Plant Physiol. (1998) 118: 965-973
Identification of the Binding and Inhibition Sites in the
Calmodulin Molecule for Ophiobolin A by
Site-Directed
Mutagenesis1
Tai Kong Au and
Pak Chow Leung*
Department of Zoology, The University of Hong Kong, Pokfulam Road,
Hong Kong
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ABSTRACT |
Ophiobolin A, a fungal toxin that
affects maize and rice, has previously been shown to inhibit calmodulin
by reacting with the lysine (Lys) residues in the calmodulin. In the
present study we mutated Lys-75, Lys-77, and Lys-148 in the calmodulin
molecule by site-directed mutagenesis, either by deleting them or by
changing them to glutamine or arginine. We found that each of these
three Lys residues could bind one molecule of ophiobolin A. Normally, only Lys-75 and Lys-148 bind ophiobolin A. Lys-77 seemed to be blocked
by the binding of ophiobolin A to Lys-75. Lys-75 is the primary binding
site and is responsible for all of the inhibition of ophiobolin A. When
Lys-75 was removed, Lys-77 could then react with ophiobolin A to
produce inhibition. Lys-148 was shown to be a binding site but not an
inhibition site. The Lys-75 mutants were partially resistant to
ophiobolin A. When both Lys 75 and Lys-77 or all three Lys residues
were mutated, the resulting calmodulins were very resistant to
ophiobolin A. Furthermore, Lys residues added in positions 86 and/or
143 (which are highly conserved in plant calmodulins) did not
react with ophiobolin A. None of the mutations seemed to affect the
properties of calmodulin. These results show that ophiobolin A reacts
quite specifically with calmodulin.
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INTRODUCTION |
Calmodulin is a small, acidic, Ca2+-binding
protein and is the major Ca2+ receptor in
eukaryotic cells. Since its discovery as a phosphodiesterase protein
activator (Cheung, 1971
), many enzymes and proteins have been found to
be capable of binding to and being regulated by calmodulin. The
structures of Ca2+-bound and
Ca2+-free calmodulin have been resolved (Babu et
al., 1988; Zhang et al., 1995). Comparison of these two structures has
provided valuable information on how the binding of
Ca2+ induces the exposure of hydrophobic surfaces
in the N and C terminals of calmodulin. This conformational change
allows Ca2+-calmodulin to form a 1:1 complex with
target proteins.
Ophiobolin A is a fungal metabolite and a phytotoxin produced by the
plant pathogen Helminthosporium maydis Nisikado and Miyake and by other members of the same genus (Hesseltine et al., 1971). In
roots of maize seedlings ophiobolin A causes a leakage of electrolytes and Glc from cells (Tipton et al., 1977). Ophiobolin A is believed to
cause the symptoms of brown spot disease in rice (Narain and Biswal,
1992). In maize roots the cytotoxic effect of ophiobolin A was well
correlated with calmodulin inhibition (Leung et al., 1985). When maize
roots were treated with ophiobolin A, less active calmodulin was
present in the root extract, indicating a possible in vivo inhibition
of calmodulin by ophiobolin A. The cytotoxicity of ophiobolin A was
attributed to its covalent binding to calmodulin. When calmodulin was
incubated with ophiobolin A, the Tyr fluorescence of calmodulin was
quenched (Leung et al., 1984). This quenching was correlated with the
loss in calmodulin activity, and indicated a direct binding between
calmodulin and the toxin. The 
-amino group of Lys residues was
implicated in the reaction with ophiobolin A (Leung et al., 1988),
although the precise location of the reactive Lys in the calmodulin
molecule is not known.
Using bovine-brain calmodulin as a model system, we report the
identification of the Lys residues in the calmodulin molecule that
react with ophiobolin A by site-directed mutagenesis. We found that
Lys-75 and Lys-148 were the ophiobolin A-binding sites. However, only
Lys-75 was responsible for the inhibitory effect of ophiobolin A. Removal of Lys-75 could confer resistance to the toxin. Lys-77 was
another binding site, but was available only when Lys-75 was deleted or
substituted by another amino acid. When Lys residues unique to plant
calmodulins were introduced, ophiobolin A did not react with
them.
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MATERIALS AND METHODS |
Ophiobolin A, snake venom 5
-nucleotidase, cAMP, malachite green,
and most of the chemicals were from Sigma. Phenyl-Sepharose CL-4B,
lysozyme, and DNase were from Pharmacia. Oligonucleotides were
synthesized on a GeneAssembler (Pharmacia) and purified according to
the recommended procedures of the manufacturer. Protein assay dye
solution was from Bio-Rad. Bovine brains were obtained from local
markets and transported to the laboratory on ice.
Protein Preparation
Calmodulin-deficient PDE from bovine brain was partially purified
according to the method of Wallace et al. (1983) and stored at
80°C. Bovine-brain calmodulin was purified to homogeneity by
phenyl-Sepharose column chromatography (Gopalakrishna and Anderson, 1982), followed by DEAE-cellulose column chromatography. Protein concentration was determined by the dye-binding method of Bradford (1976) using BSA as a standard.
Assay of PDE Activity
The activity of PDE was assayed by coupling to 5-nucleotidase as
described by Sharma and Wang (1979) with modifications as described by
Leung et al. (1984). In this assay the PDE changed the cAMP to 5-AMP
and the 5-nucleotidase changed the 5-AMP to adenosine and phosphate.
The amount of phosphate released was measured by the malachite green
method at 610 nm (Veldhoven and Mannaerts, 1987). Therefore, the
A610 represented a direct measurement of
the PDE activity.
Isolation of Bovine-Brain Calmodulin cDNA and Construction of a
Calmodulin-Expression Plasmid
The following procedures produced a calmodulin-expression plasmid
that directed the synthesis of calmodulin in bacteria. Total RNA was
isolated from bovine brain by acid guanidinium
thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi,
1987). The first strand of cDNA was synthesized from total RNA from
bovine brain using a reverse-transcription system (Promega). A cDNA
fragment of 450 bp that encompassed the entire calmodulin-coding region
was isolated using PCR from a bovine-brain first-strand cDNA
preparation. For the PCR reaction the 5 and 3 primers were
5-ACACCATGGCTGAC/TCAA/GCTGACC/TGA-3 and
5-ACAGGATCCTCAC/TTTC/TGCA/TGTCATCAT-3,
respectively. The boldface sequences are the NcoI and
BamHI sites, respectively.
Amplified cDNA was purified by agarose-gel electrophoresis. The
purified cDNA was digested with NcoI and BamHI,
and then ligated to pTrc99A vector (Pharmacia)
previously digested with NcoI and BamHI. The
ligation mixture was used to transform competent Escherichia coli JM105 cells. Transformants were selected on Luria-Bertani agar plates containing 100 µg/mL ampicillin and screened for clones that contained the calmodulin insert. The identities of the inserts were verified by DNA sequencing. One recombinant plasmid, designated pCam-Trc, contained the calmodulin cDNA sequence under the
control of the trc promoter. The distance between the
Shine-Dalgarno sequence and the translation-initiation codon ATG was
found to be eight nucleotides. The predicted amino acid sequence was
identical to the published bovine-brain calmodulin-amino acid sequence
(Watterson et al., 1980). The bacterial clone that contained
pCam-Trc was used for the expression of wild-type
calmodulin.
Site-Directed Mutagenesis
Site-directed mutations of individual Lys residues in calmodulin
were performed by the inverse PCR method of Hemsley et al. (1989). A
pair of oligonucleotide primers complementary to the calmodulin
sequence at the site of mutagenesis were designed so that they would
line up in a back-to-back fashion on opposite strands of the template.
One of the primers carried the desired mutation. The uncut
calmodulin-expression plasmid was used as the template in the PCR.
Amplified linear DNA was self-ligated to regenerate a circular plasmid
with the mutation incorporated. Three series of mutations were created
in the Lys-75, Lys-77, and Lys-148 of the calmodulin molecule. They
were the single-amino acid mutants (K75
, K75Q, K75R, K77,
K148, K148Q, and K148R), the double-amino acid mutants
(K75,77; K75,148R; and K77,148R), and the triple-amino acid
mutants (K75,77,148R; K75,77,148; K75,77,148Q; and K75,77,148R),
where stands for deletion.
For the single-amino acid mutants, K75 indicates that Lys-75 was
deleted, K75Q indicates that Lys-75 was changed to Gln, and K75R
indicates that Lys-75 was changed to Arg. The same meaning is applied
to Lys-77 and Lys-148 mutants. For the double-amino acid mutants,
K75,77 indicates that both Lys-75 and Lys-77 were deleted,
K75,148R indicates that Lys-75 was deleted and Lys-148 was changed
to Arg, and K77,148R indicates that Lys-77 was deleted and Lys-148
was changed to Arg. For the triple-amino acid mutants, K75,77,148R
indicates that Lys-75 and Lys-77 were deleted and Lys-148 was changed
to Arg; K75,77,148 indicates that Lys-75, Lys-77, and Lys-148 were
deleted; K75,77,148Q indicates that Lys-75, Lys-77, and Lys-148 were
changed to Gln; and K75,77,148R indicates that Lys-75, Lys-77, and
Lys-148 were changed to Arg.
The deletion mutation was generated by amplification with a pair of
oligonucleotide primers that spanned both sides of the Lys codon (AAA).
Therefore, the Lys codon was not included in the amplified DNA product.
For the Gln and Arg substitutions, the AAA codon was replaced by CAG
and CGT in the oligonucleotides, respectively. A silent mutation was
introduced into the K75 mutants to create a unique MscI site
for screening purposes. This silent mutation was not introduced in
subsequent mutagenesis. To obtain the double-amino acid mutants
K75,77 and K75,148R, the single-amino acid mutant K75 was used
as the PCR template. For K77,148R, K77 was the PCR template.
Likewise, the triple-amino acid mutants were obtained using single- or
double-amino acid mutants as PCR templates.
Using the triple-amino acid mutant K75,77,148R as the PCR template,
additional Lys residues were introduced at positions 86 and 143 of the
calmodulin molecule as single-addition (R86K and Q143K) and
double-addition (R86,Q143K) mutants. Thus, mutant R86K has Arg-86
changed to Lys, mutant Q143K has Gln-143 changed to Lys, and mutant
R86,Q143K has Arg-86 and Gln-143 changed to Lys, in addition to the
three mutations at Lys-75, Lys-77, and Lys-148. The CGC codon (for Arg)
at position 86 and the CAG codon (for Gln) at position 143 were
replaced by the AAA codon (for Lys) in the mutagenesis primers.
The PCR took place in a 100-µL reaction mixture containing 20 mM Tris-HCl, pH 8.8, at 25°C, 10 mM KCl, 10 mM
(NH4)2SO4,
3 mM MgSO4, 0.1% Triton X-100, 100 µg/mL BSA, 400 µM of each deoxyribonucleotide triphosphate, 5 ng of the template plasmid, 1 µM of each
oligonucleotide primer, and 1 unit of DNA polymerase (Vent, New England
Biolabs). This reaction mixture was incubated for 25 cycles (1 cycle = 94°C for 1 min, 60°C for 1 min, and then 72°C for 5 min) followed by a 10-min incubation at 72°C. The products of the
reaction were purified by agarose-gel electrophoresis. A portion of the
agarose-gel-purified PCR product was phosphorylated at the 5 ends and
self-ligated in 20 µL of 70 mM Tris-HCl, pH 7.6, at
25°C, 10 mM MgCl2, 5 mM DTT, 1 mM ATP, 5 units of T4 polynucleotide kinase (New
England Biolabs), and 200 units of T4 DNA ligase (New England Biolabs) at 15°C for 16 h. The ligation mixture was used to transform
competent E. coli JM105 cells. Transformants were selected
by plating on Luria-Bertani agar plates containing 100 µg/mL
ampicillin and then screened for the expression of calmodulin. The
presence of the desired mutations in the plasmid were confirmed by
sequencing the entire coding sequence. For each mutagenesis, more than
80% of the clones selected by ampicillin resistance contained the desired mutation.
Expression and Purification of Wild-Type and Mutated
Calmodulins
Bacterial expression and purification of wild-type and mutated
calmodulins were performed using the method of Putkey et al. (1985)
with modifications. A single colony of E. coli JM105
carrying the appropriate expression plasmid was used to inoculate 50 mL of Luria-Bertani medium containing 100 µg/mL ampicillin. After overnight growth at 37°C, 20 mL was used to inoculate 1 L of 2× YT
medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, pH
7.0) containing 100 µg/mL ampicillin. The culture was
incubated at 37°C for 2 h with shaking. Expression of calmodulin
was induced by the addition of isopropylthio-
-galactoside to a
concentration of 2 mM. The culture was incubated for
another 6 h. The cells were harvested by centrifugation
(4000g, 5 min, 4°C). The cell pellet was washed twice in
200 mL of 50 mM Tris-HCl, pH 7.5, and then resuspended in
100 mL of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. Lysozyme was added to
200 µg/mL, and the suspension was incubated at 4°C for 30 min.
MgCl2 and DNase were added to concentrations of
10 mM and 10 µg/mL, respectively. The mixture was
incubated at 4°C for another 2 h. CaCl2
was added to a concentration of 10 mM.
The lysate was then placed in a boiling-water bath for 5 min and then
on ice for 10 min. The suspension was cleared by centrifugation at
31,000g for 30 min at 4°C. The supernatant was applied to
a 5-mL phenyl-Sepharose CL-4B column equilibrated in 50 mM
Tris-HCl, pH 7.5, 1 mM CaCl2, 1 mM DTT, and 0.5 mM PMSF. The column was then
washed with 50 mL of the same buffer and then with 50 mL of the buffer
containing 0.5 M NaCl. Calmodulin was eluted from the
column with 50 mM Tris-HCl, pH 7.5, 1 mM EGTA,
1 mM DTT, and 0.5 mM PMSF. Eluted protein was
dialyzed extensively against deionized water, concentrated, and stored
at 20°C. The yields were 10 to 16 mg of calmodulin per liter of
cell culture, which is comparable to the yields of other protocols
(Putkey et al., 1985; Roberts et al., 1985; Rhyner et al., 1992).
N-terminal sequencing of the recombinant wild-type calmodulin by a
protein-sequencing system (model G1005A, Hewlett-Packard) revealed that
the recombinant wild-type calmodulin has a nonacetylated Ala as the
first residue (data not shown). This is the same first amino acid as in
natural bovine-brain calmodulin, although the Ala in the latter is
acetylated. Therefore, the same numbering system was used for
recombinant calmodulin in this study.
Binding Measurements
Binding between ophiobolin A and calmodulin was monitored by the
increase in A272. It has been shown that a
new chromophore at 272 nm is formed quantitatively when ophiobolin A
reacts with calmodulin (Leung et al., 1988). The binding experiments
were carried out in 5-mL glass culture tubes (Fisher Scientific). In these experiments the calmodulin was in 0.15 mL of 40 mM
Tris-HCl, pH 7.5, and 1 mM CaCl2.
Various concentrations of ophiobolin A were added as 1 or 5 mM solutions in methanol using a syringe (Hamilton Co.,
Reno, NV). After incubation at room temperature for 2 h, the
A272 was measured by a UV-visible
spectrophotometer (Biochrom 4060, Pharmacia LKB). The absorbance was
corrected for the absorbance of the calmodulin and that of ophiobolin A
to obtain the absorbance of the calmodulin-ophiobolin A complex. The
extent of ophiobolin A binding to calmodulin was calculated using the molar extinction (19,200 M
1
cm1 at 272 nm) of the conjugated enamine
product (Leung et al., 1988).
Recombinant DNA Methods
DNA fragments were purified by agarose gel electrophoresis and
eluted using a kit (Sephaglas BandPrep, Pharmacia). Nucleotide sequencing was performed by the dideoxy chain-termination method (T7Sequencing kit, Pharmacia). Plasmid DNA was
isolated by the alkaline lysate method, as described by Sambrook et al.
(1989).
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RESULTS |
Comparison of Bovine-Brain and Recombinant Wild-Type Calmodulin
The recombinant wild-type calmodulin we prepared is identical in
many ways to the natural bovine-brain calmodulin: it can be purified by
phenyl-Sepharose column chromatography; it has a similar UV spectrum;
and it can activate PDE and be inhibited by ophiobolin A to the same
extent. The concentration of ophiobolin A required for half-maximal
inhibition for the recombinant wild-type calmodulin (Fig. 1, middle)
was found to be 1.5 µM.
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| Figure 1.
The effects of single-amino acid mutations on the
ability to activate PDE (top), the inhibition by ophiobolin A (middle),
and the extent of ophiobolin A binding of calmodulin (bottom). Top, PDE
activation assay was done in a final reaction volume of 90 µL,
containing 0.0015 unit bovine brain PDE, 0.03 unit 5-nucleotidase, 40 mM Tris-HCl, 40 mM imidazole, 5 mM
magnesium acetate, and 0.5 mM CaCl2, pH 7.5. Wild-type or mutant calmodulins were added to the concentrations
indicated. The reaction mixture was preincubated at 30°C for 1 min
before the addition of cAMP to a final concentration of 1.2 mM to start the enzyme
reaction. After 30 min at 30°C, the reaction was stopped by the
addition of 910 µL of water and the amount of phosphate was measured.
Maximal activation is the PDE activity at 45 nM wild-type
calmodulin minus the basal activity (activity in the absence of
calmodulin). The extent of phosphodiesterase activation by lower
concentrations of wild-type or mutant calmodulins was expressed as a
percentage of the maximal activation. The measurements were separated
into two experiments. Middle, Inhibition of calmodulins by ophiobolin A
was measured by the decrease in the activation of PDE. The reaction
conditions were as in the top graph, except that the calmodulin
concentration was kept at 15 nM. Ophiobolin A was added as
a 1 or 5 mM solution in methanol to the desired
concentrations. The reaction mixtures were then incubated at 30°C for
30 min before the addition of cAMP. The PDE activity minus the basal
activity in the absence of ophiobolin A was taken as the maximal
activity. PDE activity at each concentration of ophiobolin A was
expressed as a percentage of the maximal activity. All measurements
were made in two experiments. Bottom, Extent of ophiobolin A binding to
each mole of calmodulin. Aliquots of 10 µM calmodulin in
40 mM Tris-HCl, pH 7.5, and 1 mM
CaCl2 were incubated with various concentrations of
ophiobolin A. All measurements were made in two experiments. All
experiments were repeated at least two times.  , Wild-type
calmodulin;  , K75; ×, K75Q;  , K75R;  , K77;  ,
K148;  , K148Q;  , K148R.
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Effects of Single-Amino Acid Mutations
In a previous study it was suggested that ophiobolin A may
interact with Lys residues 75 and 148 in the calmodulin molecule (Leung
et al., 1988). Therefore, we started by introducing mutations at these
two positions in the calmodulin molecule. To assess the effects of
mutations on calmodulin, three parameters were used: (a) the ability to
activate PDE, (b) the extent of inhibition by ophiobolin A, and (c) the
extent of binding by ophiobolin A. Figure
1 (top) shows the effect of these
mutations in the activation of PDE. Deletion of Lys-75 (K75) or
substitution by Gln (K75Q) or Arg (K75R) had little effect on the
activation of PDE. These mutant calmodulins could maximally activate
PDE with Kact values of 5, 8.5, and 7 nM, respectively. The mutants K148, K148Q, and K148R
could also activate PDE to a maximum with
Kact values of 8.5, 6.5, and 6.5 nM, respectively. The PDE prepared in this study could be
activated approximately 6.5-fold by natural bovine-brain calmodulin
with a Kact of 6.5 nM.
The mutants were then assayed for their extent of inhibition by
ophiobolin A. As shown in Figure 1 (middle), the K75 mutants (K75,
K75Q, and K75R) were only partially inhibited by the toxin. The
concentration of ophiobolin A required for half-maximal inhibition increased from 1.5 µM for wild-type calmodulin to 9 µM for the mutants. In contrast, the inhibition curves of
the K148 mutants (K148, K148Q, and K148R) were almost identical to
that of the wild-type calmodulin. Therefore, Lys-75 is a site for
ophiobolin A inhibition and Lys-148 is not.
The mutants were further analyzed for their ophiobolin A-binding
capacity. The results show that the wild-type calmodulin bound 2 mol of
ophiobolin A, the K75 mutants bound about 1.7 mol, and the K148 mutants
bound about 1.4 mol of ophiobolin A per mol of calmodulin (Fig. 1,
bottom). The decrease in binding in the K75 mutants was small. The
decrease in the K148 mutants was large and indicates that Lys-148 is a
binding site for ophiobolin A.
If Lys-75 were the only site of inhibition by ophiobolin A, then
removing it by deletion or substitution should have removed all of the
inhibition. However, there was still some inhibition left in the K75
mutants (Fig. 1, middle), suggesting that an inhibition site other than
Lys-75 and Lys-148 may be responsible for the inhibition left in the
K75 mutants. Lys-77, which is in proximity to Lys-75, could be a
potential site of inhibition, because in the three-dimensional
structure of calmodulin, Lys-75 and Lys-77 were shown to be in a
similar environment (Babu et al., 1988). Therefore, a Lys-77 deletion
mutant (K77) was prepared. This mutant calmodulin could activate PDE
normally (Fig. 1, top) and bound about 1.7 mol of ophiobolin A per mol
of calmodulin, like the K75 mutants (Fig. 1, bottom). The mutant could
be inhibited by ophiobolin A, like the wild-type calmodulin (Fig. 1,
middle). This result shows that Lys-77 is not a site of inhibition.
Effects of Double-Amino Acid Mutations
Next, we studied the effect of changing two Lys residues at a
time. Three mutants (K75,77; K77,148R; and K75,148R) were made. They could activate PDE to near maximum in spite of the mutations
and their Kact values were 4, 4, and 5.5 nM, respectively (Fig. 2,
top). The ophiobolin A inhibition assay
showed that mutant K75,77 was only minimally inhibited by ophiobolin
A (Fig. 2, middle). This resistance was most prominent at lower
concentrations of ophiobolin A. At 20 µM ophiobolin A the
mutant calmodulin was inhibited by less than 10%, whereas the
wild-type calmodulin was inhibited by 90%. Mutants K77,148R and
K75,148R were produced to distinguish the effect of these two
residues without the contribution of Lys-148. It was found that
K77,148R was inhibited by ophiobolin A as easily as the wild-type
calmodulin (Fig. 2, middle). Like the K75 single-amino acid mutants,
K75,148R exhibited partial resistance to the toxin, but the extent
of resistance was larger in K75,148R (Fig. 2, middle).
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| Figure 2.
Effects of double-amino acid mutations on the
ability to activate PDE (top), the inhibition by ophiobolin A (middle),
and the extent of ophiobolin A binding of calmodulin (bottom).
Experimental details are as described in the legend to Figure 1. All
measurements were made in one experiment. All experiments were repeated
at least two times. , Wild-type calmodulin; , K75,77; ×,
K75,148R; , K77,148R.
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When the ophiobolin A-binding profiles of these double-amino acid
mutants were determined, it was found that these three mutants bound
only 1 mol of ophiobolin A per mol of calmodulin (Fig. 2, bottom).
Effects of Triple-Amino Acid Mutations
To explore the combined effect of Lys-75, Lys-77, and Lys-148 in
the ophiobolin A inhibition, triple-amino acid mutants (K75,77,148R; K75,77,148; K75,77,148Q; and K75,77,148R) were produced. In these mutants the three Lys residues were removed either by deletion or
substitution. All of the mutants could activate PDE to approximately maximum. The Kact values were 4.5, 5.5, 6.5, and 7 nM, respectively (Fig. 3,
top). These mutants exhibited a great
deal of resistance to ophiobolin A inhibition (Fig. 3, middle). For
unknown reasons, mutants with deletions in Lys-75 and Lys-77 provided
more resistance than mutants with substitutions in these two positions.
In the binding experiment, the extent of ophiobolin A binding to these triple-amino acid mutants was about 0.6 mol of ophiobolin A per mol of
calmodulin (Fig. 3, bottom). This fractional binding was probably
caused by the binding of ophiobolin A to some nonspecific sites,
because the initial rate of binding was very slow. For example, at 100 µM ophiobolin A, the rate of binding of ophiobolin A to
the triple-amino acid mutant K75,77,148R was at least 30 times
smaller than the binding rate to the wild-type calmodulin (data not
shown).
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| Figure 3.
The effects of triple-amino acid mutations on the
ability to activate PDE (top), the inhibition by ophiobolin A (middle),
and the extent of ophiobolin A binding of calmodulin (bottom).
Experimental details are as described in the legend to Figure 1. All
measurements were made in one experiment. All experiments were repeated
at least two times. , Wild-type calmodulin; , K75,77,148R; ×,
K75,77,148; , K75,77,148Q; , K75,77,148R.
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Effects of Introducing Additional Lys Residues
The results presented above established that Lys-75 in the
calmodulin molecule was responsible for the inhibitory action of ophiobolin A. To determine if other Lys residues in plant calmodulin also react with ophiobolin A, additional Lys residues were introduced in positions 86 and/or 143 of the triple-amino acid mutant K75,77,148R. These two Lys residues are unique in plant calmodulins and are present
in nearly all plant calmodulins characterized so far (Poovaiah and
Reddy, 1993). As shown in Figure 4, the
mutant calmodulins (R86K, Q143K and R86, Q143K) were nearly identical
to the parental molecule (K75,77,148R) in terms of PDE-activating
ability, the extent of inhibition by ophiobolin A, and the extent of
binding by ophiobolin A. This indicates that the additional Lys
residues in the mutant calmodulins do not react with ophiobolin A.
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| Figure 4.
Effects of introducing additional Lys residues on
the ability to activate PDE (top), the inhibition by ophiobolin A
(middle), and the extent of ophiobolin A binding of calmodulin
(bottom). Experimental details are as described in the legend to Figure
1. All experiments were repeated once. , Wild-type calmodulin; ,
K75,77,148R; ×, R86K; , Q143K; , R86,Q143K.
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DISCUSSION |
We used site-directed mutagenesis to locate the ophiobolin
A-binding sites in the calmodulin molecule. This approach is based on
the idea that the removal of the reactive Lys residues should result in
a decrease in the extent of inhibition and the extent of binding by
ophiobolin A in the calmodulin molecule. Three types of mutations were
made: deletion of the Lys residue, changing Lys to Gln, and changing
Lys to Arg. Gln and Arg were chosen because both contain nitrogen in
the side chain, and this nitrogen does not form a Schiff base with
carbonyl groups like it does in Lys. Therefore, the reaction between
calmodulin and ophiobolin A was eliminated because the reaction between
calmodulin and ophiobolin A was proposed to be a Schiff-base reaction
(Leung et al., 1988).
Gln has an 
-helix-forming propensity similar to that of Lys
(Maxfield and Scheraga, 1979) and would preserve the structure at the
Lys site. With Arg, the positive charge in the side chain is preserved.
All mutant calmodulins show properties in common with those of the
natural bovine-brain calmodulin. They could be purified by
Ca2+-dependent phenyl-Sepharose chromatography,
indicating that the mutations had not affected the
Ca2+-induced exposure of hydrophobic domains in
the calmodulin molecule. The UV spectra of all mutant calmodulins were
similar to that of the natural bovine-brain calmodulin. We also looked
at the electrophoretic mobility of the mutant calmodulins under
denaturing and nondenaturing conditions, and the results did not
suggest any great change in the structure of the mutant calmodulins
(data not shown).
Using this series of mutants we confirmed the previous suggestion that
Lys-75 and Lys-148 are the binding sites for ophiobolin A (Leung et
al., 1988), and found that Lys-77 is also a binding site. Binding means
the presence of a covalent interaction between ophiobolin A and the
-amino group of the Lys residue (Leung et al., 1988). The
double-amino acid mutants K75,77; K77,148R; and K75,148R (Fig.
2, bottom) bind 1 mol of ophiobolin A per mol of calmodulin over a wide
range of ophiobolin A concentrations, showing that each of the three
binding sites can bind one molecule of ophiobolin A and has similar
affinity for the toxin.
For K75,77, ophiobolin A was bound to Lys-148; for K75,148R, it
was bound to Lys-77; and for K77,148R, it was bound to Lys-75. These
assignments are supported by the results with the triple-amino acid
mutants. When all three Lys residues were removed, the binding was
almost abolished (Fig. 3, bottom). The residual fractional binding in
the triple-amino acid mutants was probably caused by nonspecific
binding, because the rates of these fractional binding reactions were
much slower than that of the specific binding reactions with the
wild-type calmodulin. These nonspecific binding sites are presumably
the other Lys residues in the calmodulin molecule.
Although there are three specific binding sites for ophiobolin A, the
data show that only two sites are used, because the maximum number of
moles of ophiobolin A bound to each mole of wild-type calmodulin was 2. The same molar ratio was obtained for the natural bovine-brain
calmodulin (Leung et al., 1988). A possible explanation is that one of
the three sites is hindered. This conclusion is based on the fact that
the maximum binding was only about 1 mol of ophiobolin A per mol of
calmodulin for the K148 single-amino acid mutants (Fig. 1, bottom), in
which Lys-148 was removed and Lys-75 and Lys-77 remained intact in the calmodulin molecule. This binding ratio suggests that although Lys-75
and Lys-77 can bind one molecule of ophiobolin A independently, they do
not combine to give two binding sites for ophiobolin A. Lys-77 is the
most probable hindered site. Lys-75 is not the hindered site; the
inhibition assay revealed it to be the main binding site. It is
possible that the binding of ophiobolin A to Lys-75 sterically prevents
another molecule of ophiobolin A from binding to Lys-77 because Lys-75
and Lys-77 are very close to each other.
The fractional binding in the single mutants (Fig. 1, bottom) might be
explained as follows. The 1.4 value for the K148 mutants might have
been caused by nonspecific binding added to the specific binding value
of 1.0. The 1.7 value for K75 and K77 mutants might have been
attributable to some structural changes that lowered the binding
capacity of the mutants from a value of 2.0.
The binding of ophiobolin A to calmodulin does not necessarily inhibit
calmodulin. Inhibition means the loss of the ability to activate PDE.
For example, Lys-148 can bind ophiobolin A but does not seem to be an
inhibition site, because the removal of Lys-148 in each of the K148
single-amino acid mutants did not reduce the inhibition by ophiobolin
A.
The results show that Lys-75 alone was responsible for all of the
inhibition by ophiobolin A. Whenever Lys-75 was removed, either by
deletion or by substitution, the inhibition by ophiobolin A was
reduced. Also, whenever Lys-75 was present in the calmodulin molecule,
whether Lys-77 and Lys-148 were present, the calmodulin was as easily
inhibited by ophiobolin A as wild-type calmodulin.
Although Lys-77 can bind ophiobolin A, it does not seem to be a site of
inhibition. If Lys-77 were another site of inhibition, there should
have been a reduction of inhibition whenever Lys-77 was removed from
the calmodulin. No such reduction in inhibition was observed in any of
the K77 mutants when Lys-75 was intact. The mutants K77 and
K77,148R (Figs. 1 and 2, middle panels) were as easily inhibited by
ophiobolin A as the wild-type calmodulin. This result is consistent
with the finding that Dictyostelium discoideum calmodulin,
which contains a Gln at position 77 and a Lys at position 75, was as
easily inhibited by ophiobolin A as the bovine-brain calmodulin (Leung
et al., 1988). However, when Lys-75 is removed, Lys-77 seems to become
a site of inhibition. This is suggested by the partial inhibition left
in the K75 single-amino acid mutants (Fig. 1, middle), which was caused
by the binding of ophiobolin A to Lys-77, because when Lys-77 was
also removed, as in the double-amino acid mutant K75,77, the partial
inhibition was also removed and the mutant became very resistant to
ophiobolin A.
Calmodulins from many sources contain seven Lys residues and one
trimethyl-Lys residue. Of the seven Lys residues, Lys-75 and Lys-148
have been shown to react with a number of chemical reagents (Jackson
and Puett, 1984; Faust et al., 1987; Newton and Klee, 1989). The
interaction of ophiobolin A with calmodulin is in many respects similar
to that of synthetic, therapeutic phenothiazines. Both compounds bind
calmodulin in a Ca2+-dependent manner and in a
2:1 molar ratio. The phenothiazines norchlorpromazine
isothiocyanate (Newton et al., 1983) and 10-(3-propionyloxy succinimide)-2-(trifluoromethyl)phenothiazine (Faust et al., 1987) were also reported to label Lys-75 and Lys-148. It is interesting that
a natural product possesses properties similar to those of a synthetic
compound. However, ophiobolin A represents a calmodulin inhibitor of a
different chemical class (a sesterterpenoid). It can be a prototype for
further synthesis of a highly specific calmodulin inhibitor.
Although the current research was done using calmodulin from bovine
brain, the results can most likely be extended to plant calmodulins.
Ophiobolin A has been shown to inhibit spinach and maize calmodulin
(Leung et al., 1984, 1985) in the same manner as bovine-brain
calmodulin. The sequence and structure of calmodulin is conserved.
Maize, rice, and spinach calmodulins also contain Lys in positions
equivalent to Lys-75, Lys-77, and Lys-148 of bovine-brain calmodulin
(Poovaiah and Reddy, 1993). More importantly, the introduction of Lys
residues unique to plant calmodulins does not increase the inhibition
and binding by ophiobolin A (Fig. 4). The results presented in this
paper show that ophiobolin A reacts quite specifically with calmodulin
and that this could be the mechanism of action of ophiobolin A.
Although we have suggested that the introduced mutations did not cause
any large structural changes in the mutant calmodulins, small changes
in the local environment of the molecule most likely occurred. These
changes may have caused some slight variations in the interaction of
the mutant calmodulins with the PDE enzyme or ophiobolin A. If this is
true, then some of the variations between mutants may be explained by
these local molecular variations. For example, the unexpected slightly
higher resistance to ophiobolin A in K75,148R than in K75 (Figs.
1 and 2, middle panels) might have been caused by a slight variation in
the molecular environment between Arg and Lys at residue 148. The same
argument may be applied to explain why the triple mutants with Lys-75
and Lys-77 deleted were more resistant to ophiobolin A than those with
substitutions in these two positions (Fig. 3, middle).
We have not explored in detail the structural changes that may have
occurred as a result of mutagenesis and the role of hydrophobic amino
acid residues in the interaction between ophiobolin A and calmodulin.
Further studies of these two aspects will give a more complete picture
of the interaction between these two interesting molecules.
| |
FOOTNOTES |
1
This project was supported by a grant from the
Research Grants Council of Hong Kong.
*
Corresponding author; e-mail pcleung{at}hkucc.hku.hk; fax
852-2857-4672.
Received April 21, 1998;
accepted July 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Kact, the
concentration of calmodulin required for half-maximal activation of
PDE.
PDE, calmodulin-dependent cyclic nucleotide phosphodiesterase.
| |
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Abstract
Introduction