(Received for publication, November 21, 1995; and in revised form, February 10, 1996)
From the
We have previously reported specific labeling of Escherichia
coli DNA gyrase by the ATP affinity analog pyridoxal
5`-diphospho-5`adenosine (PLP-AMP), which resulted in inhibition of
ATP-dependent reactions. The analog was found to be covalently bound at
Lys and Lys
on the gyrase B subunit
(Tamura, J. K., and Gellert, M.(1990) J. Biol. Chem. 265,
21342-21349). In this study, the importance of these two lysine
residues is examined by site-directed mutagenesis.
Substitutions of
Lys result in the loss of ATP-dependent functions. These
mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a
nonhydrolysable ATP analog, 5`-adenylyl-
,
-imidodiphosphate
(ADPNP). The ATP-independent functions of gyrase, such as relaxation of
negatively supercoiled DNA and oxolinic acid-induced cleavage of
double-stranded DNA, are unaffected by these mutations, suggesting that
the mutant B subunits are assembling correctly with the A subunits.
Gyrase with substitutions of Lys
retains all activities.
However, the affinity of ATP is decreased. The DNA supercoiling
activity of gyrase A
B
tetramers reconstituted
with varying ratios of inactive mutant and wild-type gyrase B subunits
is consistent with a mechanism of DNA supercoiling that requires the
interdependent activity of both B subunits in ATP binding and
hydrolysis.
DNA gyrase is a bacterial type II topoisomerase that couples the free energy of ATP hydrolysis to the introduction of negative supercoils into closed-circular DNA. (For recent reviews, see (1, 2, 3) .) In the absence of ATP, DNA gyrase relaxes negatively, but not positively, supercoiled DNA. In contrast, eukaryotic and T-even phage type II topoisomerases, which are structurally related to DNA gyrase (4) , catalyze ATP-dependent relaxation of both positively and negatively supercoiled DNA, but do not supercoil DNA(5) . The topoisomerase reactions of all type II enzymes involve the passage of a double-stranded DNA segment through a transient double-strand break, which is then resealed to give changes in the DNA linking number in steps of two.
DNA gyrase from Escherichia coli contains two subunits, A (GyrA) ()and B (GyrB), which are assembled in an
A
B
complex(6, 7) . GyrA has a
molecular weight of 97,000 and is essential for DNA breakage and
reunion. An intermediate step in this reaction involves the covalent
attachment of Tyr
of GyrA at the broken 5`-end of each
DNA strand(8) . GyrB has a molecular weight of 90,000 and
carries a site for ATP binding and
hydrolysis(9, 10, 11, 12) . Affinity
labeling studies using the ATP analog, PLP-AMP, have identified
Lys
and Lys
as possible active site
residues(13) . The presence of Lys
at the
nucleotide binding site was later confirmed by crystallographic studies
on an N-terminal fragment of GyrB with bound ADPNP(14) , an ATP
analog which cannot be hydrolyzed by gyrase.
The mechanism of energy coupling between ATP hydrolysis and DNA supercoiling is largely unknown. Limited supercoiling of relaxed closed circular DNA occurs upon the binding of ADPNP, suggesting that conformational changes associated with nucleotide binding can induce one supercoiling event while catalytic supercoiling requires hydrolysis of ATP and dissociation of products(15, 16) . Once formed, the gyrase-DNA-ADPNP complex does not readily dissociate. Binding of ADPNP to a gyrase-DNA complex is slow, and involves cooperative interactions between the two nucleotide binding sites in the gyrase tetramer(16) . Modification of only one of these sites appears to inhibit both ATP hydrolysis and DNA supercoiling(13) .
In
this work, we introduce mutations into GyrB at Lys and
Lys
and study the effects of these changes on the
ATP-dependent and ATP-independent reactions of DNA gyrase. Evidence is
presented indicating that Lys
, which is not conserved in
the equivalent region of the eukaryotic type II topoisomerases, is
essential for the ATP-dependent activities of gyrase. Results of
supercoiling experiments combining wild-type and Lys
mutant GyrB subunits in the reconstituted enzyme are discussed in
terms of a mechanism requiring participation by both nucleotide binding
sites of the gyrase A
B
tetramer.
GyrA protein was purified from E. coli strain N4186 by the method of Mizuuchi et al.(19) . Oligonucleotides for mutagenesis and sequencing primers were synthesized using an Applied Biosystems model 380B DNA synthesizer. DNA sequencing was performed using Sequenase Version 2.0 from U. S. Biochemical Corp.
ATPase activity was measured using two methods. The first followed P
liberation from
[
-
P]ATP in a reaction containing 50 mM Hepes-NaOH, pH 7.5, 24 mM KCl, 10 mM potassium
phosphate, pH 7.5, 6 mM MgCl
, 6.5% (w/v) glycerol,
1.8 mM spermidine, 5 mM dithiothreitol (buffer 1) and
0.4 mM [
-
P]ATP. Additions to
selected assay samples included 33 µg/ml novobiocin, 15 µg/ml
linear pBR322 DNA, and 10 µg/ml GyrA protein. The reactions were
initiated by addition of 5 µg/ml GyrB protein. After a 1-h
incubation at 25 °C the reactions were terminated by the addition
of 20 mM EDTA. Hydrolysis products were separated by
thin-layer chromatography on polyethyleneimine-cellulose plates
(Bakerflex) developed in 0.8 M acetic acid, 0.8 M LiCl. The dried plates were then exposed to Kodak XAR-5 x-ray
film. The second method, for kinetic studies of DNA-dependent ATPase
activity, was an ATP-regenerating spectrophotometric assay used as
described previously(13) . The apparent values for K
and k
were determined
from double reciprocal plots of the turnover rate for ATP hydrolysis
against the concentration of ATP (0.1-1.0 mM). Prior to
the assay, 1.2 µM GyrA, 1 µM GyrB, 50
µg/ml linear pUC9 DNA, and 0.2 mg/ml bovine serum albumin were
preincubated for 1 h at room temperature in buffer 1. ATPase reactions
were initiated by the addition of 10 µl of enzyme to 90 µl of
the ATPase assay mixture. Binding studies using ADPNP (Sigma) and
[
-
P]ADPNP (ICN) were carried out as
described elsewhere(16) .
Wild-type and mutant GyrB proteins were expressed in E.
coli as described under ``Experimental Procedures.''
Cells producing the Lys GyrB mutants grew at the same
slow rate as those producing the wild-type protein. On the other hand,
cells producing the Lys
mutants grew more rapidly,
indicating that excess production of the Lys
mutants may
be less toxic to the cells than overproduction of the wild-type GyrB
protein. Expression of all of the GyrB proteins was very high,
averaging about 30% of the total cell protein. However, based on the
supercoiling assay, the specific activity of the wild-type GyrB protein
in the cell lysate was much lower than expected. Further investigation
led to the finding that wild-type GyrB protein could be resolved into
two peaks, which eluted at 0.2 and 0.3 M NaCl on a
heparin-agarose column. The first peak had a much higher specific
supercoiling activity than the second, which may largely consist of
improperly folded protein. Wild-type GyrB prepared from a strain
carrying the same plasmid as used here was previously found to contain
a large amount of low-activity protein, which showed increased activity
after renaturation from guanidine hydrochloride solution(12) .
All of the mutant GyrB proteins were similarly partitioned into two
peaks on heparin-agarose columns. DEAE-Sepharose chromatography of the
more active wild-type peak fractions produced a nearly homogenous GyrB
preparation with a specific activity close to that previously
reported(19) . This wild-type GyrB and corresponding
DEAE-Sepharose fractions containing the mutant GyrB proteins were used
for the experiments which follow.
Figure 1: Oxolinic acid-induced cleavage of DNA. DNA gyrase (5 nM) was incubated with linear pBR322 DNA (2 nM) and oxolinic acid (50 µg/ml) at 25 °C for 1 h in the presence or absence of 1.1 mM ATP as indicated. The reactions were terminated by addition of SDS followed by digestion with proteinase K, as described under ``Experimental Procedures.'' Lane 1 contains no enzyme. Gyrase tetramers were reconstituted using wild-type (WT) or mutant GyrB proteins as shown. The 0.8% agarose gel was run in 90 mM Tris borate, 1 mM EDTA. L is linear pBR322 DNA.
Figure 2:
Extent of supercoiling by wild-type DNA
gyrase and by gyrase reconstituted using K110V and K110E GyrB proteins. Lane 1 contains 2.5 nM intracellularly supercoiled
pBR322 DNA. Lanes 2-20 contain 2.2 nM relaxed
pBR322 DNA which has been incubated alone (lane 2) or with
gyrase reconstituted using wild-type (WT) or mutant GyrB
subunits at the indicated concentrations and incubation times. Gyrase
solutions (0.6 µM) containing equimolar amounts of gyrase
subunits A and wild-type or mutant B were preincubated for 30 min at 25
°C before dilution and addition to the supercoiling assay mix
containing DNA and 1.4 mM ATP. Electrophoresis was carried out
using a 1.2% agarose gel in 40 mM Tris base, 30 mM NaHPO
, 1 mM EDTA and 40 µg/ml
chloroquine. Relaxed DNA (Rel) migrates with high mobility in
this system, due to increased positive writhe in the presence of
chloroquine. The positions of intracellularly negatively supercoiled (SC) and open circular (OC) pBR322 DNAs are shown.
The arrow indicates the direction of resolution of more
negatively supercoiled topoisomers.
Figure 3:
Kinetic analysis of the ATPase activity of
wild-type gyrase and of gyrases containing mutations at Lys of GyrB. At a constant enzyme and DNA concentration, the turnover
rates for ATP hydrolysis by wild-type, K110V, or K110E gyrases were
measured in the presence of varying concentrations of ATP by an
enzyme-linked spectrophotometric assay, as described under
``Experimental Procedures.''
Using a more sensitive radioactive assay (see
``Experimental Procedures''), the effects of the various
mutations on the hydrolysis of [-
P]ATP were
studied. The K103E, K103I, or K103T GyrB proteins had very low levels
of ATPase activity, either in the presence or absence of GyrA and DNA.
This residual ATP hydrolysis was largely insensitive to novobiocin
suggesting that most, if not all, of the activity can be attributed to
contaminating ATPases in the preparations. In assays using the K110V or
K110E enzymes, DNA-dependent novobiocin-sensitive ATPase activity was
observed at levels 2-3-fold lower than with the wild-type enzyme
(data not shown).
Figure 4:
Binding of ADPNP to wild-type and mutant
GyrB proteins and to their complexes with GyrA and DNA. GyrB proteins
(0.5 µM) were preincubated alone or with 0.5 µM GyrA protein and 50 µg/ml linear pBR322 DNA in 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 10 mM potassium
phosphate (pH 7.5), 5 mM dithiothreitol, 1.8 mM spermidine, 6 mM MgCl, 0.5 mg/ml bovine serum
albumin, and 6.5% glycerol for 30 min at 25 °C. 50 µM [
-
P]ADPNP was added and the incubation
at 25 °C was continued for 5 h or 25 h. Unbound nucleotide was
removed by rapid gel filtration and stoichiometry of binding was
determined as described by Tamura et
al.(16) .
Fig. 5shows the result of the reconstitution
experiment. Included in the figure are three theoretical curves, each
predicting a different outcome depending on the mechanism. The straight
line (curve 1) is subject to two interpretations. First, curve
1 represents the anticipated results if the GyrB protein in solution is
already a dimer or if assembly is not random but strongly favors two
GyrB subunits of the same type per tetramer. Tetramers containing one
active and one inactive GyrB monomer would be unlikely to form; the
specific supercoiling activity of the wild-type enzyme would be simply
diluted. Alternately, curve 1 would also be obtained if tetramers
containing one inactive and one active B monomer are formed, but the
activity of the mixed tetramer is proportional to the active GyrB
content; that is, a mixed tetramer would have half the activity of a
tetramer containing two active B subunits. The upper curve (curve
2) predicts the results if assembly of the gyrase tetramers is
random, and catalytic supercoiling can occur with enzyme having one
active and one inactive GyrB subunit; it is assumed that an enzyme
tetramer containing only one active GyrB subunit has full catalytic
activity. This result could also be achieved if a conformational change
following ATP binding to the active GyrB subunit of a mixed tetramer
promoted ATP binding at the inactive GyrB of the tetramer despite the
inactivating Lys mutation, thus generating a second
active site. Last, the lower curve (curve 3) represents the
predicted outcome for random assembly into tetramers when enzyme
containing two active GyrB subunits is the only species capable of
catalyzing DNA supercoiling. The data points are in close agreement
with curve 3, in strong support of the model that DNA supercoiling
requires the participation of both ATP binding sites in the enzyme
A
B
complex.
Figure 5:
Correlation between DNA supercoiling
activity and percentage of inactive GyrB protein used in reconstitution
of the gyrase tetramer. Wild-type and K103I mutant GyrB proteins were
combined in 1.6 µM solutions at molar ratios of 10:0, 9:1,
4:1, 2:1, 1:1, 1:2, 1:4, 1:9, and 0:10. Mixed gyrase tetramers were
formed by adding an equimolar amount of GyrA protein to each GyrB
solution and incubating for 30 min at 25 °C. A series of dilutions
of each tetramer preparation was assayed for supercoiling activity.
Experimentally determined supercoiling activities of the mixed
tetramers () are expressed as percentages of the activity of
tetramers containing 100% wild-type GyrB protein. Theoretical curves 1, 2, and 3 are described under
``Results''.
ATP-dependent type II topoisomerases possess an amino acid
sequence motif predictive of ATP binding. This motif, a highly
conserved glycine-rich region (residues 114-119 of GyrB from E. coli) of the sequence GXXGXG, is found in
all known bacterial, phage, and eukaryotic type II topoisomerases (Table 2). This region, which is also found on a variety of other
ATP binding proteins, has been postulated to form part of a flexible
loop structure involved in conformational changes following nucleotide
binding(23, 24, 25) . A possible role in
nucleotide binding for the region of GyrB comprising amino acids
103-119 was initially proposed from the results of affinity
labeling studies, in which Lys and Lys
were
specifically modified(13) . The importance of this region was
shown in more detail in the crystal structure of a 43-kDa N-terminal
domain of GyrB containing bound ADPNP(14) . The structure shows
that residues 96-117 form a loop followed by a short helix
composed of residues 118-126. The phosphates of the bound
nucleotide are in contact with glycines 114, 117, and 119 of the
ATP-binding motif at the base of the helix. Furthermore, the
-amino group of Lys
forms a salt bridge with the
-phosphate of the bound ADPNP molecule, and Tyr
forms a hydrogen bond with N3 of the adenine ring. When the amino
acid sequence of E. coli GyrB residues 96-127 is
compared with the corresponding region of all other bacterial GyrB
proteins for which sequences have been determined, a very high degree
of conservation is found. A separate comparison of these regions from
the eukaryotic type II topoisomerases also shows very high sequence
homology. However, comparing the gyrases to the eukaryotic enzymes
reveals variations in this region which may be significant (Table 2). The sequences of residues 96-104 are quite
different in the two classes of enzyme; in particular, the Gly
and Gly
residues of gyrase, which should contribute
to the flexibility of the loop, are replaced by serine, and
Lys
, which is conserved in all GyrB species, is replaced
by asparagine in all of the eukaryotic type II topoisomerases. The
Tyr
residue found in all GyrB proteins is replaced by
lysine, glutamine or serine in the eukaryotic type II topoisomerases.
Lys
is commonly found in enzymes from both groups;
however, substitutions at this position have been identified in both
gyrases and eukaryotic type II topoisomerases. Mutation of the
corresponding lysine to alanine in yeast topoisomerase II had little
effect on the activity(26) . Thus, in eukaryotic type II
topoisomerases, neither the equivalent of Lys
nor of
Lys
is required. It is interesting that this segment of E. coli ParE, a component of bacterial topoisomerase IV, very
closely resembles GyrB in overall sequence, while the phage T2 and T4
gene 39 proteins include characteristics of both gyrase and eukaryotic
type II topoisomerase. Like gyrase, topoisomerase IV and the T-even
phage type II topoisomerases are multimers of two or more different
subunits, but they resemble the homodimeric eukaryotic type II
topoisomerases in activity.
Our present results suggest that
Lys is essential for the ATP-dependent activities of DNA
gyrase. Mutations at this site resulted in loss of these functions but
did not affect ATP-independent activities. The ADPNP binding results,
along with the implication of Lys
in nucleotide binding
from the crystal structure, support the hypothesis that it is the
ability to bind ATP which is lost. However, we have previously proposed
that ATP and its analog ADPNP initially form a rapidly reversible
complex with gyrase, followed by a conformational change to a tightly
bound state (16) . It is possible that mutations at Lys
do not altogether prevent nucleotide binding, but prevent or
alter the conformational changes that follow; our spin column assay for
ADPNP binding may not detect bound ADPNP if dissociation of the initial
complex is very fast. We can nevertheless conclude that these
substitutions at Lys
alter the nucleotide binding
behavior of gyrase in such a way that little or no tightly bound
enzyme-nucleotide complex is formed and binding cannot be demonstrated
by the methods previously used with the wild-type enzyme.
The
mutations of Lys indicate that this residue is not
directly involved in ATP binding. Enzyme reconstituted with GyrB
containing valine or glutamic acid substituted for Lys
retains all activities of gyrase. However, the increase in the
K
for ATP of these mutants, together with the
reduced level of ADPNP binding, suggests that ATP binds with lower
affinity. The K
and k
values must be interpreted with caution; cooperative binding of
two nucleotide molecules to the two binding sites in each gyrase
tetramer, leading to conformational changes in the protein prior to
hydrolysis(11, 16, 27) casts doubt upon the
validity of a steady-state approach to gyrase kinetics. However, by
using gyrase as the preformed A
B
-DNA complex
and holding the concentration of this complex constant, we obtain
apparent parameters for the wild-type and mutant GyrB proteins which
provide a useful means for comparing their relative ATPase activities.
Ali et al.(11) have proposed that the
rate-limiting step for the ATPase activity of gyrase is not nucleotide
binding, but hydrolysis and release of products. However, if the rate
of nucleotide binding is considerably slower for the Lys mutants, as suggested by Fig. 4, then ATP binding may
become rate-limiting. The binding rate of ADPNP to the wild-type
gyrase-DNA complex has recently been found to be dependent on the
topology of the substrate DNA, with binding being more rapid if the DNA
is negatively supercoiled(28) . This increase in nucleotide
binding rate at higher levels of supercoiling may be less pronounced
with the Lys
mutants. It is therefore possible that with
these mutants the ATP-independent DNA relaxation activity would make a
greater contribution to the equilibrium superhelical density (Fig. 2).
Our results on the Lys mutants are
consistent with the structural information on the 43 kDa fragment of
GyrB which shows that Lys
does not form contacts with
bound ADPNP(14) . It is probable that amino acid substitutions
for Lys
cause a slight perturbation of the protein
conformation involved with ATP binding or subunit interactions. This is
not surprising in view of the close proximity of Lys
to
residues known to interact with ATP such as Tyr
,
Gly
, Gly
, and Gly
.
The
Lys mutants have provided a unique tool for obtaining
gyrase A
B
tetramers containing a predictable
distribution of active and inactive GyrB subunits. They offer an
alternative to partial inactivation using nucleotide analogs or other
inhibitors which might themselves cause or prevent conformational
changes when bound to the protein, perhaps blocking interactions
between the active and inactivated subunits. We have exploited the fact
that the GyrB mutants at Lys
, while devoid of
ATP-dependent reactions when reconstituted with GyrA and DNA, appear to
retain the capacity to assemble into an enzyme complex capable of
carrying out ATP-independent reactions. In the analysis, it was assumed
that wild-type and mutant GyrB proteins in a mixture can assemble
randomly and equivalently into the enzyme A
B
complex. The observed supercoiling activities closely follow the
predicted theoretical curve of a mechanism in which both GyrB subunits
must be functional in ATP binding and hydrolysis to catalyze DNA
supercoiling.
The interdependent action of the two ATP binding sites in DNA supercoiling by gyrase has been previously proposed. Thermodynamic calculations of the free energy change required to decrease the DNA linking number by two at the high-supercoiling limit of gyrase action are consistent with the concerted hydrolysis of two molecules of nucleotide(29, 30, 31, 32) . Furthermore, measurements of the inhibition of ATPase and DNA supercoiling activities of gyrase following reaction with the ATP affinity reagent, PLP-AMP, indicate that modification of only one of the two ATP binding sites can lead to inactivation(13) . A cooperative model for ATP binding was proposed based on the rates of ADPNP binding in the presence of ATP(16) . This model predicts the interdependence of the GyrB subunits for catalyzing ATP-driven reactions and agrees well with the present results from the subunit mixing experiments. Lastly, there is structural information which supports the functional interaction of the two ATP binding sites. The crystal structure of the N-terminal fragment of GyrB with ADPNP bound shows that the fragment exists as a dimer with dyad symmetry(14) . Each subunit contains an N-terminal extension that goes from close proximity to one nucleotide binding site to direct contact with the bound nucleotide on the other subunit. The physical contact between the two subunits provides a possible way of coupling ATP hydrolysis at one site to the participation of the ATP binding site on the other subunit.
Related studies of the yeast type II topoisomerase have come to different though not necessarily contradictory conclusions. ATP binding to the two ATPase sites of the DNA-bound homodimer of this enzyme appears to be cooperative(26) . At low ATP concentrations, at which the coupling of ATP hydrolysis to relaxation activity is most efficient, an average of 1.9 ± 0.5 ATP molecules are hydrolyzed for each DNA transport event(26) . However, in a yeast heterodimer consisting of one wild-type subunit and one mutant allele defective in ATP binding, binding of ADPNP resulted in a concerted conformational change in both wild-type and mutant subunits(26, 33, 34) . This suggests the possibility that binding only one ATP to an enzyme dimer might suffice to drive a DNA transport event. However, strand-passage activity by the heterodimer was not studied.
Further comparison of the yeast
heterodimer with our present work is complicated because the
inactivating mutation at Gly of yeast topoisomerase II
does not correspond to Lys
of gyrase, but to Gly
in the gyrase ATP binding motif. It remains possible that in a
Gly
gyrase mutant an induced conformational change
similar to that seen in the yeast topoisomerase II mutant/wild type
heterodimer would be found.