(Received for publication, May 4, 1995; and in revised form, October 4, 1995)
From the
Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has
unique pharmacological properties, including sensitivity to the
coumarin drugs novobiocin and coumermycin, which are classical
inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit
gyrase by binding the GyrB subunit and thereby blocking the ATP-binding
site, they inhibit vaccinia topoisomerase by binding to the protein and
blocking the interaction of enzyme with DNA. Noncovalent DNA binding
and single-turnover DNA cleavage by topoisomerase are inhibited with K values of 10-25 µM for coumermycin and 350 µM for novobiocin.
Spectroscopic and fluorescence measurements of drug binding to enzyme
indicate a single binding site on vaccinia topoisomerase for
coumermycin (K
= 27 ± 5
µM) and two classes of binding sites for novobiocin, one
tight site (K
= 20
± 5 µM) and several weak sites (K
= 513 ± 125
µM; n = 4.9 ± 0.7). Addition of a
stoichiometric amount of DNA to a preformed coumermycin-topoisomerase
complex quantitatively displaces the drug, indicating that coumermycin
binding and DNA binding to topoisomerase are mutually exclusive. A
simple interpretation is that the site of drug binding coincides or
overlaps with the DNA-binding site on the topoisomerase. Both
novobiocin and coumermycin alter the susceptibility of vaccinia
topoisomerase to proteolysis with either chymotrypsin or trypsin;
similar effects occur when topoisomerase binds to duplex DNA.
DNA topoisomerases are targeted by a variety of antimicrobial and antineoplastic drugs(1, 2, 3) . In addition to their therapeutic value, these drugs provide indispensable research tools. Insights into topoisomerase mechanism are provided by inhibitors and poisons affecting specific steps in the catalytic cycle. Studies of drug effects in vivo, supported by genetic analyses of drug resistance, underscore the important role played by topoisomerases in virtually all DNA transactions. Drug-target relationships have been firmly established for bacterial DNA gyrase, which is sensitive to the quinolone and coumarin antibiotics(2, 3) ; for eukaryotic type II topoisomerases, which are blocked by epipodophyllotoxins, acridines, and quinolones(4, 5, 6, 7) ; and for eukaryotic topoisomerase I, which is susceptible to camptothecin(8, 9, 10, 11, 12) .
Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, displays
unusual pharmacological properties. The vaccinia enzyme is resistant to
camptothecin(13) , the hallmark topoisomerase I poison, yet it
is sensitive to the coumarin drugs novobiocin and
coumermycin(14, 15) , which are classical inhibitors
of DNA gyrase. Coumarin action on gyrase has been studied in
detail(3) . The coumarins bind to the GyrB subunit and inhibit
its ATPase activity, which is required for enzyme-catalyzed DNA
supercoiling. A 24-kDa subdomain of GyrB is sufficient to bind the
coumarins(16) ; moreover, specific amino acids implicated in
drug binding are defined by drug-resistant gyrase mutants that map
within this subdomain(17) . The molecular basis for coumarin
action emerges from the crystal structure of the GyrB-novobiocin
complex, in which the bound drug impinges on the binding pocket for ATP (18, 19) ()The ATP-dependent eukaryotic
type II topoisomerases are also inhibited by novobiocin and
coumermycin, albeit at much higher drug
concentrations(20, 21, 22, 23) .
Fogelsong and Bauer (14) first documented coumarin inhibition of DNA relaxation by vaccinia topoisomerase using enzyme purified from infectious virions. The reported potency of novobiocin against vaccinia topoisomerase was comparable to its efficacy against eukaryotic topoisomerase II(14, 15, 20, 21, 22, 23) . The identification of the gene encoding vaccinia topoisomerase (24) made it clear that the viral enzyme is structurally homologous to other eukaryotic type I enzymes, with no discernible similarity to DNA gyrase. Because the vaccinia topoisomerase, like other eukaryotic type I enzymes, does not require ATP binding or ATP hydrolysis for DNA relaxation, it is unclear how the coumarins might inhibit the viral protein. We now show that the coumarins target the vaccinia enzyme by a distinctive mechanism in which drug binding to the topoisomerase prevents the binding of the enzyme to DNA.
Figure 2:
Coumarin effects on covalent complex
formation (DNA cleavage). The DNA substrate consisted of an 18-mer
CCCTT-containing oligonucleotide hybridized to a 24-mer
oligonucleotide, as shown. The scissile strand was radiolabeled using
[-
P]ATP and T4 polynucleotide kinase and
then gel-purified and annealed to the unlabeled complementary strand.
Topoisomerase was preincubated with coumermycin or novobiocin for 10
min at room temperature at the concentrations indicated and then mixed
with the radiolabeled DNA as described under ``Experimental
Procedures.'' The extent of covalent adduct formation (expressed
as the percent of the input DNA) is plotted as a function of drug
concentration. (Each data point is the average of three separate
experiments.)
For this analysis, the absorption maximum at 347 nm and the
entire fluorescence emission peak area were used to calculate the
fractional intensities, where I and I are
the absorbance or fluorescence intensities of coumermycin in the
absence and presence of topoisomerase, respectively, and I
is the intensity at saturation.
In , K and K
represent the dissociation
constants, and C
= n
E
and C
= n
E
(that is,
the concentration of tight and weak binding sites, respectively).
Alternatively, the data were fit by Scatchard analysis using a
graphical method that yielded identical results(31) .
To prevent irreversible covalent binding of the
32-mer(27, 32) , we included in the reaction an 8-mer
DNA strand, 5`-ATTCTCGC, which was complementary to the 5`-overhang
generated by release of the 4-mer. Reaction mixtures (0.15 ml) were
prepared containing 10 mM MES, pH 6.3, 8% MeSO, 25
µM topoisomerase, and 25 µM coumermycin. The
mixtures were then supplemented with a 0, 5, 10, 15, 20, 25, or 37.5
µM concentration of the hairpin DNA plus a 25 µM concentration of the 8-mer strand. The samples were incubated for
15 min at 37 °C before performing ultrafiltration as described
above. An aliquot (25 µl) of the filtrate, which contained free
coumermycin (as well as free 4-mer in the samples containing DNA), was
diluted in 0.775 ml of water, and a UV absorption spectrum was taken in
the range 240-370 nm. The absorbance at 310 nm, which was due to
coumermycin and not free DNA, was used to quantitate the release of
drug from the enzyme as the concentration of DNA competitor was
increased.
Figure 1:
Inhibition of DNA relaxation by
novobiocin and coumermycin. Relaxation assays were performed as
described under ``Experimental Procedures.'' In the series to
the left, novobiocin was included at 50, 100, 200, 500, 1000, and 2000
µM final concentrations (proceeding from left to right).
Control reactions were performed without novobiocin (lane
-) or without enzyme (lane C). In the series to the
right, coumermycin A was included at 20, 50, 100, 200, 500,
and 1000 µM final concentrations (proceeding from left to
right). All reactions containing coumermycin included a 10% (v/v) final
concentration of Me
SO. Control reactions contained
topoisomerase and Me
SO, but no coumermycin (second lane
-), or topoisomerase without coumermycin or Me
SO (first lane -).
The drug effect on suicide cleavage was subject to a substantial order-of-addition effect, as shown for coumermycin in Fig. 3. In this set of experiments, preincubation of topoisomerase with coumermycin for 5 min again elicited a concentration-dependent inhibition of cleavage, with 50% inhibition at 25 µM (Fig. 3, closed circles). However, when drug and DNA substrate were premixed and the reactions were initiated by addition of enzyme, the inhibition profile was shifted significantly to the right, with 50% inhibition at 125 µM coumermycin (Fig. 3, open circles). A simple view of the order-of-addition phenomenon is that coumermycin binds to the topoisomerase and dissociates relatively slowly (resulting in higher drug potency by virtue of prebinding). Decreased efficacy of coumermycin when copresented with DNA is consistent with a slower on-rate for drug than for the DNA. (By adding the DNA substrate at various times after exposure of enzyme to 60 µM coumermycin, we determined that the order-of-addition effect was fully established within 30 s of preincubation (data not shown).)
Figure 3: Order-of-drug addition effect on DNA cleavage. Topoisomerase was preincubated for 5 min with coumermycin at the concentrations indicated (closed circles); the cleavage reaction was then initiated by addition of the labeled DNA substrate. Alternatively, coumermycin was added to the reaction mixtures along with the DNA, and the cleavage reaction was initiated by addition of topoisomerase (open circles). The extent of covalent adduct formation (expressed as the percent of the input DNA; average of three separate experiments) is plotted as a function of drug concentration.
Coumarin inhibition of suicide cleavage can be explained by either of the following: (i) the drugs directly inhibit transesterification, or (ii) the drugs block noncovalent binding of topoisomerase to the DNA. We sought to address this issue by circumventing the DNA binding step, i.e. by studying the ability of topoisomerase already bound covalently to the suicide substrate to catalyze religation to an acceptor DNA provided in trans (40, 41). The acceptor strand was a 5`-OH-terminated 12-mer complementary to the 5`-tail of the ``donor'' complex. The religation product was a 24-mer that was resolved electrophoretically from the input 18-mer strand. As noted previously(32, 38, 39) , single-turnover strand transfer was very efficient; 80% of the input substrate was religated to the exogenous acceptor (Fig. 4). Treatment of the covalently bound topoisomerase for 5 min with 2 mM novobiocin or 200 µM coumermycin (concentrations that abrogated the suicide cleavage reaction) prior to the addition of the acceptor strand had no discernible effect on strand religation.
Figure 4:
Coumarin effects on DNA strand transfer
(religation). Covalent complexes were formed in reaction mixtures
containing (per 20 µl) 50 mM Tris-HCl, pH 8.0, 1 pmol of P-labeled suicide substrate, and 2 pmol of topoisomerase.
Aliquots (20 µl) were transferred to individual tubes and adjusted
to either 2 mM novobiocin or 0.2 mM coumermycin as
indicated. Control samples received no drug. The mixtures were
incubated for 10 min at room temperature. Strand transfer was then
induced by addition of 50 pmol of a 5`-OH 12-mer acceptor strand that
was complementary to the 12-nucleotide single-strand tail of the
covalent donor complex. (The structures of the donor complex and the
acceptor strand are shown.) After incubation for 5 min at 37 °C,
the samples were adjusted to 0.2 M NaCl. Formamide was added
to 33% (v/v), and the samples were denatured for 5 min at 95 °C.
Aliquots (7 µl) were analyzed by electrophoresis through a 12%
polyacrylamide gel containing 7 M urea in TBE (90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA).
Religation of the labeled input strand to the acceptor was revealed by
the appearance of a radiolabeled 24-mer strand. The extent of
religation was quantitated by scanning the gel using a FUJIX BAS1000
Bio-Imaging Analyzer and is expressed as the percent of the input
5`-
P-labeled 18-mer oligonucleotide that was converted to
the 24-mer product.
Figure 5: Inhibition of DNA binding. A, topoisomerase was preincubated with coumermycin at the concentrations indicated and then mixed with the radiolabeled DNA ligand (60-mer) as described under ``Experimental Procedures.'' The extent of binding (expressed as the percent of the input DNA shifted to the protein-DNA complex) is plotted as a function of drug concentration (closed circles). (Each data point is the average of three separate experiments.) Alternatively, topoisomerase was incubated for 5 min at 37 °C with radiolabeled 60-mer DNA in the absence of drug and then challenged with coumermycin (5-min incubation at 37 °C) at the indicated concentrations (open circles) prior to native gel electrophoresis. DNA binding (average of two separate experiments) is plotted as a function of coumermycin concentration. B, topoisomerase was preincubated with novobiocin and then mixed with the radiolabeled 60-mer. DNA binding (closed circles; average of two experiments) is plotted as a function of novobiocin concentration. Alternatively, topoisomerase was incubated with the 60-mer DNA in the absence of drug and then challenged with novobiocin (open circles). DNA binding (average of two experiments) is plotted as a function of novobiocin concentration.
Dramatically different effects were
observed when the order of addition was reversed such that
topoisomerase was incubated with the P-labeled 60-mer DNA
prior to addition of drug. Preformed topoisomerase-DNA complexes were
refractory to coumermycin in the range 10-100 µM and
to novobiocin in the range 0.1-1 mM (Fig. 5, A and B). Thus, the topoisomerase, once bound to DNA,
was not induced to dissociate by the drugs. This finding was extended
by order-of-addition competition experiments designed to provoke
dissociation of the prebound protein by challenge with unlabeled DNA. A
control assay established that addition of unlabeled 60-mer DNA to the
binding reaction mixtures prior to addition of enzyme reduced the
extent of DNA binding in accordance with the molar ratio of unlabeled
competitor to labeled ligand (Fig. 6). When competitor was added
after preincubation of topoisomerase with the labeled 60-mer, the
protein-DNA complex was relatively resistant to competition, but could
dissociate at higher ratios of unlabeled competitor to labeled probe (Fig. 6). Addition of 200 µM coumermycin to the
preformed protein-DNA complexes had no apparent effect on enzyme-DNA
dissociation by unlabeled competitor. Thus, coumermycin did not
stabilize or destabilize the enzyme-DNA complex.
Figure 6:
Stability of the binary complex to
competitor DNA is unaffected by coumermycin. Binding of topoisomerase
(1 pmol) to P-labeled CCCTT-containing 60-bp DNA (1 pmol)
was assayed as described under ``Experimental Procedures.''
Unlabeled 60-mer DNA was added as a competitor in the amounts
indicated. The order of addition of competitor relative to enzyme was
varied as follows: open circles, competitor was included in
the reaction mixtures along with labeled 60-mer, and the binding
reaction was initiated by addition of topoisomerase; closed
circles, topoisomerase was incubated for 5 min at 37 °C with
labeled 60-mer in the absence of competitor and then challenged with
unlabeled 60-mer (5-min incubation at 37 °C) prior to native gel
electrophoresis; open squares, topoisomerase was incubated for
5 min at 37 °C with labeled 60-mer, then exposed to 200 µM coumermycin (for 5 min at 37 °C), and finally challenged with
competitor DNA (5-min incubation at 37 °C) prior to native gel
electrophoresis. The extent of binding to labeled 60-mer (percent of
the input ligand shifted to the protein-DNA complex) is plotted as a
function of unlabeled 60-mer added to the reaction. (Each data point is
the average of three separate experiments.)
These experiments make it clear that coumarins inhibit noncovalent binding of topoisomerase to duplex DNA. A simple hypothesis for this inhibition is that the coumarins themselves bind to a site (or sites) on the topoisomerase and that site occupancy by drug and DNA would be mutually exclusive (by steric hindrance at the ligand-binding site). Failure of the coumarins to dissociate the DNA-bound enzyme can be easily accounted for on this basis (i.e. exclusion of drug binding to the relatively stable topoisomerase-DNA complex). This would also account for the failure of the drugs to inhibit strand religation by preformed covalent complexes. Although these experiments do not exclude the existence of a drug ternary complex (topoisomerase-DNA complex with bound coumarin), we detected no effect of coumarins on the stability or the strand transferase activity of the binary protein-DNA complex. Given that reaction chemistry was not affected, at least not at the level of religation, and that coumarin inhibition of binding (predominantly noncovalent) to the 60-mer paralleled the inhibition of suicide cleavage, it is likely that the inhibition of suicide cleavage was caused by inhibition of precleavage binding. Taken together, these data suggest that coumarin interference with noncovalent binding of topoisomerase to DNA is sufficient to account for coumarin inhibition of DNA relaxation. Further experiments to substantiate these points are described below.
Figure 7:
Binding of coumermycin to topoisomerase.
Ultraviolet absorbance spectra (A) and fluorescence emission
spectra ( = 327 nm) (B) of 30
µM coumermycin were determined as a function of
topoisomerase concentration in the range 0-127 µM. C shows a plot of the fractional absorbance (closed
circles) and fluorescence (open circles) intensities of
coumermycin as a function of total topoisomerase (topo)
concentration. For this plot, the absorbance intensities at 347 nm and
the entire fluorescence emission peak areas were used. The line describes the nonlinear least-squares fit of the data to . A double-reciprocal plot of the data is shown in the inset. arb. units, arbitrary
units.
Figure 8:
Displacement of bound coumermycin by DNA.
Increasing concentrations of CCCTT-containing DNA were added to
solutions of topoisomerase (topo) and coumermycin as described
under ``Experimental Procedures.'' After incubation for 15
min at 37 °C, the samples were subjected to ultrafiltration, and
the absorbance of the filtrate at 310 nm was determined. The fractional
absorbance change is plotted as a function of the molar ratio of DNA to
topoisomerase (A
= 0.01). The dashed line shows the expected curve for fractional binding of
one DNA molecule to topoisomerase based on the reported K
value of 50
nM(32) .
Figure 9:
Equilibrium binding of novobiocin to
topoisomerase. A, the fluorescence of topoisomerase (25
µM) was measured as a function of novobiocin (NB)
concentration in the range 0-147 µM. The observed
fluorescence intensities were corrected for optical filtering effects
as described(29) . The fractional fluorescence intensity of
topoisomerase is plotted as a function of total novobiocin
concentration. The line describes the nonlinear least-squares
fit of the data to . The inset shows a
double-reciprocal plot of the data. B, equilibrium
ultrafiltration measurements of novobiocin binding were performed as
described under ``Experimental Procedures'' at 200 µM topoisomerase. Novobiocin concentration was varied in the range
0-1350 µM. MeSO (8%, v/v) was included
in the reactions to prevent aggregation of the drug-topoisomerase
complex. The ratio of bound novobiocin to total topoisomerase (topo) is plotted as a function of free novobiocin. The solid line is the nonlinear least-squares fit of the data to . The dashed lines show the individual curves for
the two classes of binding sites. C, shown is the Scatchard
analysis of the binding data obtained by equilibrium ultrafiltration.
The lines correspond to those shown in B. The
stoichiometries for the tight and weak binding sites are 1 ± 0.1
and 4.9 ± 0.7, respectively.
The affinity for novobiocin determined by the fluorescence method
was higher than expected based on the concentrations inhibitory for DNA
relaxation. Therefore, the binding measurements were extended to higher
concentrations of enzyme and novobiocin to ascertain whether additional
weaker novobiocin-binding sites existed on the enzyme. Because of large
optical filtering effects when high enzyme and ligand concentrations
are used, the fluorescence method is not generally suitable for
quantitation of weak binding sites. However, the method of equilibrium
ultrafiltration is not subject to these problems and was therefore used
to separate free and enzyme-bound novobiocin. This provided an
independent determination of the binding equilibria. In Fig. 9B, the data obtained by the ultrafiltration
method are shown as a plot of bound novobiocin, normalized to the total
concentration of enzyme present, against free novobiocin. The data show
two classes of binding sites. A nonlinear least-squares fit to (Fig. 9B, solid line) reveals a
single tight site (K = 20
± 5 µM; n = 1 ± 0.1) and a
class of weak sites (K
= 513
± 125 µM; n = 4.9 ± 0.7).
For illustration, the dashed lines in Fig. 9B show the individual binding curves for the two classes of sites.
Scatchard analysis of the data gave similar results (Fig. 9C). Both the stoichiometry and affinity for the
tight site are consistent with the values obtained by the fluorescence
method. The K
value for the weak binding sites is
similar to the concentrations of drug that inhibit DNA relaxation and
DNA binding, suggesting that binding of novobiocin to one or more weak
sites is necessary for enzyme inhibition.
Figure 10:
Structure probing by proteolysis with
chymotrypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated
at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures
containing coumermycin included 10% MeSO. Increasing
amounts of chymotrypsin were added (50, 100, 500, and 1000 ng,
proceeding from left to right within each series), and the samples were
digested for 15 min at room temperature. Control reactions were
incubated without chymotrypsin (lanes -). The samples
were then denatured by addition of SDS to 1% and analyzed by
electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. A
Coomassie Blue-stained gel is shown. The positions of 31- and 14-kDa
marker proteins are indicated to the left. The identities of the
predominant chymotryptic fragments are denoted in schematic form to the
left and right. A polypeptide contributed by the chymotrypsin
preparation (Chymo) is indicated by the arrow to
right.
Novobiocin (1 mM) and coumermycin (0.1 mM) both
rendered the topoisomerase resistant to chymotrypsin; a
10-20-fold higher level of chymotrypsin was required to achieve a
comparable extent of digestion of the drug-topoisomerase complex
compared with free topoisomerase. More important, however, was the
marked shift in the distribution of proteolytic fragments. Cleavage by
chymotrypsin in the presence of the coumarin drugs yielded a
polypeptide doublet at 18 kDa. Peptide sequencing after transfer of the
cleavage products to a polyvinylidene difluoride membrane showed that
this doublet consisted of a C-terminal fragment starting at Thr-147 and
a fragment derived from the N terminus of the topoisomerase. (Note that
the fragment from residues 1 to 146 has a predicted molecular mass of
17.4 kDa (Fig. 10).) Cleavage between Tyr-136 and Leu-137 within
the hinge was suppressed strongly, as reflected by the much lower
abundance of the 20- and 16-kDa chymotryptic fragments. (Note that the
drugs themselves display characteristic mobility during SDS-PAGE and
that they weakly take up Coomassie Blue dye. Hence, novobiocin appears
as a diffusely staining band at 14 kDa in Fig. 10, whereas
coumermycin, which structurally resembles a dimer of novobiocin,
appears as a diffuse band at
27 kDa.) A second key finding was
that the actual amount of cleavage by chymotrypsin at Leu-146 in the
presence of the coumarins (reflected by the abundance of the 18-kDa
C-terminal species) was increased compared with the free enzyme. These
changes in proteolysis in the drug-bound state suggest either that
topoisomerase undergoes a conformational change upon ligand binding or
that the coumarins bind directly to the protease-sensitive hinge region
of the protein. We showed previously that identical changes in the
chymotrypsin sensitivity of the topoisomerase are elicited when the
enzyme binds to duplex DNA(34) .
The shift in the protease susceptibility of the topoisomerase depended on the concentration of coumermycin or novobiocin included in the digests (Fig. 11). Acquisition of overall protease resistance (indicated by the amount of intact topoisomerase polypeptide) and the enhancement of cleavage at the secondary chymotryptic site (reflected by increased abundance of the 18-kDa doublet) occurred in parallel between 20 and 60 µM coumermycin (Fig. 11, top panel) and between 0.4 and 0.8 mM novobiocin (bottom panel). The drug concentrations for coumarin-dependent alteration of protease sensitivity correlated well with those for drug binding and for inhibition of DNA binding and DNA relaxation.
Figure 11:
Protection from proteolysis depends on
drug concentration. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, 5 µg of topoisomerase, and various
concentrations of coumermycin (top panel) or novobiocin (bottom panel) were preincubated at room temperature for 10
min. Chymotrypsin (0.5 µg) was added to each mixture. Proteolysis
proceeded for another 15 min at room temperature. The samples were then
denatured and analyzed by SDS-PAGE. The concentration of drug included
in each sample is indicated above the lanes. All reaction mixtures in
the coumermycin experiment (top panel) included 10%
MeSO. The position of the full-length topoisomerase
polypeptide (Topo) is indicated to the right. The identities
of the predominant chymotryptic fragments are denoted in schematic form
to the left and right.
It is worth pointing out that neither ATP nor AMP-PNP had any effect on the chymotrypsin sensitivity of the topoisomerase, or on the distribution of the proteolytic fragments, at nucleotide concentrations as high as 5 mM (data not shown). This is relevant because the coumarin drugs block gyrase-catalyzed ATP hydrolysis, apparently by obstructing the ATP-binding site(3) . Purified recombinant vaccinia topoisomerase has no associated ATPase(35) . Although nucleoside triphosphates at 5 mM can stimulate DNA relaxation by the vaccinia topoisomerase, this effect is mediated by the pyrophosphate moiety, not by the nucleoside(35) . Thus, there is no indication that coumarin inhibition of vaccinia topoisomerase is related mechanistically to coumarin action on DNA gyrase.
Figure 12:
Structure probing by proteolysis with
trypsin. Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 8.0, and 5 µg of topoisomerase were preincubated
at room temperature for 10 min without drug or with 1 mM novobiocin or 0.1 mM coumermycin. All reaction mixtures
containing coumermycin included 10% MeSO. Increasing
amounts of trypsin were added (1, 5, 10, 50, and 100 ng, proceeding
from left to right within each series), and the samples were digested
for 15 min at room temperature. Control reactions were incubated
without trypsin (lanes -). The samples were then
denatured and analyzed by SDS-PAGE. A Coomassie Blue-stained gel is
shown. The positions of 31- and 14-kDa marker proteins are indicated to
the left. The identities of the predominant tryptic fragments are
denoted in schematic form to the left.
The results presented above indicate that the coumarin drugs novobiocin and coumermycin inhibit the vaccinia type I topoisomerase via a distinctive mechanism. Whereas coumarins inhibit DNA gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site on GyrB, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Coumermycin binding and DNA binding to the enzyme are mutually exclusive, as judged by the ability of added DNA to displace coumermycin from a preformed drug-protein complex. The simplest interpretation of the data is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. These findings illuminate new properties of the coumarins and provide additional insights into the ligand binding properties of the vaccinia type I enzyme.
An intriguing finding is that the binding of the drugs to the topoisomerase results in protection of the interdomain bridge and hinge regions from proteolysis, the same effects observed upon binding of the enzyme to duplex DNA. Models to account for the effects of drug binding on proteolysis follow naturally from the two simple cases discussed previously for DNA binding(34) , i.e. (i) that protection by ligand from proteolysis stems from direct binding of ligand to the protected region of the enzyme or (ii) that ligand binding induces a conformational change in the topoisomerase that affects the hinge and bridge. In the second case, no assumptions are made about the location of the ligand-binding site. The bridge and hinge are separated in the linear protein sequence, but their proximity in three dimensions is not known. Although duplex DNA constitutes a relatively large ligand (the ``minimal'' substrate for covalent complex formation by vaccinia topoisomerase is an 11-bp duplex, whereas stable noncovalent binding requires a 20-bp DNA duplex(36, 37) ) compared with coumermycin (1110 kDa), it is conceivable that the binding of a single coumermycin molecule at or near the DNA site could elicit the same protective effects if the bridge and hinge are relatively close to each other in the native protein.
The structures of novobiocin and coumermycin bear little resemblance to DNA; therefore, it seems unlikely that they would make the same spectrum of contacts with the enzyme that are made by duplex DNA. A more plausible explanation for hindrance of DNA binding by coumarins is that the drugs bind to the enzyme and overlap with the DNA-binding site. This is similar to the gyrase case, where novobiocin does not bind in the ATP site, but binds adjacent to it and sterically hinders ATP binding. The binding data indicate that occupancy by one coumermycin molecule at a high affinity site is sufficient to account for inhibition of vaccinia topoisomerase, whereas novobiocin inhibition entails binding to one or more weak sites. Coumermycin, which resembles a dimer of novobiocin, may therefore extend from its tight binding site into the DNA-binding site. The present data do not address whether coumermycin and novobiocin bind to the same high affinity site on the topoisomerase (with novobiocin also binding to additional weak sites). Invoking a common site is nonetheless in keeping with the similar chemical structures and the similar dissociation constants for a tight binding site.
The increased susceptibility of the coumarin-topoisomerase complex to proteolysis at sites outside the bridge and hinge segments is consistent with a ligand-induced conformational change. The increased cleavage by chymotrypsin at Leu-146 in the presence of coumermycin or novobiocin compared with free enzyme is precisely what was observed for the topoisomerase-DNA complex(34) . The induction of multiple novel tryptic cleavage sites upon coumarin binding was more dramatic than the (primarily protective) shifts in the tryptic pattern seen with the protein-DNA complex(34) . To the extent that competitive inhibition of DNA binding by coumermycin likely involves an overlapping ligand-binding site, we view the exposure of protease-sensitive sites upon drug binding as additional indirect evidence for a conformational change upon binding of topoisomerase to DNA. Again, this does not rule out direct contact between DNA or drugs and the hinge or bridge regions.
Our understanding of the interaction of vaccinia topoisomerase with DNA would be enhanced immeasurably by a crystal structure of the enzyme, preferably in the DNA-bound state. The crystal structure of the 9-kDa N-terminal tryptic fragment of the enzyme (33) is not informative in this regard because this fragment does not bind DNA by itself. Efforts to crystallize the full sized protein alone or with DNA have not yet been successful. The binary complex of topoisomerase and coumermycin, with drug bound at or near the DNA-binding site, offers an alternative target for crystallization.