©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Binding Site for Cyclothialidine on the B Subunit of DNA Gyrase (*)

Naoki Nakada (§) , Hans Gmünder (1), Takahiro Hirata , Mikio Arisawa

From the (1)Nippon Roche Research Center, Kamakura 247, Japan and F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (AB)DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyraseDNA complex (increase in affinity: B AB ABDNA). Furthermore, displacement curves of [C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ.


INTRODUCTION

DNA gyrase is a type II DNA topoisomerase that catalyzes the negative supercoiling of DNA in prokaryotes, and its function is essential for cell growth. The enzyme is implicated in the process of DNA replication, transcription, and recombination and in a number of other cellular processes(1, 2, 3) . DNA gyrase from Escherichia coli consists of two subunits, A and B, with molecular masses of 97,000 and 90,000 Da, respectively. The active enzyme is an AB tetramer complex. The mechanism of DNA supercoiling by gyrase involves the wrapping of a segment of DNA around a protein core, cleavage of this DNA, and passage of another piece of DNA through the double-stranded break, which is then rejoined(4) . The reaction cycle normally requires the hydrolysis of ATP. However, replacement of ATP by a nonhydrolyzable ATP analogue, ADPNP,()results in limited supercoiling by gyrase, suggesting that ATP binding can promote a single round of supercoiling, but that the hydrolysis step is required to regenerate the enzyme in its active form(5) . The A subunit contains the site of the DNA breakage and rejoining of the DNA supercoiling, while the B subunit contains the site of ATP hydrolysis. Both subunits are thought to consist of two distinct functional domains. A subunit contains an N-terminal domain (58-64 kDa), thought to be involved in the DNA breakage-rejoining reactions, and a C-terminal domain involved in the DNA-subunit interactions(6, 7) . The B subunit contains a 43-kDa N-terminal domain, which involves the site of ATP hydrolysis(8) , and a 47-kDa C-terminal domain, which interacts with the A subunit and DNA(9) . The crystal structure of this 43-kDa N-terminal fragment protein complexed with ADPNP has been reported(10) . The genes for the gyrase subunits, gyrA and gyrB, have been sequenced and cloned such that the A and B subunits can be overproduced, up to 40% of soluble cell proteins(11) . DNA gyrase is known to be the target of two classes of antibiotics: the synthetic quinolones, typified by nalidixic acid and the new fluoro-quinolones, and the natural coumarins such as novobiocin and coumermycin A1. The quinolones are thought to act at the A subunit probably by interfering with the DNA-rejoining step of the gyrase-mediated DNA strand-passing reaction(12, 13, 14, 15, 16) . The coumarins are thought to act at the B subunit probably by competing with ATP for binding to the B subunit of the enzyme(5, 17, 18, 19) .

From our screening of natural products for DNA gyrase inhibitors, we isolated a novel gyrase inhibitor, cyclothialidine, from Streptomyces filipinensis NR0484(20) . Cyclothialidine contains a unique 12-membered lactone ring that is partly integrated into a pentapeptide chain(21) . Cyclothialidine exhibited the highest inhibitory activity against DNA gyrases from several bacterial species, including Escherichia coli and Staphylococcus aureus, with high selectivity in its biological activity(22) .

Our previous studies (23) indicated that cyclothialidine inhibits, under steady-state conditions, the ATPase activity of E. coli DNA gyrase competitively with a K of 6 nM. Therefore, cyclothialidine, being a B subunit inhibitor of DNA gyrase, shows the same mode of action as the coumarin antibiotics novobiocin and coumermycin A1. However, cyclothialidine was also active against a DNA gyrase resistant to novobiocin, suggesting that the residues required for novobiocin binding are not involved in cyclothialidine binding. In addition, there is no obvious structural resemblance among cyclothialidine, novobiocin, and ATP (Fig. 1). Here, we report on a further characterization of the DNA gyrase-mediated ATPase activity by cyclothialidine. We performed [C]benzoylcyclothialidine binding experiments to correlate the inhibition of the ATPase activity of DNA gyrase by cyclothialidine with binding of the compound to the B subunit. We also compared the binding of cyclothialidine with that of coumarin antibiotics and ATP analogues. For these studies we used a reconstituted E. coli DNA gyrase system that contained the A subunit, the B subunit, and relaxed ColE1 plasmid DNA. Some studies were also performed with the 43-kDa N-terminal fragment of the B subunit(8) , which contains the ATP- and the coumarin-binding site(s).


Figure 1: Structures of cyclothialidine, [C]benzoylcyclothialidine, novobiocin, and ATP.




EXPERIMENTAL PROCEDURES

Materials

Cyclothialidine was purified from a culture broth of S. filipinensis NR0484 having a >98% purity detected by HPLC analysis(21) . Novobiocin, coumermycin A1, distamycin A, ATP, ADPNP, and ATPS were purchased from Sigma. Phosphoenolpyruvate, NADH, and pyruvate kinase/lactate dehydrogenase mix were purchased from Boehringer Mannheim.

DNA Gyrase

Gyrase subunits A and B were purified separately from the E. coli overproducing strains N4186 and MK47 by the method of Mizuuchi et al.(24) . The fraction containing the gyrase A subunit, after it had been subjected to valine-Sepharose chromatography, still contained minor amounts of the gyrase B subunit. Therefore, the fraction was applied to a novobiocin-Sepharose column, as described by Staudenbauer and Orr(25) , and the flow-through fraction was collected. The gyrase B subunit was eluted from a hydroxylapatite column. Each sample was stored in 25 mM Hepes-KOH (pH 8.0) containing 1 mM dithiothreitol, 0.2 mM EDTA, and 50% (w/v) ethylene glycol at -70 °C. Purified A subunit was judged to be >90% pure by SDS-polyacrylamide gel electrophoresis; similarly, the purified B subunit was >95% pure. Gyrase activity was measured using a standard supercoiling assay(26) . One unit was defined as the minimum amount of reconstituted gyrase that will maximally supercoil 0.5 µg of relaxed ColE1 DNA at 30 °C in 30 min. The specific activities of the A subunit and B subunit were 6.4 10 units/mg and 1.2 10 units/mg, respectively. Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin as the standard.

Purification of the 43-kDa N-terminal Fragment of the DNA Gyrase B Subunit

The 43-kDa protein was purified from E. coli cells containing plasmid pAJ1 essentially as described by Jackson et al.(27) . Briefly, cells were grown at 37 °C in LB broth containing 50 µg/ml ampicillin to an OD of 0.5 and induced for 4 h by the addition of isopropyl--D-thiogalactopyranoside to 50 µM. Cells were harvested and resuspended at a concentration of 1 g/ml in 50 mM Tris-HCl (pH 7.6), 10% sucrose. The cell suspension was adjusted to 1 µg/ml RNase, 1 µg/ml DNase, and 20 µg/ml lysozyme and incubated for 30 min at room temperature. The cells were disrupted at 60 megapascals using a French press and centrifuged at 100,000 g for 60 min at 4 °C. Cell extracts were dialyzed against 50 mM Tris-HCl (pH 8.0) and applied to a DEAE-Sepharose CL 6B column (Pharmacia Biotech Inc.). After washing with 50 mM Tris-HCl (pH 8.0), the column was eluted with a 100-ml linear gradient of 0.0-0.7 M NaCl in 50 mM Tris-HCl (pH 8.0). Fractions containing the 43-kDa protein were identified by SDS-polyacrylamide gel electrophoresis, pooled, dialyzed against 50 mM Tris-HCl (pH 8.0), and loaded onto an FPLC Mono Q HR 10/10 column (Pharmacia). After washing the column with 50 mM Tris-HCl (pH 8.0), we eluted the 43-kDa protein with a 160-ml linear gradient of 0.0-0.4 M NaCl in 50 mM Tris-HCl (pH 8.0). Fractions containing the protein fragment were identified by SDS-polyacrylamide gel electrophoresis, pooled, and dialyzed against 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 10% (w/v) glycerol, frozen in liquid nitrogen, and stored at -80 °C(28) . [C]Benzoylcyclothialidine-The [C]benzoylcyclothialidine was prepared by the reaction of cyclothialidine with [C]benzoic acid (21.8 mCi/mmol) in the presence of N,N`-disuccinimidyl carbonate in acetonitrile/pyridine (1:1) having >98% purity. The labeled compound has a specific activity of 21.8 mCi/mmol. Its mobilities on TLC and HPLC are identical to those of authentic benzoyl-cyclothialidine. Benzoyl-cyclothialidine has almost the same inhibitory activity against E. coli DNA gyrase as cyclothialidine in the DNA supercoiling assay (results not shown). [C]Benzoylcyclothialidine Binding Experiment-The binding of [C]benzoylcyclothialidine was determined by a centrifugal filtration method(13) . Centrifree micropartition devices (Amicon number 4103) were used to separate [C]benzoylcyclothialidine bound to DNA gyrase from the free ligand. Reactions (400 µl) were carried out similar to that of the supercoiling assay but without the addition of ATP; the reaction mixture contained an appropriate amount of DNA gyrase and radioactive ligand in standard buffer (50 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl, 1 mM EDTA, and 1 mM dithiothreitol). After incubation for 30 min at 30 °C, the mixtures were transferred to the centrifree devices and centrifuged at 1,600 g in a Kubota KR-180B (swinging bucket rotor) for 30 min at 4 °C. The membrane disks and o-rings of the centrifree devices were placed in vials with 1 ml of standard buffer. The vials were shaken on a rotary shaker for 2 h for solubilization of the [C]benzoylcyclothialidine. Then 15 ml of liquid scintillation fluid (toluene-ethanol (1:1), 0.7% [2-(4-tert-butylphenyl)5-(4`-biphenylyl)-1,3,4-oxadiazole]) was added per vial for the measurement of radioactivity. For the determination of nonspecific ligand binding, the assay was run with excess cyclothialidine. The amount of bound ligand was calculated after subtracting the nonspecific bound radioactivity.

ATPase Assay

ATPase assays were carried out at 25 °C in the following: 300 µl in 40 mM Tris-HCl (pH 8.0), 25 mM KCl, 2.5 mM spermidine, 4 mM MgCl with phosphoenolpyruvate and NADH at 400 and 250 µM, respectively, and 3 µl of pyruvate kinase/lactate dehydrogenase mix (in ammonium sulfate solution, 3.2 M)(8) . The ATP was added at concentrations from 0.5 to 3.5 mM; the 43-kDa N-terminal fragment of the DNA gyrase B subunit at a concentration of 5 µM; and cyclothialidine at concentrations from 0.025 to 10 µM. Reactions were initiated by the addition of the protein, and the decrease in A was continuously measured as a function of time (up to 10 min). The change in absorbance was related to ADP production using = 5100, with the production of NADH stoichiometrically related to the amount of ADP released.


RESULTS

[C]Benzoylcyclothialidine Binds to the 43-kDa N-terminal Fragment of the B Subunit-Our previous studies have shown (23) that, under steady-state conditions, cyclothialidine competitively inhibits the ATPase activity of the E. coli DNA gyrase B subunit with a Kvalue of 6 nM and that [C]benzoyl-cyclothialidine binds to the DNA gyrase holoenzyme (AB tetramer) in the absence of DNA. This binding is inhibited by cyclothialidine, novobiocin, and the ATP analogue ATPS, but it is not inhibited by ofloxacin, strongly suggesting that cyclothialidine inhibits the binding of ATP to the gyrase by acting on the B subunit. However, it is not yet known whether the binding of [C]benzoylcyclothialidine requires both subunits of the DNA gyrase or only the B subunit and what effect DNA has on the binding. To investigate these aspects more precisely, we studied [C]benzoylcyclothialidine binding by using a system of reconstituted E. coli gyrase subunits plus relaxed ColE1 DNA and also the 43-kDa N-terminal fragment of the B subunit. The binding experiments were carried out by a centrifugal filtration technique. The results of the binding studies of [C]benzoylcyclothialidine to the gyrase subunits and to the plasmid DNA are shown in Fig. 2. There was significant binding of the compound to the B subunit but not to the A subunit nor to the relaxed ColE1 plasmid DNA. Furthermore, the compound bound to the 43-kDa N-terminal fragment of the B subunit, showing that the binding site of cyclothialidine as well as that for the coumarin antibiotics is located within the 43-kDa N-terminal fragment.


Figure 2: The binding of [C]benzoylcyclothialidine to A subunit, B subunit of DNA gyrase, or relaxed ColE1 DNA. Reactions were as described under ``Experimental Procedures'' except for the addition of the indicated amounts of either a 43-kDa N-terminal fragment of the B subunit (), B subunit (), A subunit (), or relaxed ColE1 DNA (). Reaction mixtures contained 1.3 10M [C]benzoyl-cyclothialidine. After 30 min at 30 °C, the levels of bound [C]benzoyl-cyclothialidine were determined by the centrifugal filtration method.



Next, to study whether the binding of [C]benzoylcyclothialidine depends only on the presence of the B subunit alone, we performed our binding experiments in the presence of 1) the B subunit, 2) the B subunit plus A subunit, and 3) the B subunit plus the A subunit plus plasmid DNA. The amount of [C]benzoylcyclothialidine bound to the B subunit was somewhat higher in the presence of the A subunit and even higher in the presence of the A subunit plus plasmid DNA (data not shown). When a 250-fold excess of unlabeled cyclothialidine was added, the amount of [C]benzoylcyclothialidine bound to the B subunit decreased to the level where it could hardly be detected under all conditions (data not shown). We confirmed that [C]benzoylcyclothialidine reversibly binds to the B subunit even in the presence of the A subunit and plasmid DNA.

Scatchard Analysis

To study the binding of [C]benzoyl-cyclothialidine to the B subunit further, including the effect of the A subunit and plasmid DNA on the binding, we performed Scatchard analysis for the four conditions. The results in Fig. 3show that the [C]benzoylcyclothialidine bound to the B subunit in the presence of the 43-kDa fragment with a K value of 2.5 ± 0.40 10M; in the presence of the B subunit with a value of 2.4 ± 0.10 10M; in the presence of the B subunit plus the A subunit with a value of 1.5 ± 0.31 10M; and in the presence of the B subunit plus the A subunit plus plasmid DNA with a value of 8.3 ± 0.12 10M. This result suggests that the binding affinity of the compound to the B subunit is dependent on the stage of the assembly of the gyraseDNA complex. The lowest binding affinity to the B subunit could be observed in the presence of the B subunit alone, and the highest binding affinity could be observed in the presence of the ABDNA complex (increase in affinity: B AB ABDNA), although the binding capacity (B) remained invariant, with approximately 1 pmol of [C]benzoylcyclothialidine bound per pmol of the B subunit.


Figure 3: Scatchard analysis of [C]benzoylcyclothialidine binding to the reconstituted E. coli DNA gyrase subunits plus DNA. Reactions were as described in the legend to Fig. 2 and under ``Experimental Procedures'' except that the incubation was done for 30 min. Reaction mixtures contained 0.11-1.6 10M [C]benzoylcyclothialidine. When added, 100 pmol of A subunit, 50 pmol of B subunit, 50 pmol of the 43-kDa fragment, and 0.5 µg of relaxed ColE1 DNA were present. Scatchard plots in the presence of the 43-kDa fragment (), B subunit (), B subunit + A subunit (), and B subunit + A subunit + plasmid DNA () were performed.



Inhibition of Binding by Cyclothialidine, ATP Analogues, and Coumarin Antibiotics

Although the structures are different (Fig. 1), cyclothialidine proved to be similar to novobiocin and coumermycin A1 in their inhibition of the DNA-dependent ATPase activity of DNA gyrase. Furthermore, there are no structural similarities between ATP and cyclothialidine. Therefore, to investigate whether the binding site for cyclothialidine on the B subunit is different from that for ATP and for the coumarin antibiotics, we examined the effects of nonlabeled cyclothialidine, ADPNP, ATPS, noboviocin, and coumermycin A1 on the binding of [C]benzoylcyclothialidine to the 43-kDa N-terminal fragment of the B subunit, to the B subunit alone, and to the B subunit in the presence of the A subunit or in the presence of the A subunit and DNA. The results are shown in Fig. 4and . The displacement curves of [C]benzoyl-cyclothialidine binding obtained after the addition of cyclothialidine had almost the same profiles, with IC values of 8.4 10M for the 43-kDa protein, 9.3 10M for the B subunit, 1.0 10M for the B subunit plus A subunit, and 8.1 10M for the B subunit plus the A subunit plus plasmid DNA, respectively. However, the displacement curves induced by ADPNP were surprisingly quite different from those induced by cyclothialidine and critically depended on the form of the B subunit present. The relative inhibitory activity of ADPNP is as follows: B subunit plus A subunit plus DNA > B subunit plus A subunit > B subunit > 43-kDa N-terminal fragment, with IC values of 8.0 10M, 4.5 10M, 1.5 10M, and >1.5 10M, respectively. The IC value of ADPNP for the ABDNA complex is 19-fold lower than that for the B subunit alone, and similar results were obtained with ATPS and coumarin antibiotics (). The coumarin antibiotic coumermycin A1, for example, inhibited [C]benzoyl-cyclothialidine binding in the order B subunit plus A subunit plus DNA > B subunit plus A subunit > B subunit > 43-kDa N-terminal fragment, with IC values of 9.5 10M, 1.8 10 2.4 10, and 6.3 10M, respectively. The IC value for the ABDNA complex is 6.6-fold lower than that for only the 43-kDa N-terminal protein.


Figure 4: Effect of cyclothialidine, ATP analogues, and coumarin antibiotics on [C]benzoylcyclothialidine binding to the each B subunit form of DNA gyrase. Reactions were as described in the legend to Fig. 3 and under ``Experimental Procedures.'' Reaction mixtures contained 1.3 10M [C]benzoylcyclothialidine and the indicated amounts of cyclothialidine (A), ADPNP (B), ATPS (C), novobiocin (D), and coumermycin A1 (E). The effect of the compounds on [C]benzoylcyclothialidine binding to the 43-kDa fragment (), B subunit (), B subunit + A subunit (), and B subunit + A subunit + plasmid DNA () was measured. After 30 min at 30 °C, the levels of bound [C]benzoylcyclothialidine were determined by the centrifugal filtration method.



Furthermore, Scatchard analysis of the competitive binding of ADPNP and novobiocin to the B subunit plus the A subunit were also carried out (Fig. 5). Although the slope of the Scatchard plot decreased in the presence of ADPNP and was dependent on its concentration, the binding capacity (B) remained invariant. The same result was obtained with novobiocin. These observations suggest that ATP and coumarin antibiotics competitively inhibit the binding of [C]benzoyl-cyclothialidine to the B subunit.


Figure 5: Scatchard analysis of [C]benzoylcyclothialidine bind-ing for the competition studies of ADPNP and novobiocin. Reactions were as described in the legend to Fig. 3 (B subunit + A subunit). A, reaction mixtures contained no ADPNP (), 3 mM ADPNP (), or 10 mM ADPNP (). B, reaction mixtures contained no novobiocin (), 0.3 µM novobiocin (), or 1.5 µM novobiocin ().



Cyclothialidine Is Not a Simple Competitive Inhibitor of the ATPase Activity of the 43-kDa N-terminal Fragment

The 43-kDa N-terminal fragment of the DNA gyrase B subunit hydrolyzes ATP and binds novobiocin and coumermycin A1(8) . We showed that cyclothialidine binds to the B subunit and inhibits the ATPase activity of DNA gyrase(23) . To test whether cyclothialidine also inhibits the ATPase activity of the 43-kDa N-terminal fragment of the DNA gyrase B subunit, we determined its inhibitory activity in steady-state kinetic experiments. Cyclothialidine was included in the ATPase assay, and the results indicate that cyclothialidine is indeed an inhibitor of the ATPase activity of this N-terminal fragment of the B subunit (Fig. 6). The V values decrease with increasing drug concentrations, indicating that cyclothialidine is not a simple competitive inhibitor. The calculated K (mM ATP) and V (nM ADP/µM 43 kDa s) from these data are as follows: 2.1 mM and 30.0 nMM s for no cyclothialidine, 2.1 mM and 26.7 nMM s for 1 µM cyclothialidine, and 2.4 mM and 17.4 nMM s for 5 µM cyclothialidine. The values determined for the ATPase activity of the 43-kDa protein in the presence of novobiocin are K = 0.68 mM and V 18.5 nMM s for no novobiocin and K = 0.51 mM and V = 10.5 nMM s for 6 µM novobiocin(8) . These results are consistent with the results of the displacement of the [C]benzoyl-cyclothialidine binding by the compounds, shown in Fig. 4, B and C, in that the binding of the labeled compound to the 43-kDa N-terminal fragment of the B subunit is hardly inhibited by the ATP analogues ADPNP and ATPS.


Figure 6: ATPase activity of the 43-kDa N-terminal fragment of DNA gyrase B subunit (5 µM) in the presence of cyclothialidine. Reactions were as described under ``Experimental Procedures.'' The K (mM ATP) and V (nM ADP/µM 43 kDa s) are as follows: 2.1 mM and 30.0 nMM s for no cyclothialidine (), 2.1 mM and 26.7 nMM s for 1 µM cyclothialidine (), and 2.4 mM and 17.4 nMM s for 5 µM cyclothialidine ().




DISCUSSION

The characterization of the mode of inhibition of DNA gyrase by cyclothialidine promises to yield important information on the mechanism of DNA supercoiling and especially on the role of the ATPase activity of DNA gyrase during this process, since we have shown that cyclothialidine inhibits the ATPase activity of the B subunit. One possible means of characterizing the mode of inhibition would be to study the binding of [C]benzoylcyclothialidine to the reconstituted E. coli DNA gyrase with the centrifugal filtration method.

Our data presented here (Fig. 2) show that [C]benzoyl-cyclothialidine binds to the B subunit alone and also to the 43-kDa N-terminal fragment of this subunit. Scatchard analysis, carried out with the reconstituted gyrase subunits plus DNA ( Fig. 3and ) showed that the affinity (Kvalue) for the binding of [C]benzoylcyclothialidine to the B subunit depended on the association state of the gyraseDNA complex (increasing affinity: B AB AB DNA). This suggests that the B subunit undergoes conformational changes as it becomes part of the complete gyraseDNA complex. The affinity of cyclothialidine (K = 8.3 ± 0.12 10M) with the binding site on the gyraseDNA complex (ABDNA) was also in good agreement with the inhibition of the DNA-dependent ATPase activity of gyrase (K = 6 10M). Therefore, there is a close correlation of the inhibition of the ATPase activity of gyrase by cyclothialidine with the amount of [C]benzoylcyclothialidine bound to the B subunit in the gyraseDNA complex.

Furthermore, our observations agreed well with the ``model of ATP hydrolysis by DNA gyrase'' as described by Maxell and Gellert (29) in which they say that ATP hydrolysis requires the binding of DNA to two sites on the enzyme and that when both DNA binding sites on the enzyme are occupied by DNA the enzyme is proposed to undergo a conformational change whereby it becomes an active ATPase. It is possible that the conformation of the ATP binding site is changed more dramatically than those for cyclothialidine and for the coumarin antibiotics (Fig. 4).

The mechanism of inhibition of DNA gyrase by cyclothialidine may be almost the same as those by coumarin antibiotics(23) . However, two important differences were observed between cyclothialidine and coumarin antibiotics. 1) Cyclothialidine is much more selective toward DNA gyrase than are coumarin antibiotics(22) . 2) Cyclothialidine is active against a DNA gyrase isolated from a novobiocin-resistant E. coli gyrB mutant strain(23) . Furthermore, our data presented in Fig. 4and showed that the profile of displacement curves of [C]benzoylcyclothialidine binding induced by ADPNP or coumermycin A1 were significantly different from that induced by nonlabeled cyclothialidine. However, from the Scatchard analysis for the competition studies shown in Fig. 5, ADPNP and novobiocin competitively inhibited the binding of [C]benzoylcyclothialidine to the B subunit in the AB form. These results suggest that cyclothialidine, coumarin antibiotics, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ. Maxwell et al. described (8) that the ADPNP inhibition against the ATPase activity of the 43-kDa fragment is different from those of ADP and novobiocin because in the presence of ADPNP the protein behaves as a dimer whereas in the presence of ADP or novobiocin the enzyme is a monomer. The result shown in Fig. 6also might reflect the artifactual nature of the protein fragment, because in the subunit structure of ABDNA cyclothialidine competitively inhibits the ATPase activity of DNA gyrase. It is possible that the differential competition seen with the 43-kDa fragment of the B subunit is due to the effects of cyclothialidine and coumarin antibiotics on the dimerization of the protein fragments. As shown in Fig. 4, the inhibition by nonlabeled cyclothialidine of [C]benzoylcyclothialidine binding to each B subunit form (B, AB, and ABDNA) is essentially the same in each case. These findings are reasonable because the affinities of [C]benzoylcyclothialidine (ligand) and nonlabeled cyclothialidine (inhibitor) change in parallel.

Wigley et al. reported on the crystallization of the 43-kDa N-terminal fragment of the E. coli DNA gyrase B subunit, which comprises positions 2-393 of the intact protein in the presence of ADPNP(10) . They have described the 43-kDa N-terminal fragment containing two distinct subdomains: an N-terminal subdomain (residues 2-220) containing the bound ADPNP and a C-terminal subdomain (residues 221-393), forming the sides of a proposed DNA-binding site. Ali et al.(8) have reported on steady-state ATPase experiments by using the 43-kDa N-terminal fragment of the B subunit; their results are consistent with the hypothesis of a noncompetitive mechanism for the inhibition of the ATPase activity of the B subunit by the coumarin antibiotics novobiocin and coumermycin A1. This indicates that coumarin antibiotics bind close to the ATP binding site of the protein with low affinity for ATP. We also suggest that, similar to coumarin antibiotics, cyclothialidine binds close to the ATP-binding site of the gyrase B subunit and stabilizes a conformation of the protein that is unable to bind ATP. However, our results further suggest that cyclothialidine recognizes a site that is also different from that recognized by the coumarin antibiotics.

To sum up, two models with the currently available data are proposed as follows: 1) ATP, cyclothialidine, and coumarins bind to the same site, but the precise interactions that are important for ATP binding are different from those that are involved in cyclothialidine or coumarin binding. 2) The binding sites for ATP, cyclothialidine, and coumarins are completely separate but interactive and, to some extent, exclusive. Thus occupancy of the cyclothialidine site would prevent binding of ATP (or coumarins) and vice versa. The connectivity between the sites is dependent on the tertiary structure of the protein, and one could easily imagine that it would be influenced by the oligomeric state of the protein. The three-dimensional structure of the 43-kDa N-terminal fragment of the B subunit together with cyclothialidine should provide precise clues about the binding site of cyclothialidine and should determine whether cyclothialidine recognizes the same amino acids as do ATP or the coumarin antibiotics.

Gilbert et al. have reported that the production and properties of a 24-kDa N-terminal fragment of the B subunit (residues 2-220) is shown to contain the coumarin antibiotics-binding site(30) . Lewis et al.(31) have recently reported on their succeeding in the co-crystallization of the 24-kDa N-terminal fragment and novobiocin and of the 24-kDa fragment and GR122222X, an inhibitor that is structurally related to cyclothialidine. Our results are consistent with those findings, meaning that the binding sites of cyclothialidine and novobiocin probably lie within this 24-kDa N-terminal fragment of the B subunit.

However, our results did not allow us to determine whether conformational changes of the binding site of cyclothialidine on the B subunit or in its vicinity occur before cyclothialidine binding or after. By using the electric dichroism method, as previously reported by Rau et al.(32) , we may obtain the necessary information for determining the time point of the structural changes.

  
Table: Competition of substrate analogues and gyrase inhibitors for [C]benzoylcyclothialidine binding



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Nippon Roche Research Center, 200 Kajiwara, Kamakura-City, Kanagawa Pref. 247, Japan. Tel.: 467-47-2214; Fax: 467-47-2247.

The abbreviations used are: ADPNP, 5`-adenylyl---imidodiphosphate; ATPS, adenosine-5`-O-(thiotriphosphate); HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Malcolm Page for critical reading of the manuscript, Dr. Martin Gellert for providing the E. coli strains N4186 and MK47, and Dr. F. Hermann for carrying out the purification of the 43-kDa N-terminal fragment.


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  10. Wigley, D. B., Davies, G. J., Dodson, E. J., Maxwell, A., and Dodson, G.(1991) Nature35, 624-629
  11. Hallett, P., Grimshaw, A. J., Wigley, D. B., and Maxwell, A.(1990) Gene (Amst.) 93, 139-142 [CrossRef][Medline] [Order article via Infotrieve]
  12. Sugino, A., Peebles, C., Kreuzer, K., and Cozzarelli, N. R.(1977) Proc. Natl. Acad. Sci. U. S. A.74, 4767-4771 [Abstract]
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  14. Shen, L. L., Kohlbrenner, W. E., Weigl, D., and Baranowski, J.(1989) J. Biol. Chem.264, 2973-2978 [Abstract/Free Full Text]
  15. Shen, L. L., Baranowski, J., and Pernet, A. G.(1989) Biochemistry28, 3879-3885 [Medline] [Order article via Infotrieve]
  16. Shen, L. L., Mitscher, L. A., Sharma, P. N., O'Donnell, T. J., Chu, D. W. T., Cooper, C. S., Rosen, T., and Pernet, A. G.(1989) Biochemistry28, 3886-3894 [Medline] [Order article via Infotrieve]
  17. Gellert, M., O'Dea, M. H., Itoh, T., and Tomizawa, J.(1976) Proc. Natl. Acad. Sci. U. S. A.73, 4474-4478 [Abstract]
  18. del Castillo, I., Vizán, J. L., Rodrguez-Sáinz, M. C., and Moreno, F.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 8860-8864 [Abstract]
  19. Contreras, A., and Maxwell, A.(1992) Mol. Microbiol.6, 1617-1624 [Medline] [Order article via Infotrieve]
  20. Watanabe, J., Nakada, N., Sawairi, S., Shimada, H., Ohshima, S., Kamiyama, T., and Arisawa, M.(1994) J. Antibiotics47, 32-36 [Medline] [Order article via Infotrieve]
  21. Kamiyama, T., Shimma, N., Ohtsuka, T., Nakayama, N., Itezono, Y., Nakada, N., Watanabe, J., and Yokose, K.(1994) J. Antibiotics47, 37-45 [Medline] [Order article via Infotrieve]
  22. Nakada, N., Shimada, H., Hirata, T., Aoki, Y., Kamiyama, T., Watanabe, J., and Arisawa, M.(1993) Antimicrob. Agents Chemother.37, 2656-2661 [Abstract]
  23. Nakada, N., Gmünder, H., Hirata, T., and Arisawa, M.(1994) Antimicrob. Agents Chemother.38, 1966-1973 [Abstract]
  24. Mizuuchi, K., Mizuuchi, M., O'Dea, M. H., and Gellert, M.(1984) J. Biol. Chem.259, 9199-9201 [Abstract/Free Full Text]
  25. Staudenbauer, W. L., and Orr, E.(1981) Nucleic Acids Res.9, 3589-3603 [Abstract]
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  27. Jackson, A. P., Maxwell, A., and Wigley, D. B.(1991) J. Mol. Biol.217, 15-17 [Medline] [Order article via Infotrieve]
  28. Bates, A. D., and Maxwell, A.(1989) EMBO J.8, 1861-1866 [Abstract]
  29. Maxwell, A., and Gellert, M.(1984) J. Biol. Chem.259, 14472-14480 [Abstract/Free Full Text]
  30. Gilbert, E. J., and Maxwell, A.(1994) Mol. Microbiol.12, 365-373 [Medline] [Order article via Infotrieve]
  31. Lewis, R. J., Singh, O. M. P., Smith, C. V., Maxwell, A., Skarzynski, T., Wonacott, A. J., and Wigley, D. B.(1994) J. Mol. Biol.241, 128-130 [CrossRef][Medline] [Order article via Infotrieve]
  32. Rau, D. C., Gellert, M., Thoma, F., and Maxwell, A.(1987) J. Mol. Biol.193, 555-569 [Medline] [Order article via Infotrieve]

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