(Received for publication, December 20, 1996, and in revised form, March 28, 1997)
From the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
ParA is an essential P1 plasmid partition protein. It represses transcription of the par genes (parA and parB) and is also required for a second, as yet undefined step in partition. ParA is a ParB-stimulated ATPase that binds to a specific DNA site in the par promoter region. ATP binding and hydrolysis by ParA affect ParA activities in vitro. ATP and ADP binding stimulate ParA DNA binding and dimerization; however, ATP hydrolysis has a negative effect on DNA binding. Our current experiments reveal that ATP binding and hydrolysis affect ParA conformation and ParA sensitivity to ParB. Nucleotide binding assays show that ParA binds ATP better than ADP (Kd values of 33 and 50 µM, respectively). Interaction with these nucleotides as well as ATP hydrolysis by ParA alter ParA conformation as established by CD and ParA sensitivity to heat denaturation. Finally, we show that ParB stimulates ParA DNA binding. This stimulation requires ATP hydrolysis in vitro, suggesting that one role for ATP hydrolysis in vivo is to make ParA repressor sensitive to ParB. Our observations lead to the suggestion that ATP binding and hydrolysis have separable roles in ParA repressor function and perhaps in ParA partition functions as well.
ParA is an essential component of the P1 plasmid partition system (1). The prophage of bacteriophage P1 exists as an autonomously replicating plasmid, and its copy number is about the same as that of the bacterial chromosome (2). The P1 partition system, Par, directs proper segregation and thus stable maintenance of the plasmid. The mechanism of partition is unknown, but can be thought of as a positioning process that via interaction with the Escherichia coli host ensures proper distribution of the plasmid. Par encodes two essential trans-acting proteins, ParA and ParB, and contains a cis-acting DNA site called parS (1). In addition, the E. coli integration host factor, IHF,1 also participates, although it is not absolutely required (3). ParB and IHF bind to parS to form the partition complex (3-5). Assembly of this complex is assumed to be an early step in the partition pathway. Formation of this complex does not require the action of ParA (3-5), and we infer that ParA acts during a later step in the partition process.
ParA has at least two roles in partition. First, ParA represses transcription of its own gene and parB from a promoter upstream of parA (6). ParA repressor activity is stimulated by ParB; however, ParB has no effect on par gene expression on its own. A second role for ParA in partition is inferred from genetic data that show a requirement for ParA in partition even when its regulatory role is bypassed (6, 7). Therefore, we consider ParA's repressor activity its "regulatory" function, and this second, as yet undefined role as ParA's "partition" function because we think it reflects a direct role for ParA in the positioning reaction. The latter function requires that ParA interact, directly or indirectly, with the ParB-IHF-parS partition complex.
ParA is an ATPase and a site-specific DNA-binding protein (8). The ATPase is stimulated by ParB and nonspecific DNA (8). ParA is one of the better characterized proteins in a superfamily of ATPases defined by a modified Walker type A motif in the protein sequence (9-11). This superfamily includes other plasmid partition proteins such as F SopA, as well as plasmid and chromosomally encoded proteins from various bacteria species whose functions have not yet been determined (10-12). Many of the plasmids and bacterial chromosomes also encode a ParB homolog adjacent to the ParA homolog. The similarities of these ParA-like proteins with P1 ParA and F SopA have led to the suggestion that these homologs are also involved in plasmid or chromosome segregation (10, 11).
ParA binds to the par promoter region called
parOP (8), and this binding is thought to mediate
ParA repressor activity in vivo. The recognition site for
ParA is likely a large inverted repeat in the par promoter
region, since (i) several mutations in this repeat reduce or eliminate
ParA repression in vivo (13) and (ii) in DNase I
footprinting experiments, protection centers over the inverted repeat,
especially at low ParA concentrations (8, 14). ParA DNA binding
activity is strongly stimulated by ATP (8, 14). ATP hydrolysis is not
required for this stimulation; in fact, it appears to be inhibitory
since ATPS, AMPPNP, and ADP stimulate ParA DNA binding about 5-fold
better than ATP (14). In addition, ATP and ADP promote ParA
dimerization (14), suggesting that ParA prefers to bind DNA as a dimer.
We are interested in how ParA performs both its regulatory and partition roles. Mutation of the ParA nucleotide binding motif A interferes with both ParA repressor and partition functions in vivo (7). However, the steps at which ATP binding and hydrolysis modulate ParA function are not understood. We have further probed ParA interaction with ATP, ADP, and ParB, by examining their effects on ParA structure and function in vitro, to address these questions. We show that ATP binding and hydrolysis alter ParA conformation and that these conformational changes can be correlated to changes in ParA activity. In addition, we demonstrate that ParB stimulates ParA site-specific DNA binding activity in vitro, consistent with ParB's role as a corepressor in vivo. ParB modulation of ParA DNA binding activity requires ATP hydrolysis. We conclude that ATP binding and hydrolysis have different roles in repression and perhaps partition, presumably mediated by their effects on ParA conformation.
Sources for reagents were as follows:
Mono Q and Mono S columns, GTP, Sephadex G-25 M, Pharmacia
Biotech Inc.; bovine serum albumin (FrV), ATP, and ADP, Sigma; ATPS
and bovine pancreatic DNase I, Boehringer Mannheim; heparin-agarose,
Bio-Gel P-6, dithiothreitol (DTT), and Bradford protein assay dye,
Bio-Rad; and [
-32P]dATP, [
-32P]ATP,
and [2,8-3H]ADP, DuPont NEN. Restriction enzymes and
E. coli DNA polymerase I large fragment were purchased from
New England Biolabs.
Buffer A contained 10 mM Tris acetate, pH 7.5, 30 mM NaCl, and 5 mM MgCl2. Buffer B was 50 mM HEPES, pH 7.5, 0.1 mM Na2EDTA, and 20% glycerol. Dilution buffer was 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10% glycerol.
ProteinsParA was prepared as described (14) except that
the glycerol concentration of FrIV was adjusted to 50% (v/v) and the
protein was stored at 20 °C. We consistently maintain ParA in
buffers containing 10 mM DTT (14), because we have observed
that at lower DTT concentrations ParA oxidizes and forms disulfide
cross-linked multimers, particularly after long term storage (data not
shown).
ParB (FrIV) was prepared as described (3, 4) except that ParB FrIII was concentrated using a 1-ml Mono S column prior to gel filtration. ParB FrIII was diluted to 50 mM KCl in buffer B containing 2 mM DTT and then loaded on a Mono S column equilibrated in the same buffer. ParB was eluted in one step with 600 mM KCl in buffer B containing 2 mM DTT. Fractions containing ParB were then purified by gel filtration (4).
ParB (FrIV) occasionally contained a contaminating ATPase activity that
could potentially interfere with the ParA assays. This ATPase activity
was removed by chromatography through a heparin-agarose column. ParB
(FrIV) in buffer B containing 50 mM KCl and 2 mM DTT was applied to a 5-ml column equilibrated in the
same buffer and eluted with a 100-ml, 50 mM to 1 M KCl gradient in buffer B. ParB (FrV) was stored at
80 °C.
For DNA binding assays, ParB and/or ParA were equilibrated in dilution buffer by gel filtration through either 1-ml Sephadex G-25M or Bio-Gel P-6 columns.
Nucleotide BindingHummell and Dreyer equilibrium gel
filtration experiments were used to measure ATP and ADP binding by ParA
(15, 16). Binding was determined as the amount of ATP and/or ADP that
co-eluted with ParA through a gel filtration column. The column was
equilibrated in the same buffer and [32P]ATP and/or
[3H]ADP concentration as the ParA sample. A 5-ml Bio-Gel
P-6 column was equilibrated in buffer A containing
[-32P]ATP (10-20 µCi/µmol) and/or
[2,8-3H]ADP (10 µCi/µmol). Two hundred microliters of
ParA in the same buffer with the same concentration of nucleotide was
applied to the column. Four drop fractions were collected (150-180
µl), and the radioactivity in 100 µl of each fraction was measured
by liquid scintillation counting. The amount of ParA in the peak
fractions was determined by Bradford assay, using a ParA standard whose concentration was determined by amino acid analysis (see "Protein Concentration Determination"). The amount of ATP bound by ParA was
determined as the concentration of ATP in the peak fractions minus the
background concentration of ATP (in the buffer). Experiments were
performed with buffers containing 0.5, 2, 5, 10, 15, 20, 30, or 50 µM ATP using either 31 µM or 62 µM ParA in the sample loaded on the column. At least two
experiments were performed at each concentration of ATP. Concentrations
used in the ADP binding assays are in Table I.
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Protein concentration was routinely measured by Bradford assay (17). The accuracy of this assay for ParA was determined by comparing the measurement of protein concentration by amino acid analysis to the measurement by Bradford assay. For amino acid analysis, ParA was equilibrated to 20 mM NH4HCO3 by gel filtration through a 1-ml Bio-Gel P-6 column. ParA hydrolysis and amino acid analysis were performed at the HSC Biotech Service Center (University of Toronto). ParA molar concentration is expressed as a monomer and ParB as a dimer.
DNA Binding AssayParA DNA binding to
parOP was measured using DNase I protection assays
(14). The substrate was the 388-base pair
HindIII-XhoI fragment from pMD6 (14) and was
labeled at HindIII on the 3 end with
[
-32P]dATP using DNA polymerase I large fragment (18).
A typical binding assay contained between 0.1 and 30 pmol of ParA
(monomer), 10 fmol of pMD6 HindIII-XhoI fragment,
50 nmol of CaCl2, and 1 µg of sonicated salmon sperm DNA
in 15 µl of buffer A. Nucleotide, when added, was present at 1 mM. The mixtures were incubated for 10 min at 30 °C,
1.25 µg of bovine pancreatic DNase I was then added, and the mixtures
were incubated for an additional 60 s at 30 °C. DNase I
digestion was stopped by the addition of 5 µl of "stop" solution
(98% formamide, 10 mM Na2EDTA, pH 8, 0.025% xylene cyanol, 0.025% bromphenol blue (18)). The samples were heated
to 90 °C for 3 min and loaded on a 6% acrylamide urea gel. After
electrophoresis, the gel was dried on Whatman DE81 paper and exposed to
a PhosphorImager screen (Molecular Dynamics) and/or to Kodak XRP
film.
DNase I protection assays were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics). We defined binding as protection from DNase I digestion of the inverted repeat in parOP by ParA. To quantify this binding in each lane, we drew a box in the area that corresponded to the inverted repeat sequences and another box around a set of bands outside of the protected region but in the same lane. The volume ("counts") of the box corresponding to the inverted repeat was divided by the volume of the box outside of the footprint to yield the relative DNase I sensitivity of the inverted repeat. The sensitivity values obtained were normalized so that the assays that did not contain ParA had a DNase I sensitivity of 1. As the sensitivity to DNase I decreases, the protection or binding by ParA increases.
Since ATP affects ParA activity,
we decided to directly measure ATP binding by ParA. We used equilibrium
gel filtration (15, 16), which measures ATP binding as the amount of
ATP that co-elutes with protein over a gel filtration column. Since
this is an equilibrium technique, one can deduce ATP stoichiometry as
well as binding affinity. ParA was loaded on a Bio-Gel P-6 column that
had been equilibrated in the same [32P]ATP concentration
as the ParA sample. ATP that bound to ParA co-eluted with the ParA
peak, and there was a corresponding trough of ATP concentration after
ParA eluted (Fig. 1A) (15). ParA concentration was determined by amino acid analysis (see
"Experimental Procedures"). A typical gel filtration experiment is
shown in Fig. 1A. The experiment was repeated multiple
times, varying ATP concentration from 0.5 to 50 µM and
using two different concentrations of ParA (31 or 62 µM).
The ratio of ATP bound per monomer of ParA was calculated for each
experiment, and each experiment represents one point on a binding curve
(Fig. 1B) or a Scatchard curve (Fig. 1C) (19). We
were unable to measure ATP binding by ParA to saturation (i.e. at higher nucleotide and protein concentrations)
because of limitations of ParA solubility at high concentration.
Regression analysis on the Scatchard curve results in a
Kd of 33 µM ATP and 0.8 ATP binding
sites (n) in a ParA monomer. This Kd is
close to the Km for ParA ATPase (50 µM; Ref. 8).
Mixing experiments indicate that ParA has a slightly lower affinity for
ADP than for ATP. When [-32P]ATP and
[2,8-3H]ADP were present at equal concentrations in an
equilibrium gel filtration experiment, ParA bound approximately 1.5 times less ADP than ATP (Table I), suggesting that the
ParA affinity for ADP is about 1.5 times weaker than ParA affinity for
ATP. When the ADP concentration was 1.5 times that of ATP,
approximately equal amounts of each nucleotide were bound by ParA
(Table I). In the presence of ADP, less ATP was bound by ParA than when
the same concentration of ATP alone was used, suggesting that ATP and
ADP are binding to the same site. We estimated a Kd of 50 µM for ADP, approximately 1.5 times lower than the
Kd for ATP (33 µM). The different
affinities of ParA for ATP and ADP indicate that ParA can distinguish
between adenine nucleoside diphosphates and triphosphates.
Adenine nucleoside di-
and triphosphates strongly stimulate ParA DNA binding (8, 14); however,
ADP and ATPS are better cofactors than ATP (14). To test whether the
ability of adenine nucleotides to modulate ParA activity was a
consequence of their effects on ParA conformation, we examined ParA
stability with and without nucleotide. We found that although ParA was
quite stable at 0 °C (for at least 4 days), it was rapidly
inactivated by short heat treatments at 52 °C (Fig.
2), and we used this treatment to detect
nucleotide-dependent changes in ParA conformation.
ATP, ATPS, and ADP stabilize ParA.
A, activity of heat-treated ParA, assessed by DNase I
footprinting at the parOP site. ParA was heated for 3 min at 52 °C in buffer A containing 100 mM NaCl, 10%
(v/v) glycerol, 100 ng of bovine serum albumin/µl, and 1 mM of the indicated nucleotide (ATP, ATP
S, ADP, or GTP; above each set of lanes). Following the heat treatment, ParA was diluted and titrated into DNA binding assays containing 1 mM ATP at 30 °C, as described under "Experimental
Procedures." Each lane is labeled with the amount of ParA in the DNA
binding mixture. Note that this ParA scale varies; ParA concentration
was lower in assays containing ATP or ATP
S than ones containing ADP
or GTP. The labels and boxes on the
left indicate the
10 and
35 transcriptional signals, the
ribosome binding site (RBS), and the parA start
codon (ATG). The imperfect inverted repeat is delineated by
the inverted arrows. B, ParA DNA binding activity
was quantified from the data in A as described under
"Experimental Procedures." The relative sensitivity to DNase I
cleavage of the inverted repeat sequences in each lane was plotted
against the amount of ParA (pmol), heated in buffer containing 1 mM ATP (
), 1 mM ATP
S (
), 1 mM ADP (
), or 1 mM GTP (
). C,
DNase I footprints of ParA following a heat treatment with (+) or
without (
) 1 mM GTP. ParA was heated for 1 min at
52 °C in buffer A containing 100 mM NaCl, 100 ng of
bovine serum albumin/µl, 10% (v/v) glycerol with or without 1 mM GTP before measuring DNA binding. ParA DNA binding
was assayed in the presence of 1 mM ATP at 30 °C as
described under "Experimental Procedures." The heating step was
shorter in this experiment than in A because ParA is less
stable in these conditions. The positions of the DNA sequence
elements are represented as described in A.
ParA stability at 52 °C was measured in the presence of various
nucleotides (Fig. 2). When ParA was heated to 52 °C for 3 min in
buffer containing 1 mM ATP or ATPS, the protein retained significantly more DNA binding activity than when it was heated in
buffer with 1 mM GTP (Fig. 2, A and
B). ADP (1 mM) also stabilized ParA, although
not to the same extent as ATP and ATP
S stabilized ParA (Fig. 2,
A and B). After this heat treatment, ParA with
GTP was essentially inactivated (Fig. 2, A and
B). After a shorter heat treatment (1 min), ParA with GTP
retained the same activity as ParA that was heated without any
nucleotide (Fig. 2C), consistent with other assays that
indicate that ParA does not bind GTP (8, 14). Therefore, ATP, ADP, and
ATP
S stabilize ParA, suggesting that ParA interaction with these
nucleotides alters ParA conformation. However, ADP is less effective
than ATP or ATP
S.
In these experiments, ParA was heated with different nucleotides and
then assayed for DNA binding activity in 1 mM ATP. Although different adenine nucleotides stimulate ParA DNA binding to different extents (14), the differences in DNA binding activity observed here
were not due to carryover of nucleotide from the heating step into the
DNA binding step for the following reasons. First, the nucleotide
specificity for stability (Fig. 2) differed from the nucleotide
specificity for DNA binding (14). Second, when ParA was incubated at
0 °C rather than 52 °C in buffer containing 1 mM ATP,
ADP, or ATPS and then assayed for DNA binding activity in 1 mM ATP, there was no difference in activity compared with each other or to untreated ParA (data not shown). Finally, when ATP was
added to ParA after, rather than before, incubation at 52 °C, there
was no increase in DNA binding activity compared with no nucleotide.
Therefore, we have measured the effect of nucleotide on protein
stability, not on DNA binding activity, in these assays (Fig. 2).
ATPS and ATP stabilized ParA to the same extent in this assay,
suggesting that ATP hydrolysis was not required for maximum ParA
stability under these conditions. Because some ATPases can hydrolyze
ATP
S, we tested whether ParA hydrolyzes ATP
S at 52 °C. We did
not detect any ParA-dependent hydrolysis of ATP
S at
52 °C under conditions where we could detect 1% or more hydrolysis
(data not shown). Under the same conditions, 2-5% of ATP (at 1 mM) was hydrolyzed, which is approximately 2-5 fold more
than the rate of hydrolysis at 30 °C (data not shown). The
similarity between ATP and ATP
S was also not due to ATP
contamination of ATP
S. By Mono Q chromatography, no ATP was detected
in the ATP
S sample (as little as 1% could be detected; data not
shown).
These experiments show that adenine nucleotides stabilize ParA; however, the triphosphate-bound forms are more stable than the diphosphate-bound form. This difference may reflect the difference in affinity for adenine nucleoside diphosphates and triphosphates, but may also reflect a difference in conformation. These observations suggest that ParA interaction with adenine nucleotides alter ParA conformation, resulting in increased stability.
ATP Binding and Hydrolysis Alter ParA ConformationWe tested
the conclusion that adenine nucleotides alter ParA conformation using
circular dichroism (CD). Addition of 1 mM ATP elicited a
decrease in ParA molar ellipticity () indicating that ATP alters
ParA conformation (Fig. 3). ADP or ATP
S (1 mM) each had a smaller, but measurable effect in this assay
(Fig. 3). AMP, which does not stimulate ParA DNA binding activity (14), had no effect on ParA conformation (Fig. 3). The high absorbance of
light in the samples containing nucleotide prevented accurate measurement of ellipticity below 219 nm. In this assay we used AMP
rather than GTP as a negative control so that the absorption spectra of
all nucleotides in the buffers were identical. The decrease in molar
ellipticity at 222 nm indicated an increase in helicity upon ParA
interaction with nucleotides (20) (Table II). This
increase in helicity corresponded to approximately 16 peptide bonds
changing conformation in a ParA monomer (398 amino acids) with ATP and
approximately 8 peptide bonds changing conformation with ADP or
ATP
S. ParA, therefore, assumes three different conformations as
detected by CD: ParA without nucleotide, ParA bound to nonhydrolyzable nucleotide (ADP or ATP
S), and ParA bound to hydrolyzable nucleotide (ATP). We conclude therefore that ParA interaction with nucleotide alters ParA structure and that hydrolysis further alters that structure.
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We also used circular dichroism to examine the effects of adenine
nucleotides on ParA stability and confirmed our initial results (Fig.
2). We measured temperature-dependent changes in ParA
conformation by monitoring ParA molar ellipticity at 220 nm between
25 °C and 65 °C (Fig. 4). Since ParA precipitated
when it was heated, denaturation was not reversible and we cannot make any conclusions about the thermodynamics of ParA conformation in
response to heat, such as the free energy change. However, we can
estimate the temperatures at which ParA denatured with and without
nucleotide (Table III). As in the initial stability assay, adenine nucleotides increased ParA stability (Fig. 4, Table III). However, in this assay ATPS did not stabilize ParA as well as
ATP stabilized ParA (Table III). This result suggests that ATP hydrolysis modifies ParA conformation, as was also suggested by the ATP
hydrolysis-dependent increase of ParA helical content (Fig.
3). However, the ADP-bound form of ParA was still less stable than both
adenine nucleoside triphosphate-bound forms.
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ParB stimulates ParA
repressor activity in vivo; however, ParB has no repressor
activity on its own (6). Simplistically one might expect that ParB
stimulates ParA repressor activity in vivo via a direct
interaction of ParB with ParA at parOP. We predicted
that such an interaction would influence ParA DNA binding to the
operator, either by increasing ParA affinity for
parOP or by altering ParA activity, making it a more
effective block to RNA polymerase (resulting, for example, in a change
in the DNase I footprint). We show here that a large molar excess
(10-fold as ParB dimers to ParA monomers) of ParB over ParA stimulated ParA DNA binding activity up to 5-fold in the presence of ATP (Fig.
5A). ParB did not protect
parOP from DNase I cleavage on its own (Fig.
5B), nor did ParB alter the pattern of DNase I cleavage in
the presence of ParA (e.g. compare the last lane
(1.8 pmol of ParA) without ParB to the fifth lane (.37 pmol
of ParA) with ParB in Fig. 5A). This implies that ParB does
not directly contact the promoter and that ParB probably acts by
increasing ParA affinity for parOP in the presence of
ATP. At low ParB to ParA ratios, ParB stimulation of ParA DNA binding
was only observed when ParA and ParB were preincubated together before
assaying for DNA binding. Less stimulation of ParA DNA binding by ParB
was observed under these conditions (about 2-fold; Fig.
5B).
In previous experiments (8, 14) no ParB effect on ParA DNA binding was detected. We (14) had used the lower levels of ParB with no preincubation step. Under these conditions, we saw identical results in our current study: Without preincubation, lower ParB levels did not stimulate ParA DNA binding (data not shown). Since ParB stimulated ParA DNA binding at lower ParB to ParA ratios after preincubation, we think it reasonable that the initial requirement for a large excess of ParB over ParA is the result of a weak ParB-ParA interaction rather than a high stoichiometric ratio.
Nucleotide Specificity of ParB Stimulation of ParA DNA Binding ActivityParB stimulated ParA DNA binding activity only in the
presence of ATP. We did not detect any stimulation of ParA DNA binding by ParB in the presence of ADP (Fig. 6A) or
in the absence of nucleotide (Fig. 6B). In the absence of
nucleotide, ParA binds parOP weakly, about 10-fold
less well than ParA-ATP binds parOP (14). Because a
large amount of protein was required in these assays and because
nonspecific DNA binding interfered, we could not add a 10-fold molar
excess of ParB to ParA DNA binding assays without nucleotide. Instead,
less ParB was used and the two proteins were preincubated on ice before
assaying for ParA DNA binding activity (as in Fig. 5B).
Under these conditions, ParB did not affect ParA DNA binding activity
(Fig. 6B). In the presence of ADP or ATPS (but without
ParB), ParA binds to parOP very well, about 5-fold
better than in the presence of ATP (14). When a 10-fold molar excess of
ParB was added to ParA DNA binding assays in the presence of 1 mM ADP, no effect of ParB was detected (Fig. 6A). ParB also did not stimulate ParA DNA binding in the
presence of ATP
S (data not shown).
Since ParA-ADP binds DNA better than ParA-ATP binds DNA (14) and ParB stimulates ParA ATPase (8), one explanation of our current results is that ParB stimulates ParA DNA binding via ParB stimulation of ParA ATPase and hence production of ADP. This seemed a remote possibility since approximately 0.1 mM ADP is required to stimulate ParA DNA binding (14), ParA has a lower affinity for ADP than for ATP (Table I), and previous measurements of ParA ATPase with a large molar excess of ParB (although at a lower ATP concentration than used in our assays) indicate that insufficient ADP was produced to stimulate ParA DNA binding (8). Nevertheless, we measured ATP hydrolysis by ParA under DNA binding conditions with a 10-fold molar excess of ParB. In agreement with previous measurements of ParA ATPase (8), insufficient ADP was produced (final concentration, less than 1 µM; data not shown) to account for ParB stimulation of ParA DNA binding activity.
We suggest therefore that ParB stimulates ParA DNA binding via a direct interaction with ParA. Interestingly, ParB stimulated ParA DNA binding (with ATP) to the same extent that ADP does over ATP without ParB (up to 5-fold; Ref. 14) (Fig. 5A). Since ParB stimulates ParA ATPase (8) and ATP hydrolysis by ParA is inhibitory to ParA DNA binding (14), we propose that ParB stimulates ParA DNA binding by circumventing the negative action of ATP hydrolysis on ParA DNA binding activity.
P1 ParA binds (Fig. 1) and hydrolyzes ATP (8), and one or both of these activities are essential for both its regulation and partition activities (7). Nucleotide binding experiments (Fig. 1) suggest one ATP binding site per ParA monomer, which agrees well with the presence of one putative ATP binding site defined by sequence analysis (10), and the observation that deletion of this region of the protein destroys ATPase activity (7). We have used different adenine nucleotide cofactors to probe the structure and function of ParA. Our results suggest that ATP binding and ATP hydrolysis by ParA induce different conformations of ParA that in turn have different roles in ParA activity. In addition, we suggest that ATP binding and hydrolysis play separable roles in ParA activities in vitro and consequently in partition and par gene expression in vivo.
ParA Conformation with and without NucleotideUpon
interaction with ATP, ADP, and ATPS, ParA changes conformation (Fig.
3) and ParA dimerization and DNA binding are stimulated (8, 14). The
nucleotide specificities of the changes in ParA helical content
detected by CD and of the stimulation of ParA DNA binding are the same,
implying that the effects of ATP binding and hydrolysis on ParA DNA
binding activity are mediated by their effects on ParA conformation.
However, the nucleotide specificities for dimerization and helical
content are different, suggesting that dimerization is not the only
factor determining the change in ParA conformation upon interaction
with nucleotides. The concentration of intracellular nucleotide pools
is sufficiently high (e.g. ATP is in the millimolar range;
Ref. 21) that ParA would rarely be free of nucleotide in
vivo. However, differences between ParA-ATP and ParA-ADP or the
perhaps the interchange between these conformations are likely to be
important for ParA function in partition.
We found it intriguing that ADP and ATPS behaved
similarly in our assays, such as site-specific DNA binding (14) and
conformation (Fig. 3). This raised the formal possibility that ParA did
not discriminate between nucleoside di- and triphosphates, except via
the act of hydrolysis. Here, measurements of nucleotide binding indicate a slightly better affinity for ATP than for ADP, and a large
difference in the ability of adenine nucleoside tri- and diphosphates
to stabilize ParA. The differences in stability may be a function of
the differences in affinity for nucleotide, or due to differences in
ParA conformation that we cannot detect by CD. Differences in ParA
stability mediated by nucleotide could also potentially play roles in
partition or regulation.
ParA consistently behaves differently when bound to a hydrolyzable nucleotide than when bound to a nonhydrolyzable nucleotide. The ATP-bound form of ParA has a lower affinity for DNA (14), it has a different conformation than the other forms (Figs. 3 and 4), and it is the only form detectably modulated by ParB (Figs. 5 and 6). Our current results provide further support that ATP hydrolysis plays roles in ParA function that are distinct and separable from ATP binding.
ParA-ParB InteractionsParB stimulates ParA binding to
parOP, but only in the presence of ATP. ParB did not
stimulate ParA DNA binding in the absence of nucleotide (Fig.
6B), a form that binds parOP weakly (14). We detected no ParB effect on ParA-ADP or ParA-ATPS DNA binding (Fig. 6A; data not shown), forms that bind to DNA about
5-fold better than ParA-ATP binds to DNA (14). ParB stimulates ParA-ATP DNA binding activity to the same level as ParA-ADP DNA binding activity
with or without ParB. Since ATP hydrolysis by ParA in the absence of
ParB is inhibitory to DNA binding (14), we think that the simplest
explanation is that association with ParB prevents the inhibitory
action of ATP hydrolysis on ParA DNA binding. This model also explains
the observation that ParB can stimulate both ParA ATPase and ParA DNA
binding, since one might otherwise expect increased ATP hydrolysis by
ParA to further interfere with ParA DNA binding. Mutations have been
isolated in other ATPases that can bind but not hydrolyze ATP (22, 23).
Our hypothesis predicts that a similar ParA mutant would repress
transcription from parOP as well as wild-type ParA
represses in the presence of ParB (6), and that this repression would
be independent of ParB.
Why is ATP hydrolysis required for ParB stimulation of ParA DNA binding? One possible explanation is that ATP hydrolysis is required for physical association of ParA and ParB. Preliminary co-immunoprecipitation experiments suggest that ParA and ParB associate in the absence of ATP,2 so we favor other interpretations of these results. Perhaps a ParA-ParB complex forms regardless of the nucleotide present but is detectable by our DNA binding assay only in the presence of ATP hydrolysis. ParA has no detectable kinase activity (8),2 so we think it unlikely that phosphorylation of either protein by ParA is required for activity.
Roles of ATP in par Gene Regulation and PartitionThe ATP site in ParA is essential for both ParA's regulation and partition activities in vivo (7). One role of ATP binding is to promote ParA binding to parOP for ParA repressor activity (8, 14). A role for ATP hydrolysis in repression is suggested by its ability to make ParA DNA binding, and thus ParA repressor, sensitive to ParB levels. Alternatively, one might argue that the role of ParB in repression is to make ParA DNA binding insensitive to ATP hydrolysis. We do not yet know what other roles ATP binding and hydrolysis have for ParA function in vivo. It is possible, for example that ATP hydrolysis is necessary only for partition and not regulation. Intuitively, we think it likely that ATP hydrolysis is required for the positioning reaction, in particular to fulfil the energy requirement inherent in the partition process. For example, conformational changes induced by ATP binding and hydrolysis in some proteins are thought to mediate the displacement of other structures in the cell, such as transport across membranes (MalK; Ref. 24), passage of one DNA strand through another (topoisomerase II ; Ref. 25), or movement along microtubules (kinesin; Ref. 26). Perhaps the conformational changes induced in ParA by ATP binding and hydrolysis result in the movement or orientation of the partition apparatus.
ParA-ParB interactions are likely important for ParA's activity in the positioning reaction. Host factors might interact with ParA, ParB, or both proteins. These host factors are still undefined, although they are not DNA sites on the host chromosome (27). We assume that they are proteins or membrane components that position partition complexes and/or time positioning events with respect to the cell division cycle. A careful analysis of ParA-ParB interactions at parS, including the roles of ATP binding and hydrolysis in these interactions, as well as identification of host factors, are crucial to the dissection of the P1 plasmid partition pathway.
We are very grateful to Alan Davidson (A. D.) for use of his CD spectrometer and his and Ariel Di Nardo's assistance in performing the CD experiments. In addition, we thank A. D. for very helpful discussions and advice, and A. D. and Marc Perry for critical reading of the manuscript.