From the Department of Microbiology and Molecular Genetics,
University of Texas Medical School, Houston, Texas 77030
To gain further insight into the structural
relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell
division protein, we tested the effect of tubulin assembly inhibitors
on FtsZ assembly. Common tubulin inhibitors, such as colchicine,
colcemid, benomyl, and vinblastine, had no effect on
Ca2+-promoted GTP-dependent assembly of
FtsZ into polymers. However, the hydrophobic probe
5,5'-bis-(8-anilino-1-naphthalenesulfonate) (bis-ANS) inhibited FtsZ
assembly. The potential mechanisms for inhibition are discussed.
Titrations of FtsZ with bis-ANS indicated that FtsZ has one high
affinity binding site and multiple low affinity binding sites. ANS
(8-anilino-1-naphthalenesulfonate), a hydrophobic probe similar to
bis-ANS, had no inhibitory effect on FtsZ assembly. Because tubulin
assembly has also been shown to be inhibited by bis-ANS but not by ANS,
it supports the idea that FtsZ and tubulin share similar conformational
properties. Ca2+, which promotes GTP-dependent
FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesting
that Ca2+ binding induces changes in the hydrophobic
conformation of the protein. Interestingly, depletion of bound
Ca2+ with EGTA further enhanced bis-ANS fluorescence. These
findings suggest that both binding and dissociation of Ca2+
are capable of inducing FtsZ conformational changes, and these changes
could promote the GTP-dependent assembly of FtsZ.
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INTRODUCTION |
The key bacterial cell division protein FtsZ is widespread among
all prokaryotes. It is essential for cell division and assembles into a
ring-like structure at the site of cytokinesis (1-8). Despite its
actin-like behavior in the cell, FtsZ is biochemically quite similar to
tubulin. FtsZ proteins share limited primary sequence homology with
tubulin, but more importantly, they can bind and hydrolyze GTP and
assemble into polymers, such as sheets of tubulin-like protofilaments
and minirings as determined by electron microscopic analysis (9-13).
FtsZ polymers have not yet been visualized in vivo, making
it impossible to verify the physiological relevance of such structures.
Secondary structural prediction suggests that FtsZ and tubulin share
70-80% similarity, with the exception of their short carboxyl termini
(14). Therefore, the overall three-dimensional structures of FtsZ
monomers and tubulin are likely to be very similar.
Because many inhibitors of tubulin assembly exist, investigating the
effects of tubulin inhibitors on FtsZ assembly should provide important
information for FtsZ structure and function. Tubulin inhibitors have
been classified into four categories: (i) colchicine and its structural
analogues, such as colcemid and podophyllotoxin; (ii) vinblastine and
its analogues vincristine and maytansine; (iii) the metal ions
Ca2+, Cu2+, and Hg2+ (15, 16); and
(iv) aminonaphthalenes, such as
bis-ANS1 (17). Different
inhibitors bind to tubulin at different sites and presumably arrest
tubulin assembly by different mechanisms (17).
We previously developed a simple in vitro assay for FtsZ
assembly using a FtsZ-green fluorescent protein fusion (FtsZ-GFP) (18).
By using this assay, we demonstrated that FtsZ is capable of
microtubule-like dynamic assembly and can self-assemble into structures
that are similar to microtubule asters. This assembly is strictly
dependent on GTP. In addition, there is a striking difference in the
effects of Ca2+ on FtsZ and tubulin assembly. Whereas
tubulin assembly into microtubules is strongly inhibited by
Ca2+, millimolar concentrations of Ca2+
specifically promote assembly of FtsZ into polymer networks. The
polymers are composed of bundles of protofilaments that are structurally similar to those observed by electron microscopic analysis, and their ability to grow and interconnect in a
GTP-dependent manner suggests that they are physiologically
relevant structures. The stimulatory effect of Ca2+
suggests that Ca2+ interacts with FtsZ, but with different
effects on protein conformation than with tubulin. The mechanism of
Ca2+ stimulation of FtsZ assembly is unknown, as is any
possible role of Ca2+ for FtsZ assembly in the cell.
Here, we use the fluorescent assembly assay and other analytical
techniques to test the effects of some tubulin inhibitors on FtsZ
assembly. We show that colchicine, colcemid, benomyl, and vinblastine
have no effect on Ca2+-induced FtsZ polymerization,
suggesting that FtsZ interacts with these drugs in a manner distinct
from that of tubulin. However, we show that another tubulin inhibitor,
bis-ANS, effectively inhibits FtsZ polymerization, and its possible
mechanism of action is investigated and discussed. Because bis-ANS is a
fluorescent probe that measures protein hydrophobic surface properties,
we used bis-ANS and a related compound, ANS, to probe directly for FtsZ
conformational changes induced by Ca2+ binding and
dissociation.
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EXPERIMENTAL PROCEDURES |
Reagents--
Bis-ANS was obtained from Molecular Probes, Inc.
ANS, colchicine, colcemid, vinblastine, and GTP were purchased from
Sigma, and radiolabeled GTP was from Amersham Pharmacia Biotech.
Benomyl was obtained from DuPont. Other chemicals were of analytical
grade or better. Molecular mass markers were from Life Technologies, Inc. The concentrations of ANS and bis-ANS in stock solution (dissolved in H2O) were determined using
350 = 4900 and
385 = 16,790 M
1cm
1, respectively.
Protein Purification--
FtsZ and FtsZ-GFP proteins were
overexpressed and purified from strains WM688 and WM617, respectively,
as described previously (18). The glycerol and EDTA in the protein
preparation were removed by loading the protein onto a small
DEAE-Sephacel column; washing with 20 volumes of 50 mM
Tris, pH 7.5, 0.1 M KCl; and eluting with the same buffer
containing 1 M NaCl. The protein peak was then pooled and
dialyzed against the above buffer. Protein concentration was determined
with the bicinchoninic acid method (19) using bovine serum albumin as a
standard. The protein concentration of the same FtsZ sample was also
determined by quantitative amino acid analysis on an Applied Biosystems
420A analyzer. The measured amino acid composition is consistent with
the composition predicted from the DNA sequence. When two protein
dilutions were measured in duplicate assays, the bicinchoninic acid
method gave a concentration of 0.997 ± 0.002 mg/ml, whereas the
quantitative amino acid analysis gave a concentration of 1.14 ± 0.03 mg/ml. Therefore, a factor of 1.14 was used to calibrate the
bicinchoninic acid assay.
FtsZ Assembly--
The fluorescent microscopic assay for FtsZ
polymerization was described previously (18). Briefly, FtsZ-GFP or a
mix of FtsZ and FtsZ-GFP at 5.7 µM was incubated in
assembly buffer (50 mM Tris, pH 7.5, 1 mM
MgCl2, 1 mM GTP, 10 mM
CaCl2) at 37 °C for 5-10 min. In some cases, the GTP
concentrations were varied. Four µl of sample were then loaded onto a
glass slide and visualized with an Olympus BX60 fluorescence microscope
equipped with a × 100 oil immersion plan fluorite objective
(numerical aperture = 1.3), a 100-W mercury lamp, a standard
fluorescein isothiocyanate filter set, and an Optronics DEI-750 cooled
video camera. Images were digitized with a Scion LG3 video card,
manipulated with Adobe Photoshop, and printed on a Tektronix Phaser 400 dye sublimation printer. For light scattering assays, 5.7 µM FtsZ in 1 ml of the same buffer without
CaCl2 was incubated at room temperature for 3 min, and then
CaCl2 was added to initiate the polymerization. Light
scattering at 600 nm was recorded continuously for 10 min. To test the
effect of tubulin inhibitors on FtsZ polymerization, the compounds at
different concentrations were added to the polymerization buffer.
Binding of ANS and Bis-ANS to FtsZ--
The binding of bis-ANS
or ANS to FtsZ protein was monitored by bis-ANS or ANS fluorescence
intensity and the shift of
max. Unless otherwise
specified, protein at 1.14-22.8 µM was incubated with
different amount of bis-ANS or ANS (up to 100 µM) in 50 mM Tris, pH 7.5, 0.1 M KCl for 30 min. The
bis-ANS or ANS fluorescence emission spectra were recorded on a Photon
Technology International spectrophotometer. Both excitation and
emission bandwidths were 2 nm. The excitation wavelengths for bis-ANS
and ANS were 390 and 381 nm, respectively.
The stoichiometry and affinity of bis-ANS binding to FtsZ were
determined using a double titration method (20, 21) and Scatchard Plot
analysis (22). Briefly, bis-ANS at several fixed concentrations was
titrated with different concentrations of FtsZ and vice
versa. The common intersection points on the double reciprocal plots of the fluorescence intensities versus the
concentration of the varied components gave the values of
Kd/n or Kd, where
n is the number of bis-ANS binding sites and
Kd is the dissociation constant. The fluorescence
emission was recorded at a fixed wavelength of 480 nm. The observed
fluorescence intensities were corrected for the low background
fluorescence of bis-ANS in buffer and for the inner filter effect of
varying concentrations of bis-ANS (22). For Scatchard plot analysis,
the concentration of bound bis-ANS was determined from the relationship
[bis-ANS]B = F/Fmax, where
Fmax is the theoretical fluorescence of a molar solution if all of the ligand were bound to FtsZ.
Fmax was calculated by titration of 1 µM bis-ANS with different concentrations of FtsZ. The
intercept of the double reciprocal plot of fluorescence intensity
versus FtsZ concentration gave the reciprocal of
Fmax. The data thus generated were analyzed by
the Scatchard method.
Photoincorporation Methods--
Photoincorporation of bis-ANS to
FtsZ was performed as described previously (23). Briefly, FtsZ at
1.14-5.7 µM was incubated with 10-50 µM
bis-ANS in 50 mM Tris, pH 7.5, and 0.1 M KCl
for 30 min on ice, 2 cm from a UV light source. Protein samples were then subjected to SDS-polyacrylamide gel electrophoresis. Fluorescent bands representing bis-ANS bound to FtsZ were photographed by an IS1000
digital imaging system (Alpha Innotech Corp.) with a UV light box. The
UV cross-linking of GTP to FtsZ was performed as described previously
(18).
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RESULTS |
Inhibition of FtsZ Polymerization by Bis-ANS--
To study the
structural and functional similarity between FtsZ and tubulin, we
assessed the effect of tubulin inhibitors on FtsZ polymerization.
Initially, the most common tubulin inhibitors, colchicine, colcemid,
benomyl, and vinblastine, were tested for their effects on
Ca2+-promoted FtsZ assembly into protofilament bundle
networks. These drugs, even at concentrations up to 500 µM or higher, had no significant effect on assembly under
our conditions (data not shown). This suggests that the interaction of
these drugs with FtsZ is distinct from their interaction with
tubulin.
The effects on FtsZ assembly of another group of tubulin inhibitors,
such as ANS and bis-ANS, was also examined. Whereas FtsZ formed polymer
networks in the absence of bis-ANS (Fig.
1A), 50 µM
bis-ANS was sufficient to prevent polymer formation (Fig.
1B). It should be noted that polymer networks shown in Fig.
1A were also typical of those observed after addition of the
tubulin inhibitors tested above. Because of the complex topology of
FtsZ polymers formed in this assay, the distribution of polymers in the
glass slide is typically nonuniform. As a result, it is very difficult to use this technique quantitatively to evaluate inhibition of assembly. To circumvent this problem, we used the concentration of
inhibitor required to block the formation of visible polymers under the
fluorescence microscope as a standard to evaluate the inhibition. For
bis-ANS, this concentration was determined to be 36 µM in
the standard polymerization buffer.

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Fig. 1.
Inhibition of Ca2+-mediated FtsZ
assembly by bis-ANS, as detected by fluorescence microscopy. 2 µM FtsZ-GFP and 5.7 µM FtsZ were incubated
in 50 mM Tris, pH 7.5, 1 mM MgCl2,
1 mM GTP, and 10 mM CaCl2 with 50 µM bis-ANS (B) and without bis-ANS
(A) at 37 °C for 10 min.
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To rule out the possibility that the inhibition by bis-ANS was due to
the GFP tag on the FtsZ protein used for fluorescence detection, the
effect of bis-ANS on FtsZ polymerization was also examined by light
scattering. Fig. 2A shows the
time course of FtsZ polymerization in the absence (trace 1)
and presence (trace 2) of 50 µM bis-ANS as
detected by light scattering at room temperature. The apparent velocity
of the increase of light scattering in the presence of different
concentrations of bis-ANS is shown in Fig. 2B. The
IC50 was around 14 µM under these conditions.
The results suggest that bis-ANS is a specific inhibitor of FtsZ
polymerization.

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Fig. 2.
Inhibition by bis-ANS of
Ca2+-mediated FtsZ assembly into protofilament bundles as
detected by light scattering. A, 5.7 µM FtsZ
was incubated in 50 mM Tris, pH 7.5, 1 mM
MgCl2, and 1 mM GTP with 50 µM
bis-ANS (trace 2) and without bis-ANS (trace 1)
at room temperature for 5 min. CaCl2 was added to a final
concentration of 10 mM to initiate polymerization. Light
scattering was recorded at 600 nm on a Photon Technology International
spectrophotometer. B, effect of bis-ANS concentration on the
apparent velocity of FtsZ assembly at room temperature. The apparent
velocity was calculated from time courses similar to that shown in
A.
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The inhibition of FtsZ polymer formation by bis-ANS prompted us to
determine whether it could promote FtsZ depolymerization. Because
assembly of FtsZ in our assay occurs dynamically in solution, it was
possible to analyze the polymerization state in solution over time in
order to address this question. FtsZ-GFP at 5 µM was
incubated in assembly buffer at 37 °C for 10 min, and aliquots of
the sample were checked by fluorescence microscopy to verify the
formation of fluorescent polymers. Then, bis-ANS was added to the
sample to make a final concentration of 50 µM. After a 20-min incubation, no polymers could be detected by fluorescence microscopy (data not shown). This result suggests that bis-ANS, in
addition to inhibiting FtsZ polymerization, is also able to depolymerize FtsZ polymers. The possibility that bis-ANS can
depolymerize microtubules has not been reported.
The bis-ANS analog ANS (8-anilino-1-naphthalenesulfonate) has been
reported to be much less effective at inhibiting tubulin assembly than
bis-ANS (17, 24). Using the fluorescence assay, we found that ANS had
no detectable effect on FtsZ polymerization, even when the ANS
concentration was as high as 1.5 mM (data not shown). We
can conclude that the effects of bis-ANS and ANS on FtsZ assembly are
very similar to their respective effects on tubulin assembly,
suggesting that FtsZ and tubulin may interact similarly with these two
compounds.
Binding of Bis-ANS to FtsZ--
Because bis-ANS specifically
inhibits polymerization of both tubulin and FtsZ, it was reasonable to
propose that FtsZ may have a hydrophobic surface arrangement similar to
that of tubulin. It has been reported that tubulin has one high
affinity bis-ANS binding site and 6 (20) to 40 (25, 26) low affinity
binding sites. The high affinity binding site has been proposed to be responsible for the inhibition of tubulin assembly (24). To characterize the interaction between bis-ANS and FtsZ in more detail,
we analyzed bis-ANS-FtsZ binding. Evaluation of multiple binding sites
is extremely difficult using spectroscopic techniques, because the
binding of bis-ANS at different sites may not have the same quantum
yield (26). Nevertheless, we took advantage of the extensive studies of
the binding of bis-ANS to tubulin (20, 24-26) to apply spectroscopic
techniques to bis-ANS-FtsZ binding. As shown in Fig.
3A, the presence of FtsZ
greatly enhances bis-ANS fluorescence (trace 2) over the
baseline (trace 1) with a blue shift of
max
from 530 nm (data not shown) to 480 nm, suggesting strong binding of
bis-ANS to FtsZ. Fig. 3B shows an example of a Scatchard
plot for an FtsZ concentration of 2.28 µM. These data indicate that FtsZ has a high affinity binding site for bis-ANS with a
Kd of 1.33 µM and 3.59 low affinity
binding sites, each with a Kd of 22.92 µM. Interestingly, these Kd values for
both high and low affinity binding sites are similar to those
previously reported for tubulin (20, 24-26). The number of low
affinity binding sites for FtsZ is smaller than six, the smallest
number reported for tubulin. To rule out the possibility that this
difference was a result of the analytical method employed, the same
double titration method as described by Prasad et al. (20)
was applied to FtsZ. The data confirmed that the number of low affinity
binding sites is 3.66 and the Kd is 19.2 µM (Fig. 4). The data
derived from the same method strongly suggest that the bis-ANS binding
sites of FtsZ are very similar to those of tubulin, except that tubulin
may have additional sites. It is reasonable to propose from this result
that FtsZ and tubulin may have a similar pattern of hydrophobic patches
on their surfaces.

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Fig. 3.
Enhancement of bis-ANS fluorescence by
FtsZ. A, 5 µM bis-ANS was incubated with
(trace 2) and without (trace 1) 2.28 µM FtsZ in 50 mM Tris, pH 7.5, 0.1 M KCl for 10 min. The excitation wavelength was 390 nm.
Both the excitation and emission bandwidths were 2 nm. B,
Scatchard analysis of the binding of bis-ANS to FtsZ. 2.28 µM FtsZ in 50 mM Tris, pH 7.5, 0.1 M KCl was titrated with different amounts of bis-ANS. The
absence of GTP prevented FtsZ from polymerizing in this buffer. Other
conditions are described under "Experimental Procedures."
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Fig. 4.
Double-reciprocal plots for the binding of
bis-ANS to FtsZ. A, plot of the inverse of the fluorescence
intensity versus the inverse of bis-ANS concentration at
various fixed concentrations of FtsZ. The various FtsZ concentrations
were 2.28 ( ), 5.7 ( ), 11.4 ( ), and 22.8 ( )
µM. B, plot of the inverse of the fluorescence
intensity versus the inverse of FtsZ concentration at
various fixed concentrations of bis-ANS. Each line
corresponds to a fixed bis-ANS concentration of 2 ( ), 3 ( ), 5 ( ), or 12 ( ) µM. Excitation was at 390 nm, and
emission was at 480 nm, with a bandwidth of 2 nm.
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Ca2+ Changes the Binding of Bis-ANS and ANS to
FtsZ--
Ca2+ is a key factor for FtsZ polymerization
under our conditions. Because binding of Ca2+ is likely to
have an important role in conformational changes in FtsZ leading to its
assembly, and because bis-ANS is a probe for hydrophobic surface
arrangement, it was logical to include Ca2+ in the study of
bis-ANS-FtsZ interactions and to determine the effect of
Ca2+ on the binding of bis-ANS to FtsZ. The data in Fig.
5A show a 30% increase in
bis-ANS fluorescence upon addition of Ca2+ to FtsZ,
suggesting that Ca2+ moderately increases bis-ANS
binding.

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Fig. 5.
Effect of the binding and dissociation of
Ca2+ on the enhancement of FtsZ-dependent
bis-ANS and ANS fluorescence. A, enhancement of bis-ANS
fluorescence. Traces 1-4 represent 2.28 µM
FtsZ and 5 µM bis-ANS in 50 mM Tris, pH 7.5, 0.1 M KCl in the presence of 0, 2.5, 5, and 20 mM CaCl2, respectively. Traces 5 and
6 represent the same samples as in trace 4 with
the addition of 4 and 6 mM EGTA, respectively.
B, enhancement of ANS fluorescence. Traces 7-10
represent 6.84 µM FtsZ and 120 µM ANS in 50 mM Tris, pH 7.5, 0.1 M KCl in the presence of
0, 5, 20, and 40 mM CaCl2, respectively.
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Either a change in bis-ANS binding or a change in quantum yield of the
bis-ANS-FtsZ complex could explain the changes in fluorescence. To rule
out the second possibility, the binding of bis-ANS to FtsZ was also
examined by photoincorporation of bis-ANS to FtsZ. In this assay, the
bis-ANS was first cross-linked to FtsZ protein, followed by detection
of photolabeled protein after denaturation and separation by
SDS-polyacrylamide gel electrophoresis. As detected by UV
cross-linking, Ca2+ appeared to increase the binding of
bis-ANS (Fig. 6B). Although FtsZ protein was stable in the presence of bis-ANS (data not shown), UV-specific protein degradation was often observed in our experiments in the presence of bis-ANS, especially with longer UV light exposure and high bis-ANS concentrations. To address this problem, the Coomassie-stained protein bands and bis-ANS fluorescent bands from the
same gel were quantified, and bis-ANS fluorescence was normalized to
the amount of protein in the bands. The effect of different
concentrations of Ca2+ on bis-ANS binding is summarized in
Fig. 7A. Ca2+
concentrations between 5 and 10 mM enhanced bis-ANS binding
by approximately 2-fold, as detected by UV cross-linking. This effect was greater than that detected by bis-ANS fluorescence spectra (Fig.
5A) and could be attributed to a lower quantum yield of the
additional exposed binding site(s) induced by Ca2+. Another
explanation for this apparent increase is the predicted block of
bis-ANS dissociation from FtsZ due to covalent cross-linking. Therefore, the photoincorporation method may give higher numbers for
binding and as a result amplify the difference in binding affinities.

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Fig. 6.
Effect of Ca2+ on the
photoincorporation of bis-ANS into FtsZ. 5.7 µM FtsZ
was incubated with 16 µM bis-ANS in 50 mM
Tris, pH 7.5, 0.1 M KCl containing different concentrations
of CaCl2, 2 cm from a UV light for 30 min at 4 °C, and
then subjected to SDS-polyacrylamide gel electrophoresis. Lane
m contains a protein molecular mass ladder representing 220, 160, 120, 100, 90, 80, 70, 60, 50, 40, 30, 25, and 20 kDa. Lanes
1-5 contain Coomassie Blue-stained FtsZ protein bands
representing incubations with Ca2+ at concentrations of 0, 1.25, 2.5, 5, and 10 mM, respectively. B shows
the same gel corresponding to lanes 1-5 in A,
imaged for fluorescence prior to Coomassie Blue staining.
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Fig. 7.
Effect of Ca2+ (A)
and GTP (B) concentration on the photoincorporation of
bis-ANS into FtsZ. Experimental conditions were the same as
described in the legend to Fig. 6 and are described in detail under
"Experimental Procedures."
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Because bis-ANS inhibits FtsZ assembly, bis-ANS itself is likely to
induce FtsZ conformational changes. As a result, changes in bis-ANS
fluorescence in the presence of Ca2+ may be due to a
combination of both Ca2+ and bis-ANS effects on FtsZ
conformation. To test whether Ca2+ alone induces changes of
FtsZ hydrophobic surface properties in the absence of bis-ANS, we
examined the effect of Ca2+ on the enhancement of ANS
fluorescence by FtsZ. This was feasible because, as described earlier,
ANS has no detectable effect on FtsZ polymerization. The data in Fig.
5B demonstrate that Ca2+ increases ANS
fluorescence up to 2.5-fold, suggesting that Ca2+ is
sufficient to induce conformational changes in the FtsZ protein.
To test whether the effect of Ca2+ on bis-ANS binding to
FtsZ was reversible, EGTA was added to the assembly reaction to chelate Ca2+. When the Ca2+ concentration was below 5 mM, the bis-ANS fluorescence decreased back to the level
observed in the absence of Ca2+ if sufficient EGTA was
added (data not shown). When the Ca2+ concentration was 7.5 mM or greater, addition of 4 mM EGTA slightly decreased the bis-ANS fluorescence, consistent with a decrease in
available Ca2+ (Fig. 5A, trace 5). However, when
EGTA concentration was increased to deplete Ca2+ from the
solution, bis-ANS fluorescence did not decrease but instead increased
to a level greater than reached during stimulation by Ca2+
(Fig. 5A, trace 6, and data not shown). This result is
surprising, but it is consistent with the previous finding that a
critical concentration of Ca2+ at 7 mM is
required to trigger FtsZ assembly into visible fluorescent polymers
(18). One possible explanation is that when Ca2+ in the
solution reaches the critical concentration, the dissociation of
Ca2+ from certain binding sites leads to a further exposure
of hydrophobic surfaces, which may be essential for FtsZ to assemble in
the presence of GTP. In a control experiment, EGTA itself had no effect
on the fluorescence of the bis-ANS-FtsZ complex (data not shown) in the
absence of Ca2+. In another control, Ca2+ and
EGTA had no effect on the enhancement of bis-ANS fluorescence by bovine
serum albumin. This suggests that the observed effects of depleting
Ca2+ on bis-ANS fluorescence are specific under our
conditions. Moreover, ANS fluorescence was also enhanced by
EGTA-mediated depletion of Ca2+ (data not shown). This
result supports the idea that additional EGTA-mediated FtsZ
conformational changes were in fact due to Ca2+
dissociation and were not merely due to a specific effect of bis-ANS.
GTP Inhibits Bis-ANS Binding to FtsZ--
Although it is well
established that bis-ANS inhibits tubulin polymerization (24), the
mechanism of inhibition is not understood. However, the similar binding
behavior of bis-ANS to FtsZ and tubulin observed here suggested that
its mechanism of inhibition of FtsZ and tubulin assembly might be
similar. FtsZ polymerization under our conditions specifically requires
GTP, although GDP is capable of supporting FtsZ polymerization mediated
by DEAE-dextran and cationic lipid monolayers (11, 12). Because GTP is
essential for both FtsZ and tubulin assembly, it was important to
determine whether GTP binding could be affected by bis-ANS binding and
vice versa.
Fig. 8A shows that
preincubation of FtsZ with 1 mM GTP greatly decreased the
binding of bis-ANS to FtsZ. The maximum decrease of fluorescence by
increasing GTP concentration is about 50% of that in the absence of
GTP, suggesting that GTP may inhibit bis-ANS binding to certain binding
sites but not to other sites. This result was also consistent with the
titration data described earlier, which demonstrated that FtsZ has
multiple bis-ANS binding sites. When bis-ANS was preincubated with FtsZ
for 1 min, the addition of GTP caused a slow decrease of bis-ANS
fluorescence, with the apparent rate of fluorescence decrease dependent
on GTP concentration (Figs. 8B). This suggests that the
dissociation of bis-ANS from FtsZ is slower than the binding of bis-ANS
to FtsZ. The tubulin signature sequence GGGTGTG, which is likely
involved in the interaction of FtsZ with GTP, has been proposed to form
a hydrophobic pocket (27). The rapid binding of bis-ANS to the GTP
binding site and its slow dissociation is consistent with this
model.

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Fig. 8.
Effect of GTP on the enhancement of bis-ANS
fluorescence by FtsZ. A, 5.7 µM FtsZ was
incubated without (trace 1) and with (trace 2) 1 mM GTP in 50 mM Tris, pH 7.5, 0.1 M
KCl for 10 min, followed by addition of bis-ANS to a final
concentration of 16 µM. The fluorescence spectra were
recorded after another 5 min of incubation. B, 4.56 µM FtsZ was incubated with 20 µM bis-ANS in
50 mM Tris, pH 7.5, 0.1 M KCl for 1 min,
followed by addition of 0.2 (trace 1), 1 (trace
2), and 2 (trace 3) mM GTP to the sample.
Fluorescence at 480 nm was recorded immediately. Other conditions are
described under "Experimental Procedures."
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To investigate further the inhibition of bis-ANS binding by GTP, a
photoincorporation experiment was performed. As in the Ca2+
experiment, GTP was also shown to inhibit photoincorporation of bis-ANS
to FtsZ (Fig. 7B). The effect of GTP on bis-ANS binding seems to be slightly greater than that detected by fluorescent spectra.
As suggested earlier, this phenomenon may be due to amplification of
the difference in binding by UV cross-linking.
Effects of Bis-ANS on GTP Binding--
The inhibition of bis-ANS
binding by GTP binding suggested that the GTP binding site might also
overlap with at least one bis-ANS binding site. The effect of bis-ANS
on GTP binding is shown in Fig.
9A. At a low GTP concentration
(1 µM), the IC50 of bis-ANS is approximately
4 µM, but when GTP concentration increases to 10 µM, the IC50 increases to approximately 17 µM. The inhibition of GTP binding by bis-ANS and
inhibition of bis-ANS binding by GTP suggest that GTP and bis-ANS
compete for the same site on FtsZ.

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Fig. 9.
Effect of bis-ANS on the binding of GTP to
FtsZ. UV cross-linking of GTP to FtsZ was performed as described
previously (18) in the presence of bis-ANS, as indicated. A,
effect of bis-ANS concentration on the binding of a fixed concentration
of GTP to FtsZ. The fixed GTP concentration was 1 ( ) and 10 ( )
µM (400 Ci/mmol). B, the graph depicts the
ratio of GTP binding in the presence versus absence of 50 µM bis-ANS (y axis) at different
concentrations of GTP at 40 Ci/mmol (x axis).
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To determine whether the inhibition of FtsZ polymerization might
be due to competitive binding of bis-ANS to the GTP binding site, we
determined the effect of 50 µM bis-ANS on GTP binding to
FtsZ at different GTP concentrations. Because a bis-ANS concentration of 50 µM was sufficient to block the formation of visible
fluorescent polymers (Fig. 1), this low inhibitor concentration was a
good starting point to investigate its effect on GTP binding as a
function of GTP concentration. When the GTP concentration was greater
than 200 µM, 50 µM bis-ANS had little
effect on GTP binding (Fig. 9B). However, 50 µM bis-ANS inhibited FtsZ assembly even when the GTP concentration was as high as 2 mM (data not shown),
suggesting that the bis-ANS-mediated inhibition of FtsZ assembly is
caused by noncompetitive or uncompetitive inhibition of GTP
binding.
Any competitive binding of GTP and bis-ANS to FtsZ would be predicted
to be most apparent at low GTP concentrations. The bis-ANS concentration required to completely block the formation of visible fluorescent FtsZ polymers decreased slightly, from 36 to 30 µM, when the GTP concentration was reduced from 1 mM to 50 µM (data not shown). Therefore, it
is possible that at low GTP concentrations, competitive binding of
bis-ANS to the GTP binding site may have some role in the inhibition of
FtsZ assembly.
 |
DISCUSSION |
Recent direct and indirect evidence strongly suggests that the
FtsZ ring marks the plane of cytokinesis in all prokaryotic cells, even
chloroplasts (8, 28). Despite this apparently actin-like role, FtsZ
clearly has structural and biochemical similarity to tubulin, including
the ability to bind and hydrolyze GTP (9, 13, 29) and to self-assemble
in vitro into protofilament bundles that have dimensions
similar to that of tubulin (11, 30) and that have microtubule-like
dynamic and morphological properties (18). Our aim is to establish a
biochemical foundation for FtsZ in order to clarify the differences and
similarities between FtsZ and tubulin. A deeper understanding of these
key proteins should help elucidate more about their evolutionary
relationship and the precise function of FtsZ in prokaryotic cell
division. In addition, because tubulin is the target of such a wide
variety of inhibitors, FtsZ may also be a potentially good target for antimicrobial compounds. Hence, understanding structural differences between FtsZ and tubulin may eventually facilitate design of FtsZ-based antimicrobials that are modeled on anti-tubulin drugs.
In this paper, we report that common tubulin inhibitory compounds did
not inhibit FtsZ assembly but that the widely used hydrophobic probe
bis-ANS can both inhibit FtsZ assembly and also serve as a useful probe
to measure FtsZ conformational changes. These findings are a first step
in defining structural and functional differences between FtsZ and
tubulin. One obvious difference between the two proteins is the
C-terminal domain, both in primary sequence and in predicted secondary
structure (14). This difference may be responsible for the different
Ca2+ effects, because a C-terminal truncation of tubulin is
Ca2+-resistant (31, 32). Interestingly, when FtsZ and
tubulins are aligned, it can be seen that FtsZ is missing the region in Neurospora crassa tubulin that contains the mutation for
benomyl resistance (33). This could explain why colchicine, colcemid, and benomyl have no effect on FtsZ polymerization.
The similar inhibition of FtsZ and tubulin assembly by bis-ANS suggests
that bis-ANS interacts similarly with both proteins. The titrations of
FtsZ with bis-ANS and vice versa, using the same methods
that were previously applied to tubulin, suggest that FtsZ has a high
affinity bis-ANS binding site and multiple low affinity binding sites,
with Kd values similar to those of tubulin. This
analysis implies that the hydrophobic surface properties of FtsZ and
tubulin are similar. The inhibition of bis-ANS binding by GTP binding,
and vice versa, suggests that GTP binding sites and bis-ANS
binding sites overlap. Because bis-ANS binds selectively to protein
hydrophobic surfaces, this result provides evidence that the GTP
binding site is hydrophobic, in support of a previous proposal based on
epitope mapping (27). At low GTP concentrations, competition for GTP
binding by bis-ANS could play a role in its inhibition of FtsZ
assembly. Because the inhibition of GTP binding by bis-ANS can be
overcome by increasing GTP concentration, whereas the inhibition of
FtsZ assembly cannot, it is likely that noncompetitive or uncompetitive
binding by bis-ANS is also responsible for its inhibitory effect.
Hydrophobic interactions have been implicated in tubulin assembly, and
our evidence is consistent with an analogous role for such interactions
in FtsZ assembly. The fact that Ca2+ increases bis-ANS and
ANS binding to FtsZ strongly suggests that Ca2+ induces
FtsZ conformational changes. Based on our results, we propose that
bis-ANS binding inhibits FtsZ assembly by blocking FtsZ intermolecular
hydrophobic interactions. We further propose that Ca2+
binding may induce stronger intermolecular hydrophobic interactions that result in the stimulation of FtsZ assembly. This idea is in accord
with the GTP-dependent stimulation of FtsZ assembly by
7-20 mM Ca2+ and the GTP-independent
aggregation of FtsZ at higher concentrations of Ca2+ (18).
Such changes in hydrophobic properties, therefore, could be independent
of GTP or could influence or be influenced by GTP binding. For example,
GTP binding has been shown to influence the ability of tubulin to
interact with hydrophobic substrates (34).
It is likely that changes in hydrophobic surface properties of FtsZ are
involved in the interaction between FtsZ and its natural protein
inhibitors MinC and SulA. There is good evidence that SulA interacts
directly with FtsZ, and it was proposed that this interaction prevents
a GTP-induced conformational change that normally leads to
polymerization (35). This is consistent with the dispersal throughout
the ftsZ gene of mutations that result in SulA resistance
and the variable effects of these mutations on GTP binding and
hydrolysis (36). These findings are completely consistent with the idea
proposed here that hydrophobic interactions drive FtsZ polymerization.
In fact, it is tempting to speculate that the inhibition mechanisms of
bis-ANS and SulA may be similar. Future in-depth comparisons of bis-ANS
and SulA inhibition of FtsZ assembly, as well as investigation of the
effects of bis-ANS on SulA-resistant FtsZ proteins, should prove
fruitful in understanding the molecular details underlying FtsZ
assembly.
We thank J. Putkey, F. Cabral, and K. A. Borkovich for reagents and the Department of Biochemistry and Molecular
Biology, University of Texas Medical School, for the use of their
spectrophotometer. Benomyl was a generous gift from DuPont.