INTRODUCTION

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DNA gyrase is a type II topoisomerase responsible for the manipulation of the topological state of DNA in bacteria (reviewed in Ref. 1). Catalysis by type II topoisomerases requires the hydrolysis of ATP and involves the passage of a segment of DNA through a double-stranded break in another segment held open by the enzyme. Gyrase is distinct in its catalytic mechanism from the other enzymes of its class in that conventional type II enzymes (such as yeast topoisomerase II) cannot discriminate between DNA segments to be transported, thus favoring DNA relaxation or intermolecular strand passage (catenation/decatenation reactions). By contrast, gyrase is able to dictate the direction of strand passage and, by so doing, is the only topoisomerase able to perform DNA supercoiling.

Gyrase consists of two proteins which combine to form an A₂B₂ complex that binds approximately 128 bp¹ of DNA (2) in a positive superhelical sense around its core (3). When the A protein (GyrA, 97 kDa) is treated with either trypsin or chymotrypsin two large fragments are generated with approximate masses of 64 and 33 kDa (4). The C-terminal 33-kDa fragment, comprising the residues from 572 to 875, has been shown to bind DNA in positive superhelical sense but is unable to catalyze any of the reactions of gyrase (5). The N-terminal fragment contains the active site tyrosine and fragments in the size range 58-64 kDa have been shown to possess catalytic activity (6). When complexed with the B protein (GyrB), the N-terminal domain is able to perform ATP-dependent relaxation and increased DNA decatenation compared with the full-length enzyme, thus behaving like a conventional type II topoisomerase (7). Therefore, the positive wrapping of DNA caused by the 33-kDa domains appears to be largely responsible for the unique properties of gyrase.

GyrB (90 kDa) also consists of two domains: a 43-kDa N-terminal and a 47-kDa C-terminal domain (1). In the presence of GyrA, the 47-kDa domain cannot supercoil DNA but is able to perform all the ATP-independent reactions of gyrase (8). The 43-kDa N-terminal domain has been produced as a separate gene product and was found to hydrolyze ATP (9). This domain has been crystallized in the presence of a non-hydrolyzable ATP analog, ADPNP, and the structure has been solved at 2.5-Å resolution (10). As suggested by the crystal structure as well as by biochemical data (9, 11), the protein forms a dimer in the presence of ATP. This dimerization appears to be a key step in the mechanism of supercoiling. Briefly, gyrase binds DNA and wraps it in an interaction mainly supported by the 33-kDa domains of GyrA. This positions the transported segment in the right orientation for strand passage. Binding of ATP causes dimerization of the B proteins which captures the transport segment and directs it through the double-stranded break made by the enzyme on the gate segment. ATP is then hydrolyzed allowing the enzyme-DNA complex to return to its starting conformation.

Gyrase is inhibited by a number of antibacterial agents including the quinolones (12). Addition of a protein denaturant, e.g. sodium dodecyl sulfate (SDS), to a quinolone-arrested gyrase-DNA complex results in DNA cleavage (13, 14). Cleavage does not require ATP, but ATP or ADPNP can change the efficiency of cleavage and alter the pattern of preference between cleavage sites (15, 16). Efficient DNA cleavage by gyrase can also occur in the absence of quinolones if Mg²⁺ is substituted by Ca²⁺ (4).

The application of limited proteolysis can often provide useful information about the conformational changes resulting from the interaction of a protein with a substrate or effector molecule. Structured regions of a protein are usually very resistant to attack by low concentrations of endopeptidases while cleavage of the peptide backbone can occur in less structured regions or in loops linking structural domains of the protein. Using this approach it is often possible to separate functional domains of a protein and investigate their activity, e.g. Refs. 4, 17-19. The susceptibility of a region of a protein to cleavage can be altered when a conformational change occurs, resulting in cleavage sites being protected or new sites being revealed. Comparing the proteolytic pattern of a protein in the presence or the absence of a ligand it is possible to investigate conformational changes associated with this interaction (20, 21). We have used this approach to study the interaction of DNA gyrase with quinolones and ADPNP, and we have identified characteristic proteolytic signatures for these complexes.

	EXPERIMENTAL PROCEDURES

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Enzymes and DNA-- GyrA, GyrB, GyrA^Ser-122, GyrA^Trp-83 (gift of C. Willmott), the 59-kDa N-terminal domain of GyrA (GyrA59, gift of C. Smith), and the 64-kDa N-terminal domain of the same protein (gift of S. Critchlow) were purified as described previously (6, 22). Negatively supercoiled and relaxed forms of plasmid pBR322 were provided by A. J. Howells (University of Leicester). Linear pBR322 was prepared by digestion of the supercoiled form with EcoRI. A 147-bp fragment containing the major gyrase cleavage site of plasmid pBR322 (23) was prepared by the polymerase chain reaction using pBR322 as a template.

Limited Proteolysis-- Samples contained 0.3 mg/ml GyrA and/or GyrB, 0.4 mg/ml linear pBR322 DNA (where indicated) and various effector molecules (as described in the figure legends) in 50 mM Tris·HCl (pH 7.5), 50 mM KCl, 4 mM MgCl₂, 4 mM dithiothreitol, 6.5% (w/v) glycerol. After incubation for at least 1 h at 25 °C, protease was added and the reaction was allowed to proceed at 37 °C. Samples were taken at various times and quenched by adding an equal volume of 62 mM Tris·HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 5% (w/v) -mercaptoethanol, 0.001% (w/v) bromphenol blue, and boiling for up to 15 min. The products were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).

Enzyme Assays-- Quinolone- or Ca²⁺ induced cleavage assays were performed as described previously (4). ATPase rates were determined by a linked enzyme assay (9, 24) in the presence of excess GyrA or GyrA59, and linear pBR322 DNA. [³H]CFX (Amersham) and [³²P]ADPNP (ICN) binding assays were performed in 50 mM Tris·HCl (pH 7.5), 50 mM KCl, 4 mM MgCl₂, 4 mM dithiothreitol, 6.5% (w/v) glycerol at 25 °C and enzyme-bound and free ligand molecules were separated by gel filtration through a spin-column (Nick spin-columns, Pharmacia) (11, 25).

Secondary Structure Prediction-- Secondary structure and solvent accessibility predictions were performed using the programs PHDsec and PHDacc (B. Rost, EMBL, Heidelberg, Germany (26, 27)).

	RESULTS

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The Gyrase-DNA Complex (Complex I)-- To study the conformational changes associated with the interaction of DNA gyrase with a number of effector molecules a selection of endopeptidases was used: trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, giving a wide range of sequence-specific cleavage sites. Trypsin is a serine protease that cleaves at the C-terminal side of arginine and lysine residues. Chymotrypsin cleaves proteins at the C-terminal side of tyrosine, phenylalanine, and tryptophan, while V8 cleaves C-terminal to glutamic acid residues.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   The ADPNP-induced conformational change. GyrA, GyrB, A2B2, or the gyrase-DNA complex were incubated for 1 h at 25 °C, with 2 mM ADPNP added to the samples where indicated. The samples were then treated with 10 µg/ml trypsin for 1 h at 37 °C and the products analyzed by SDS-PAGE. On the right is a diagrammatic representation of the gyrase fragment corresponding to each band. The shaded part represents the position of the protected fragment in the respective subunit. The letter denotes the subunit (A or B) and the number indicates the approximate size of the fragment in kDa.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   The quinolone-induced conformational change. A, GyrA, GyrB, and gyrase with or without DNA were incubated at 25 °C for 2 h with 100 µM CFX added to the samples indicated. The samples were then treated with 10 µg/ml trypsin for 1 h at 37 °C and the products analyzed by SDS-PAGE. Explanation for the diagram on the right is given in Fig. 1. B, the effect of quinolones on an active-site mutant. Wild-type gyrase and gyrase reconstituted with GyrASer-122 were treated as above in the presence and absence of CFX (100 µM).

Dimerization of GyrB Is Indicated by Protection of the 43-kDa Domains (Complex II)-- An important step in the catalytic cycle of gyrase is the dimerization of the B subunits in the presence of ATP (9, 11). When the non-hydrolyzable ATP analog ADPNP is used the two B subunits are locked in the dimeric form (10). When GyrB or the A₂B₂ complex were incubated with ADPNP prior to treatment with 10 µg/ml trypsin, a protection of the N-terminal 43-kDa domain of GyrB was observed (Fig. 1). With time the 43-kDa fragment was slowly converted to a smaller 33-kDa product. This fragment was identified by peptide sequencing as the N-terminal part of the 43-kDa domain (N-terminal sequencing revealed the sequence SNSYDSSSIK for both fragments). When chymotrypsin was used instead of trypsin, the 43-kDa domain was very stable to further digestion (data not shown). There was no observable difference in the ADPNP-induced fingerprint between the gyrase-DNA complex and the DNA-free enzyme (Fig. 1). Earlier experiments with the 43-kDa domain alone have shown that in the presence of ADPNP the protein is cleaved by trypsin at lysine 307 to give an N-terminal 33-kDa fragment which is protected from further digestion (11). It appears that residue 307 is not readily accessible by proteases when the full B protein is present since digestion of the 43-kDa domain to give a 33-kDa fragment occurs only in later time points or higher concentrations of protease.

Quinolone Binding Induces a Conformational Change Which Protects the 47-kDa Domain of GyrB (Complex III)-- When the enzyme-DNA complex was incubated with 100 µM ciprofloxacin (CFX) prior to treatment with protease, one more fragment was produced, with an approximate size of 47 kDa (Fig. 2A). Peptide sequencing identified this fragment as the C-terminal 47-kDa domain of the B protein starting at residue 395 (KGALD). It is interesting to note that the quinolone-resistant mutations associated with the B subunit map to the same domain of the protein (29). When gyrase was incubated with CFX in the absence of DNA no protection of the 47-kDa domain was observed (Fig. 2A), consistent with the fact that very little binding of quinolones to gyrase is observed in the absence of DNA (30-32).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   The effect of a quinolone-resistant mutation on the quinolone characteristic fingerprint. Wild-type gyrase (panel A) and gyrase reconstituted with GyrATrp-83 (panel B) were incubated with the indicated concentrations of CFX for 2 h at 25 °C and then treated with 10 µg/ml trypsin for 1 h at 37 °C. The gyrase-DNA complex was formed with 0.3 mg/ml of each subunit and 0.4 mg/ml linear pBR322. The unproteolyzed A2B2 complex is shown in the leftmost lane of panel A.

Calcium-induced Cleavage-- When gyrase and DNA are incubated with calcium instead of magnesium ions in the absence of quinolones and the reaction is stopped by the addition of SDS and proteinase K, the DNA is cleaved (4). We have tried to distinguish between calcium- and quinolone-induced cleavage using limited proteolysis. DNA and gyrase were incubated for 2 h under conditions that give calcium cleavage (4 mM Ca²⁺), and then the mixture was treated with 10 µg/ml trypsin at 37 °C. Before the addition of protease, aliquots were removed from the proteolysis reactions and treated with SDS and proteinase K to reveal calcium-induced DNA cleavage (data not shown). Although under these conditions the level of Ca²⁺-induced cleavage was comparable to that caused by quinolones, we have been unable to detect any protection of the 47-kDa domain in the presence of either linear or supercoiled DNA (Fig. 4). This suggests that the quinolone-gyrase-DNA complex (complex III) has a different conformation than the calcium-enzyme-DNA complex, in support of the above results suggesting that complex III fingerprint does not reflect a cleaved DNA complex but a quinolone-associated conformational change.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Probing the gyrase-Ca2+-DNA complex with proteolysis. The complex between full-length gyrase (A2B2) and DNA was formed in the presence of Mg2+ or Ca2+. After 30 min at 25 °C, 200 µM CFX was added to the samples indicated and the incubation was continued for 1 h. An aliquot was then removed from each sample and treated with 0.1% SDS and 0.1 mg/ml proteinase K for 30 min at 37 °C to reveal DNA cleavage (data not shown) while the rest of the sample was treated with 10 µg/ml trypsin for 1 h at 37 °C and the samples analyzed by SDS-PAGE.

Quinolones Do Not Prevent Dimerization of the B Subunits (Complex IV)-- Formation of the enzyme-DNA-quinolone complex (complex III) and incubation with ADPNP prior to protease treatment reveals protection of the 43-kDa but not the 47-kDa domains (Fig. 5). Similarly, preincubation of the gyrase-DNA complex with ADPNP followed by addition of quinolones results in the same tryptic fingerprint. The ability of gyrase to bind quinolones in the presence of ADPNP was tested using rapid-gel filtration in the presence of a 147-bp DNA fragment based around the preferred 990 pBR322 cleavage site (23). The gyrase-DNA-CFX complex was compared with the complex formed when ADPNP was incubated with the enzyme after incubation with CFX and it was found that both of these complexes bound the same amount of CFX (Fig. 6A). The stoichiometry of binding was close to two quinolone molecules per enzyme complex, as observed previously (28). When the gyrase-DNA complex was incubated with ADPNP prior to addition of the quinolone, binding of the drug was slightly reduced (Fig. 6A). When ADPNP was added to the gyrase tetramer in the absence of DNA, to induce closure of the ATP-operated clamp, and DNA and CFX were then added to the complex, it was found that gyrase could still bind the drug, albeit with low efficiency (Fig. 6A).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Quinolones do not prevent B subunit dimerization induced by ADPNP. The complex between gyrase and DNA was formed as described under "Experimental Procedures" and 200 µM CFX or 2 mM ADPNP was added to the samples indicated. The reactions were incubated for 1 h at 25 °C and then further ADPNP or CFX was added to the samples as indicated. The incubation was then continued for 1 h at 25 °C for all samples. Trypsin was then added at 10 µg/ml and after 1 h at 37 °C the reactions were stopped and analyzed by SDS-PAGE.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Binding of CFX and ADPNP to the gyrase-DNA complex. A, CFX binding. The reactions were carried out in the presence of 5 pmol of 147-bp fragment, 15 pmol of A2B2, 2 mM ADPNP, and 25 µM [3H]CFX at 25 °C, but the order of addition of the ligands or DNA was varied as indicated. The gyrase-DNA complex was incubated with CFX alone for 2 h (sample labeled CFX) or, 1 h with CFX prior to addition of ADPNP and further incubation for 1 h (sample labeled CFX before ADPNP) or, 1 h with ADPNP prior to addition of CFX and a further 1-h incubation (sample labeled ADPNP before CFX). Another sample contained gyrase that had been incubated for 1 h with ADPNP without DNA and then DNA and CFX was added and incubated for 1 h further. B, ADPNP binding. Reactions were carried out at the same concentrations of enzyme and DNA as above with 200 µM CFX and 2 mM [32P]ADPNP. The binding to gyrase and the gyrase-DNA complex was measured after a 2-h incubation with the nucleotide at 25 °C. The gyrase-DNA-CFX complex was allowed to form at 25 °C for 1 h and then radiolabeled nucleotide was added and the reaction was incubated for 1 more hour. Similarly, the gyrase-DNA-ADPNP complex was formed first and then CFX was added. Both incubations were at 25 °C for 1 h.

Gyrase Can Hydrolyze ATP in the Presence of Quinolones-- Since the gyrase-DNA-quinolone complex has not lost its ability to close the N-terminal clamp in the presence of ADPNP, it is likely that it can still do ATP hydrolysis. Indeed, we found that gyrase that has been preincubated with quinolones and DNA is still able to hydrolyze ATP albeit at a lower rate than that of the drug-free enzyme-DNA complex (Fig. 7). This could be due to either the gyrase-quinolone complex having different kinetic characteristics of ATPase, or the quinolones completely abolishing ATP hydrolysis but trapping only a fraction of the enzyme molecules. This second possibility was explored using the truncated form of the GyrA protein. A59₂B₂ (a version of gyrase deleted for the 33-kDa domains) does not exhibit DNA-stimulated ATPase, probably due to its weaker binding to DNA, but can perform quinolone-induced cleavage almost as efficiently as the full-length enzyme (6, 7). When the A59₂B₂-DNA-quinolone complex is formed it can turn over ATP at a rate similar to that of the full-length enzyme in the presence of the drugs (Fig. 7). These results indicate that the quinolone-arrested gyrase-DNA complex has adopted a conformation which still enables it to hydrolyze ATP but the kinetic characteristics of this reaction are different from those of the reaction of the drug-free enzyme.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   The gyrase-quinolone complex can hydrolyze ATP. ATPase reactions contained 1.5-fold excess of GyrA or GyrA59 over GyrB and 800 bp of linear pBR322 per enzyme heterotetramer. The concentration of enzyme on the abscissa is determined by the limiting concentration of GyrB. The assay was performed at 2 mM ATP and 200 µM CFX was added where indicated. The complexes were allowed to form for 2 h at 25 °C prior to the addition of ATP and the rates were measured at 25 °C. The turnover numbers of the various complexes were measured to be, 0.81 s1 for the A2B2-DNA complex, 0.51 s1 for the A2B2-DNA-CFX complex, 0.13 s1 for the A592B2-DNA complex, and 0.35 s1 for the A592B2-DNA-CFX complex.

	DISCUSSION

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Limited proteolysis has previously been used in gyrase for the identification of functional domains in GyrA (4). Two domains were identified and their properties are now well established (5, 7, 34). In vivo GyrB is found to be proteolyzed into two fragments that have been shown to be different functional domains (1). When applied to eukaryotic topoisomerase II, limited proteolysis revealed a conformational change associated with the binding of ADPNP (20, 21). This result was considered as evidence of the dimerization of the N-terminal domains of topoisomerase II in the presence of ATP. The mechanism of supercoiling by type II topoisomerases involves the translocation of a DNA segment through an enzyme-stabilized DNA gate. This is accomplished by passing the DNA through the enzyme itself (35, 36). This mechanism requires the enzymes to undergo significant conformational changes. Therefore, we decided to use limited proteolysis to identify conformational states that are involved in the catalytic cycle of gyrase as well as proteolytic fingerprints for inhibitor-trapped enzyme conformations.

Complex I: The Enzyme-DNA Complex-- The first step was to establish the proteolytic signatures of the individual subunits and the holoenzyme. Treatment of GyrA with trypsin yielded only one product fragment. Due to the much higher protease concentrations used in these experiments than used previously (4), the 33-kDa domain of GyrA appeared only in earlier times of the reaction and was quickly degraded. Presumably due to the same reason, the N-terminal tryptic site was at lysine 17 rather than arginine 6, as found previously (4). The size of the product fragment was estimated to be ~62 kDa, supposing that the C-terminal tryptic site is at arginine 571, as suggested before (4). Nevertheless, it is possible that due to the higher concentration used, trypsin was able to cleave at the stretch of arginine and lysine residues at the region 561-567. This would have resulted in a fragment with size a of 61 kDa. Indeed, in some cases the band corresponding to this fragment appeared as a doublet in SDS-PAGE, possibly representing a mixture of the 62- and 61-kDa fragments (Figs. 1-5). It seems unlikely that cleavage could have occurred at any other Arg or Lys residue because this would have resulted in a significant reduction in the size of this fragment which would have been evident by SDS-PAGE.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Summary of the proteolytic signatures and their relationship to the gyrase catalytic cycle. On the left, a diagrammatic representation is given of the various fragments produced by the tryptic treatment of gyrase in the presence of ligands. On the right, a summary is shown of the four conformational states identified in these experiments together with a representation of their proteolytic signatures. The relationship of these complexes with the gyrase catalytic pathway or the non-productive cycle undergone by the enzyme in the presence of the drugs is also demonstrated.

Complex II: A Nucleotide-dependent Conformational Change-- An essential subsequent step in the mechanism of supercoiling involves the closure of the ATP-operated clamp and the trapping of the transported segment inside the protein tetramer. This is effected by the dimerization of the B subunits caused by ATP binding. The DNA gate then opens and the DNA segment is transported through the double-stranded break. ATP hydrolysis is not required for this translocation since stoichiometric supercoiling can be observed in the presence of the non-hydrolyzable ATP analog ADPNP (16, 38). ADPNP binding is very tight and results in the trapping of the protein clamp in the dimerized form (10, 11, 25). This conformation of the enzyme, which is a major intermediate in the catalytic cycle, we term complex II (Fig. 8). ATP hydrolysis is then required to allow the enzyme to return to its native conformation (complex I). The proteolytic fingerprint of complex II is consistent with that seen before with only the 43-kDa domains of GyrB (11). Indeed, dimerization of the 43-kDa proteins gave a resistant 33-kDa fragment which is also present in the case of the holoenzyme (A₂B₂). However, in the case of the holoenzyme the whole 43-kDa domain is protected as a result of the dimerization, and the 33-kDa fragment is produced at later times. Presumably the presence of the rest of GyrB restricts access to the tryptic site at residue 307. There was no significant difference in the fingerprint of the gyrase-DNA-ADPNP and the DNA-free gyrase-ADPNP complexes. This is in agreement with previous observations suggesting that DNA is not necessary for the dimerization of the B subunits (9) and that the presence of DNA does not affect the rate or the stoichiometry of ADPNP binding to the gyrase holoenzyme (25). DNA cleavage is not required for the dimerization of the B subunits since the active-site mutant A₂^Ser-122B₂, that is unable to cleave DNA, can still reveal the complex II characteristic fingerprint.

Complex III: A Quinolone-induced Conformational Change-- Trapping of the gyrase-DNA complex by quinolones gives rise to a characteristic proteolytic fingerprint. This could be due to either a conformational change or a direct protection of a proteolytic cleavage site by the drug. The latter seems unlikely since all three proteases used in this study were able to reveal this characteristic protection. Being that a total of six different amino acids could be targeted by all three enzymes it seems quite unlikely that a molecule the size of a quinolone (~360 Da) could have such a direct effect. Moreover, this protection was observed with more than one member of this class of compounds (CFX and oxolinic acid). The quinolone-protected fragment seems to comprise the whole C-terminal domain of GyrB since it runs next to purified 47-kDa domain on SDS-PAGE (data not shown). Nevertheless, it is still possible that cleavage could have occurred close to the C terminus, after lysine 798, releasing a 650-Da fragment. A difference of that size would be quite difficult to determine by SDS-PAGE. This characteristic protection of the 47-kDa domain does not appear to be complete. This is evident both by the lower relative intensity of the 47-kDa band and the high proportion of 25-kDa fragment still present (Fig. 2, A and B). Since the 25-kDa fragment derives from the 47-kDa domain, protection of this domain would result in decreased intensity of the 25-kDa band. Although significant reduction in the intensity of this band is observed (Fig. 2B), this never completely disappears. It is possible that a proportion of inactive protein exists in the GyrB preparation and does not participate in the formation of the gyrase-DNA-quinolone complex.

Complex IV: A Strand Passage Incapable Intermediate-- Quinolone binding does not prevent dimerization of the ATP-operated clamp. When ADPNP is used to trap the gate in the closed form, a fingerprint similar to that of complex II is revealed. The lack of the expected protection of the 47-kDa domain is not due to any weakening of quinolone binding, as manifested by the ability of gyrase to bind the drug in the presence of the nucleotide (Fig. 6A). The small reduction in the level of quinolone binding observed when the complex was preincubated with ADPNP cannot account for the complete disappearance of the protection of the 47-kDa domain. This reduction in binding can be attributed to the trapping of DNA-free gyrase molecules by ADPNP. Such molecules would not be able to bind DNA efficiently, thus reducing the overall number of molecules able to bind quinolones. Indeed, closing of the clamp by ADPNP prior to the addition of DNA to the complex resulted in significant reduction in the level of quinolone binding (Fig. 6A). Similar results were obtained when quinolone-induced DNA cleavage was studied. Cleavage was not affected by addition of ADPNP while little reduction in cleavage efficiency was observed when ADPNP was added to the gyrase-DNA complex before the drug. Closing of the ATP-operated clamp does not abolish binding and cleavage of a linear DNA fragment, although the efficiency of this process is low. Such a result is consistent with the finding of Roca and Wang (39) that topoisomerase II could bind and cleave linear DNA even when preincubated with ADPNP. It has been proposed that in this case the DNA threads through a hole in the enzyme and finds its way to the binding site surrounding the active-site tyrosines (39).

Mechanistic Implications-- The blocking of supercoiling by quinolones contrasts with their ability to induce DNA cleavage. It has been proposed that in mammalian type II topoisomerases, compounds related to quinolones act by inhibiting DNA religation (41), but this does not seem to be the case with quinolones and DNA gyrase. Quinolone-directed DNA cleavage is sequence-specific in that under certain conditions full cleavage occurs only at certain sites while cleavage at other locations takes place with less efficiency (15, 16, 42). It appears that there would be complexes formed between gyrase and certain DNA sites in which the cleavage religation equilibrium in the presence of the quinolones is shifted to the ligated form. If the above proposal was correct, there would be no reason that these complexes could not perform strand passage. The drugs have to fulfill the dual role of blocking strand passage from all these complexes but allowing the DNA to be in the cleaved form in some of them. We believe that the mechanism of inhibition of gyrase by quinolones has its basis on the conformational change seen here. Quinolones could block supercoiling by stabilizing a conformation of the enzyme where, irrespective of whether the DNA is cleaved or not, the DNA gate cannot open sufficiently to allow strand passage. We believe that complexes III and IV reflect such a conformation. It is possible that in the presence of quinolones the 47-kDa domains of GyrB come close together, stabilizing the closed DNA gate conformation and blocking strand passage. Such a conformational change could result in the proteolytic protection of these domains. The fact that DNA cleavage is not required for quinolones to induce this conformational change explains how quinolones block supercoiling from all gyrase-DNA complexes, irrespective of the state of DNA cleavage at those sites.

Conclusions-- We have been able to identify a number of proteolytic signatures of DNA gyrase. These fingerprints have been interpreted in terms of four different complexes each representing a different conformation of the enzyme. Two of these complexes are part of the normal catalytic cycle of gyrase while the other two are part of an non-productive cycle undergone by the enzyme in the presence of quinolone drugs (Fig. 8). Quinolone binding stabilizes a conformational state of the enzyme that is responsible for the inhibition of its activity. DNA cleavage is not required for the binding of the drugs or the induction of this conformational change. We suggest that inhibition of supercoiling by quinolones is the consequence of a drug-induced conformational change involving the 47-kDa domains of GyrB and that DNA cleavage is a subsequent slower step. Further evidence for this proposal is presented in the accompanying paper (40).