obtained by soaking p[NH]ppG into ParM–GDP crystals [99].
The ParM–ADP, ParM–GDP and ParM–p[NH]ppG structures are
essentially identical in their conformations, which suggests that
nucleotide binding stabilizes the closed conformation.
Although actin is a well-known filament-forming protein,
several actin family members have roles unrelated to filament
or polymer formation. For example, DnaK functions in protein
folding, and FtsA is involved in the regulation of cell division
[137–139]. Thus sequence homology between ParM and actin did
not necessarily dictate filament formation as a mechanism
for ParM action. To address the function of ParM, numerous
cellular, biochemical and EM analyses have been carried out
[42– 45,128,129]. More recent studies by other groups have
provided additional insights into the structure–function dynamics
of ParM. These studies have firmly established that not only does
ParM form filaments, but also that these filaments function
directly in plasmid segregation [99,140–141]. Initial hints that
ParM forms filaments were obtained from immunofluorescence
microscopy studies, which revealed that ParM forms highly
dynamic polymeric structures that extended along the E. coli
cell. In addition, ParM was shown to self-assemble in vitro when
ATP is present [129]. EM studies on these assemblies of R1
ParM filaments revealed the presence of ordered filaments that
were initially described as actin-like [129]. More recently, two
low-resolution models of ParM filaments have been constructed
based on EM data [99,130]. Specifically, Orlova et al. [130] used
the high-resolution crystal structures of R1 ParM to carry out
image reconstructions of cryo-EM data of R1 ParM filaments.
c The Authors Journal compilation c 2008 Biochemical Society
14
M. A. Schumacher
Figure 9 EM reconstructions of R1 ParM filaments and type II insertional
polymerization model
Top: comparison of EM reconstructions from Orlova et al. [130] (left) and Popp et al. [99]
(right). Left: reprinted by permission from Macmillan Publishers Ltd: Nature Structural and
Molecular Biology. Orlova, A., Garner, E., Galkin, V. E., Heuser, J., Mullins, R. D. and Egelman,
E. H. (2007) The structure of bacterial ParM filaments. Nat. Struct. Mol. Biol. 14, 921–922.
Copyright 2007. http://www.nature.com/nsmb/. Right: reprinted by permission from Macmillan
Publishers Ltd: EMBO Journal. Popp, D., Narita, A., Oda, T., Fujisawa, T., Matsuo, H., Nitanai, Y.,
Iwasa, M., Maeda, K., Onishi, H. and Maeda, Y. (2008) Molecular structure of the ParM
polymer and the mechanism leading to its nucleotide-driven dynamic instability. EMBO J. 27,
570–579. Copyright 2008. http://www.nature.com/emboj/ ParM molecules fitted into the model
are numbered in the Orlova model. Bottom: model of R1 plasmid partition (from Campbell
and Mullins [141]). Reproduced from The Journal of Cell Biology , 2007, 179: 1059–1066.
Copyright 2007 The Rockefeller University Press. The proposed steps in the model are numbered
1–5. Step 1 involves nucleation of filaments. After nucleation, one end of the filaments attaches
to one partition complex, and the other end of the filament searches for the partition complex
located on another plasmid. In step 2, the plasmids diffuse in the cell until they encounter a
second plasmid. Once in close proximity, in step 3, both ends of the filaments are bound by
a plasmid, preventing catastrophic collapse. In step 4, because the filaments are now stable,
they continue to grow and push the plasmids to opposite cell poles. In step 5, the plasmids are
finally pushed into the cell poles, the force of which destabilizes the interactions of the ParM
filaments with the plasmid. The plasmid is then released, at which point the free end of the
filament is destabilized and rapidly depolymerizes.
Subsequently, Popp et al. [99] used EM, TIRF (total internal
reflection fluorescence) microscopy, high-pressure SAXS (smallangle X-ray scattering), and X-ray fibre diffraction to construct
three-dimensional images of ParM–p[NH]ppG filaments and
obtain insights into filament dynamics [99] (Figure 9). The
resulting models from these two studies agree that ParM filaments
are left-handed rather than right-handed like F-actin. However, the
two low-resolution ParM filament models differ quite markedly in
detail [99,130]. For example, in the model by Orlova et al. [130],
c The Authors Journal compilation c 2008 Biochemical Society
the ParM molecules within the filaments adopt a conformation
that is even more open than the apo ParM conformation. In
contrast, in the filament model proposed by Popp et al. [99],
ParM adopts a closed conformation like that found in the crystal
structures of R1 ParM with bound nucleotides. Despite these
differences in detail, both ParM filament models are similar in
overall shape and dimension and thus both studies support the
insertional polymerization model whereby ParM filaments insert
between plasmids to propel them towards opposite cell poles
(Figure 9).
The insertional polymerization model has also recently
received strong support from both in vitro reconstitution
assays and in vivo time-lapse fluorescence microscopy analyses
[46,140,141]. Moreover, these studies have provided additional
insight into this process. The in vivo studies, which followed
R1 plasmid dynamics in real time, revealed that ParM filaments
are short-lived and are stabilized only when each end of the
filament is bound to a ParR–parC-associated plasmid [141]. The
rates of in vivo ParM polymerization were found to be similar
to the timing required for plasmid segregation [46]. The fact
that, in vitro, purified ParR and ParM proteins and parC-labelled
magnetic beads alone could drive bead separation and the strong
similarities between the in vitro and in vivo filament-mediated
separation of plasmids and parC-labelled beads also strongly
supports the idea that host factors are not required for type II
plasmid segregation. The in vitro reconstitution study also demonstrated the important finding that the filament grows by insertion
of additional ParM–ATP molecules at the ParR–parC interface.
This insertional polymerization causes the ParM filaments to
extend bidirectionally, moving the two plasmids to opposite
cell poles. The in vivo studies suggest that, after reaching the
poles, the force of colliding into the ends of the cell lead to
dissociation of the filament ends from ParR–parC, prompting
the rapid depolymerization of the filament [141] (Figure 9).
Indeed, it has been demonstrated that pressure applied to a
filamentous structure such as actin can lead to loss of noncovalent interactions, bound nucleotides and susceptibility to
depolymerization [142]. This knowledge provides additional
insight into how the filaments depolymerize at the correct time
after plasmid separation. Specifically, ParR is known to stimulate
the ATPase activity of ParM. Because ParR is in contact with
ParM, it will hasten the formation of ParM–ADP (or ParM–GDP)
within the filament. However, as long as ParR is bound to the ends
of the filaments, it strongly stabilizes the polymer form of ParM.
But, after separating, the plasmids will be forced against the cell
poles [141]. This force will dissociate the ends of one or both
ParM filaments from its interaction with the partition complex on
the plasmid. The end subunit of ParM, which had previously been
in contact with ParR, will probably exist in the NDP-bound state.
Release of the protective partition complex contacts will allow
release of the NDP molecule. According to the recent ParM–
GDP and ParM–p[NH]ppG structures, the NTP-bound and NDPbound states of ParM are in the same conformation, which is
distinct from the apo conformation [99]. Thus release of the NDP
and subsequent conversion into the apo form will lead to domain
rotation, which in turn will cause the loss of one subunit from the
end, stimulating the disruption of the entire filament.
One problem with the insertional polymerization model is that
it necessitates that each end of the ParM filament interacts with
an identical ParR–DNA partition complex. However, structural
information indicates that ParM filaments exhibit polarity,
meaning they have a plus end and a minus end. To examine
how the different ends of the ParM filament may interact equally
with an identical partition complex, we docked the ends of the R1
ParM filament model obtained from EM on to the pSK41 partition
Structure and function of plasmid partition proteins
Figure 10
15
Molecular model of type II segregation
(A) Model of pSK41 segrosome assembly. To construct this model, the pSK41 partition complex structure and the Orlova (for which co-ordinates were deposited) ParM filament model were used
[90,130]. As can be seen from this model, the ParM filament can be embraced within the partition complex pore without steric clash and can favourably interact with the C-terminal flexible domains
of the ParR molecules, found at high concentrations within the pore. (B) In this pSK41 segregation model, the ParM filament is captured between two partition complexes on different plasmids.
The filament and partition complexes are drawn roughly to scale. Centromere DNA is coloured brown, the ParR molecules are magenta, green, blue and yellow, and ParR molecules participating in
spreading are cyan. ParR flexible C-domains, which interact with the ParM filament, are represented as half circles. The ParM filament is represented as a yellow surface rendering. Modelled using
PyMOL (DeLano Scientific).
complex superstructure [90,130]. Remarkably, this modelling
revealed that the filament can readily fit within the partition
complex pore (Figure 10). Further modelling shows that this is
true even when accounting for the addition of the ParR C-terminal
domains, which were not present in the structure [90]. As can
been seen in Figure 10(A), ParM filament polarity would not be a
problem because each end of the filament would encounter ParR
molecules in multiple orientations (Figure 10). Furthermore, the
C-terminal region of ParR, which interacts with ParM, is highly
flexible, thereby permitting it to dock on to ParM molecules
that are arranged in any orientation. An additional feature of the
partition complex structure that is likely to be important for its
interaction with ParM filaments is that its pore region displays a
very high local concentration of ParR C-terminal tails which are
perfectly poised for ParM filament interaction and capture [90].
ParR spreading could help further encase the ParM molecule and
increase the likelihood of productive ParM binding.
Could this proposed partition complex capture mechanism
apply to other par systems?As noted, although ParR/ParB proteins
show little sequence identity, recent structures of the type Ib
and type II centromere-binding proteins have revealed a striking
homology in that both contain RHH DNA-binding domains. In
fact, the RHH fold is an excellent partition complex-forming
motif because binding to tandem centromere sites would promote
its co-operative polymerization and the formation of higherorder superstructures. The combined structural data on type II
ParR proteins and partition complexes, including EM data on
the R1 partition complex, the crystal structure of pB171 ParR
and the crystal structure of the pSK41 partition complex suggest
that type II partition complexes form highly similar superhelical
structures. It might be anticipated that type Ib centromerebinding proteins, which also contain RHH motifs, form related
superstructures. We speculate that the specific conformation of
each of the partition complex and segrosomal superstructures is
likely to be dictated by the polymer structure of the motor protein
bound by each partition complex.
CONCLUSIONS
DNA segregation is an essential process for the survival of all
species. The segregation of plasmid DNA requires only three
components and thus is an ideal model system to study the detailed
mechanisms involved in this process at the atomic level. The
structures of bacterial chromosomal and plasmid partition proteins
and their DNA complexes that have been obtained over the last
5 years have shed significant light on the molecular mechanisms
of segregation of several types of partition systems. In fact,
studies on the type II system have revealed that three components,
the centromere-binding protein, ATPase and centromere site,
can orchestrate DNA separation without the addition of cellular
factors. It has also been established unequivocally that ParM forms
filaments in an ATP- or GTP-dependent manner, and formation
of these filaments drives DNA partition. Stabilization of these
filaments requires interaction of filament ends with the partition
complex superstructure, which is formed by the binding of ParR
molecules to the centomere. The first high-resolution structure
of a higher-order partition complex, that of the type II pSK41
partition complex, revealed that the ParR–centromere complex is
a superhelical structure with dimensions suitable for capture of
the ParM filaments [90]. The position of the ParM-binding sites
within the flexible C-terminal region of ParR indicates how it can
bind the dissimilar ends of the ParM filament.
Despite the substantial progress in delineating a molecular understanding of type II plasmid partition, many questions remain.
For example, how do ParR and other centromere-binding proteins
mediate the seemingly opposing functions of ATPase stimulation
and polymer stabilization? Structural studies on centromere-binding proteins bound to their requisite motor proteins should help
clarify this paradox. Although many of the steps of type II
partition are understood at the molecular level, much less is
known about type I segregation. Unexpected structural homology
revealed between the type II and type Ib centromere-binding
proteins suggests that the type Ib centromere-binding proteins
may form similar superstructures when interacting with their
centromere sites; however, this remains to be demonstrated. Also,
the types of partition complexes formed by the type Ia proteins
are, at this time, completely unknown. The presence of conserved
HTH and motif B domains hints at a mechanism involving
DNA spreading as mediated by the combination of these motifs.
Perhaps the most unresolved issue regarding type I partition is
the molecular mechanism(s) utilized by type I motor proteins in
DNA separation. In fact, no structures are available for a plasmid
type I motor protein and thus how they may form polymers and
c The Authors Journal compilation c 2008 Biochemical Society
16
M. A. Schumacher
bind nucleotides remains a mystery. Mounting evidence suggests
that polymer formation by type I motor proteins is important
in segregation. If this is the case, do these proteins utilize an
insertional polymerization mechanism similar to that of type II par
systems? Support for a common mode of polymer-driven plasmid
separation comes from recent findings that putative type III and
IV par systems employ a tubulin-like GTPase and a coiled-coil
protein respectively for partition. Thus the future challenges are
to fill in the missing pieces of the puzzle left in the type II partition
reaction and to determine the molecular mechanism(s) used by the
type Ia and Ib systems, as well as newly identified par systems, to
form partition complexes and drive plasmid separation. Structural
studies combined with biochemical and cellular analyses will be
needed to address these issues and, hopefully, in so doing, provide
atomic-level snapshots of each step in the partition reaction.
I acknowledge support from the Burroughs Wellcome (Burroughs Wellcome Career
Development Award 992863), the University of Texas M. D. Anderson Cancer Center
Trust Fellowship and the National Institutes of Health. I thank Professor Finbarr Hayes for
helpful discussions and comments. I apologize to those whose work was not discussed.
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