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J. Biol. Chem., Vol. 276, Issue 38, 35546-35551, September 21, 2001
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From the
Received for publication, March 1, 2001, and in revised form, July 12, 2001
The four salt bridges
(Asp222-Arg281,
Arg233-Glu288,
Arg234-Glu274, and
Asp242-Arg265) linking domains I and II
in Cry1Aa were abolished individually in During sporulation, Bacillus thuringiensis produces a
parasporal crystal body composed of one or more proteins that are
toxic to a number of insect larvae (1) or to other invertebrates (2).
After solubilization in the insect midgut and activation by intestinal
proteases, these proteins bind to specific receptors at the surface of
the apical brush border membrane of epithelial columnar cells, insert
into the membrane, and form pores that disrupt midgut cellular
functions (3-5).
Elucidation of the crystal structure of the coleopteranspecific
Cry3A toxin (6) and the lepidopteran-specific Cry1Aa toxin (7) revealed
a similar three-domain structure for both proteins. Domain I, composed
of eight amphipathic These domains are closely packed together with the largest number
of interdomain contacts found between domains I and II (6, 7). In
Cry1Aa, domains I and II are linked by four salt bridges: Asp222-Arg281,
Arg233-Glu288,
Arg234-Glu274, and
Asp242-Arg265 (7) (Fig.
1). Three salt bridges, structurally
equivalent to Arg233-Glu288,
Arg234-Glu274, and
Asp242-Arg265, are also found in Cry3A (6, 7).
In addition, for all four salt bridges at least one of the amino acid
residues forming the bridge is located within block 2, a sequence that
is highly conserved among Cry toxins (1, 5, 36). However, only in
the case of the Asp242-Arg265 salt bridge are
both amino acids located within block 2 (Fig. 1). Salt bridges appear
to play an important role in toxin stability and function. Mutations
preventing the formation of the Asp242-Arg265
(37, 38), Arg233-Glu288 (38), or
Arg234-Glu274 (38) salt bridges in Cry1Ab
resulted in substantial losses of stability or activity. The importance
of these salt bridges is further supported by the observation that
domain I exchanges between different Cry1 proteins can lead to inactive
recombinant proteins (39), possibly caused by the absence of one or
more essential salt bridges. In contrast, disulfide bond engineering experiments with Cry1Aa have demonstrated that pore formation requires
domain I to swing away from the rest of the molecule (14), implying
that the interdomain salt bridges must be broken during toxin insertion
into the membrane.
In the present study, each of the four salt bridges in Cry1Aa was
eliminated by site-directed mutagenesis. Because others have shown that
Cry1A mutations at Asp242 or Arg265 may result
in unstable proteins (37, 38), various amino acid alterations were
created at Asp242 including a double mutant D242A/R265A.
Because the highly alkaline pH of the lepidopteran midgut, which
ranges between 8 and 12 (40, 41), is thought to play an important role
in toxin function (5), all stable mutants were tested at pH values of
7.5 and 10.5. Although all mutations caused a substantial loss in
toxicity, several mutants retained a capacity to form pores in midgut
brush border membrane vesicles that was comparable with that of the
respective parental toxins.
Mutagenesis--
Cry1Aa mutants were created by
oligonucleotide-directed in vitro mutagenesis using the
Altered Sites II kit (Promega, Madison, WI) as recommended by the
manufacturer. Mutated genes were subcloned in the pBU4
Escherichia coli-B. thuringiensis shuttle vector
(42) and electroporated in the B. thuringiensis HD-1
acrystalloferous strain Cry Toxin Activation and Purification--
Wild-type Cry1Aa and
Cry1Aa mutant protoxins were produced as parasporal crystals in
B. thuringiensis grown at 27 °C in BP medium (45)
containing 10 mg/ml glucose. Crystals were purified with Renografin
gradients, solubilized, and trypsin-activated as described previously
(46). Wild-type Cry1Ac and Cry1Ac mutant protoxins were produced as
insoluble inclusions in E. coli and solubilized and
activated as described (44). Activated toxins were purified by fast
protein liquid chromatography using a Mono Q ion exchange column
(Amersham Pharmacia Biotech), and bound toxin was eluted with a 50-500
mM NaCl gradient in 40 mM carbonate buffer, pH
10.5 (44). The purity and integrity of all proteins were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (47), and
protein concentrations were determined by the Bradford method (48)
using bovine serum albumin as a standard.
Bioassays--
Manduca sexta fertilized eggs were
obtained from the Carolina Biological Supply Company (Burlington, NC).
Toxicity assays were performed on neonate larvae with trypsin-activated
toxins. Toxins were diluted in phosphate-buffered saline (8 mM Na2HPO4, 2 mM
KH2PO4, and 150 mM NaCl, pH 7.4).
Toxin samples (100 µl) were layered onto artificial diet in
2-cm2 wells and allowed to dry. A single larva was placed
in each well and was reared at 27 °C and 70% relative humidity with
a photoperiod of 12 h of light and 12 h of darkness. Five
groups of at least 30 larvae were tested at each toxin concentration.
Mortality was recorded after 7 days and adjusted for mortality of
control larvae that were reared in the absence of toxin. Cry1Aa and
Cry1Ac were tested at five different toxin concentrations ranging from
25 to 200 ng/ml for Cry1Aa and from 12.5 to 75 ng/ml for Cry1Ac, and
the data were analyzed by probit analysis (49). Mutant proteins were
tested at 2 µg/ml and depending on the mortality rate observed at
this concentration, the tests were repeated at either 50 ng/ml or 50 µg/ml.
Preparation of Brush Border Membrane Vesicles--
Whole midguts
were isolated from fifth-instar M. sexta larvae and freed of
the attached Malpighian tubules. The midguts were then transected
longitudinally to remove the peritrophic membranes and gut contents,
rinsed thoroughly in ice-cold 300 mM sucrose, 5 mM EGTA, and 17 mM Tris/HCl, pH 7.5 and stored
at Light-scattering Assay--
Brush border membrane permeability
was analyzed with an osmotic swelling assay (51). At least 1 h
prior to the experiments, the vesicles were further diluted to a final
concentration of 0.40 mg of membrane protein/ml by the addition
of 10 mg/ml bovine serum albumin to the buffer in which they were
equilibrated. Toxins were assayed after a 60-min preincubation period
with the vesicles to analyze the properties of the pores after having
ample time to form or without preincubation to monitor the rate at
which the toxins increased membrane permeability. The assays were
initiated by rapidly mixing the vesicles with an equal volume of 10 mM Hepes/KOH, pH 7.5 or CAPS/KOH, pH 10.5, 1 mg/ml bovine
serum albumin, and 150 mM KCl or 300 mM sucrose
or raffinose using a Hi-Tech Scientific (Salisbury, U K) stopped-flow
rapid kinetics apparatus. For rate assays, 150 pmol of toxin/mg of
membrane protein were added to the KCl substrate solution before mixing
with the vesicles. Scattered light intensity was monitored at an angle
of 90° at 23 °C in a Spex Fluorolog CM-3 (Jobin Yvon Horiba,
Edison, NJ) spectrofluorometer with monochromators set at 450 nm.
Data Analysis--
Percent volume recovery was defined as 100 (1 Protoxin Expression and Activation--
Whereas the production and
trypsin-activation of most mutants were essentially equivalent to those
of the respective parental toxins, the Cry1Ac mutants D242K and D242E
could not be expressed in sufficient quantities for bioassays or
light-scattering experiments. It was also found that the Cry1Aa single
mutant R265A and the double mutant D242A/R265A were almost immediately
degraded upon exposure to trypsin. Therefore, these mutants could not
be further analyzed.
Toxicity--
In agreement with previous studies (52, 53), both
Cry1Aa and Cry1Ac were strongly toxic to M. sexta larvae
with Cry1Ac being twice as toxic as Cry1Aa (Table
I). In comparison, all mutants
tested were substantially less toxic (Table
II). The loss of toxicity was most
evident for the Cry1Aa mutant D242A and the Cry1Ac mutant D242P and
least pronounced for the Cry1Aa mutant D222A and the Cry1Ac mutant
D242N.
Pore-forming Ability--
The ability of each mutant to form
channels in the apical membrane of the insect midgut was analyzed with
a light-scattering assay (51). When brush border membrane vesicles,
previously incubated for 60 min with either of the mutant toxins, were
rapidly mixed with a hypertonic KCl solution, two distinct responses
were observed (Fig. 2). Following a rapid
shrinking of the vesicles (due to the hypertonic shock as evidenced by
a rapid increase in scattered light intensity) the vesicles either
swelled rapidly, at a rate dependent on toxin concentration as was the
case for D222A (Fig. 2A), or at a rate comparable with that
observed for control vesicles, independent of toxin concentration as
observed for D242A (Fig. 2B). Although a contribution of
buffer efflux during vesicle shrinking cannot be excluded, rapid
swelling demonstrates a substantial increase in the KCl permeability of
the vesicles when exposed to an active toxin.
To summarize the results, percent volume recovery after 3 s
(derived from traces like those shown in Fig. 2) was plotted as a
function of toxin concentration (Fig. 3).
In contrast with D242A, the other Cry1Aa mutants, D222A, R233A, and
R234A, as well as the Cry1Ac mutants, D242N and D242P, caused a
dose-dependent increase in the permeability of the vesicles
comparable with that observed with the respective parental toxins (Fig.
3). Similar results were obtained at pH values of 7.5 and 10.5, although at the latter a slightly but significantly higher permeability
than with Cry1Aa was observed with the three Cry1Aa active mutants at
the lower toxin concentrations.
All active toxins tested at 150 pmol of toxin/mg of membrane protein
also permeabilized the membrane for sucrose and raffinose (Table
III). Cry1Aa and its three active mutants
caused a greater increase in the permeability of the vesicles to
sucrose at pH 10.5 than at pH 7.5 (Fig.
4, A and C). This
was also true for Cry1Ac but not for its two mutants (Fig. 4,
B and D). A similar pattern was observed in the
presence of raffinose although percent volume recovery at both pH
values only became significantly different for Cry1Aa, Cry1Ac, and the
Cry1Aa mutants when measured after more than ~5 s (Fig. 4,
E-H).
Rate of Pore Formation--
To investigate the kinetics of pore
formation following toxin exposure, vesicles were mixed simultaneously
with toxin and KCl. Under these conditions the vesicles began to swell
after a short delay (Fig. 5). Cry1Ac
caused the vesicles to swell more rapidly and after a shorter delay
than Cry1Aa. For both toxins, however, increasing the pH from 7.5 to
10.5 resulted in a slightly longer delay and reduced swelling rate. The
Cry1Aa mutants caused a pH-dependent increase in the rate
of vesicle swelling relative to that observed in the presence of
Cry1Aa. Although at pH 7.5 a significantly higher swelling rate
was observed only for D222A (Fig. 5A), at pH 10.5 all three
mutants caused a significantly more rapid swelling of the vesicles than
Cry1Aa (Fig. 5C). On the other hand, at both pH values the
rate of vesicle swelling was similar in the presence of Cry1Ac or
either one of its mutants, D242N and D242P (Fig. 5, B and
D).
The objective of the present study was to investigate the role of
the salt bridges linking domains I and II in Cry1Aa and Cry1Ac.
Although these two toxins, along with Cry1Ab, share a high level of
homology and are therefore expected to have similar pore-forming
properties, mutations preventing the formation of the
Asp242-Arg265 salt bridge had diverse effects
on expression, stability, and function. Whereas the Cry1Aa mutant D242A
could be produced normally but had very poor toxicity and pore-forming
ability, mutants in which the arginine at position 265 was replaced by
an alanine residue in the same protein were extremely sensitive to
degradation by trypsin. These results correlate well with those from
previous studies showing that mutations at either Asp242,
Arg265, or both positions in Cry1Ab resulted in unstable
and inactive mutant proteins (37, 38).
Cry1Ac was modeled using the published Cry1Aa (7) and Cry3A (6)
three-dimensional structures as templates (54). Interestingly, although
Cry1Aa possesses four salt bridges in the interdomain region, there
were only two salt bridges in Cry1Ac located in the same positions as
those in Cry1Aa; no salt bridge was apparent between residues
Asp222 and Arg281 or between residues
Arg234 and Glu274. The predicted atomic
distances of the Cry1Ac salt bridges are as follows:
Arg233-Glu288, 2.85 Å, and
Asp242-Arg265, 2.27 Å and 2.96 Å. The former
salt bridge is identical to that found in Cry1Aa, whereas the latter
appears to be stronger.
The properties of the Cry1Ac Asp242 mutants depended
greatly on the nature of the mutation. Whereas D242E and D242K
could not be produced, D242N and D242P retained a wild-type
pore-forming ability in brush border membrane vesicles. Although an
effect on gene expression cannot be excluded, the extremely poor
accumulation of D242E or D242K in the bacterial cell was likely due to
a structural defect causing these mutant proteins to be rapidly
degraded by endogenous proteases. In the case of D242K, normal
polypeptide chain folding would result in the juxtaposition of two
positive charges at a position normally occupied by a salt bridge. The replacement of the aspartic acid residue by a glutamic acid residue in
D242E, however, does not introduce a modification in the charge of this
residue but simply increases the length of its side chain by a single
methylene group. This modification appears to be sufficient either to
prevent the formation of the salt bridge by improper folding of the
protein or, if the salt bridge is indeed formed, to introduce an angle
or a gap between The fact that D242N and D242P retained good pore-forming activity was
surprising because a D242N mutant in Cry1Ab was among those previously
reported to be unstable (37). Moreover, the introduction of a
helix-interrupting proline residue at this position was expected to
have profound effects on the structure of the protein by destabilizing
the structure of Mutations that prevented the formation of any single salt bridge
linking domains I and II in Cry1Aa or the
Asp242-Arg265 salt bridge in Cry1Ac clearly
resulted in a substantial decrease in toxicity of the protein. At 2 µg of toxin/ml, a concentration 32-fold higher than the
LC50 measured for Cry1Aa and 61-fold higher than that of
Cry1Ac, only D222A and D242N were able to kill more than 50% of the
larvae. The toxicity level retained by the Cry1Aa mutants was directly
related to the position of the abolished salt bridge relative to the
hinge region linking domains I and II (7). Removal of the salt bridge
located farthest from the hinge region (D222A, Fig. 1) resulted in a
toxin with the highest toxicity, whereas elimination of the salt bridge
located closest to the hinge region (D242A) rendered the mutant protein
essentially non-toxic. R233A and R234A, in which the salt bridges
located near the middle of Except for D242A, a reduction in toxicity could not be attributed to a
loss in the pore-forming ability of the mutants. The increase in
vesicle permeability to KCl, sucrose, and raffinose observed with each
of the active mutants was comparable with that observed with the
respective parental toxins following a 60-min preincubation of the
vesicles with the toxin. Moreover, the rate at which membrane
permeability increased following exposure to the toxin was at least as
high for the mutants as for the wild-type toxins. These results
contrast with those of another study in which the R233A mutant of
Cry1Ab was found to have a significantly reduced ability to inhibit the
short circuit current through the epithelium of midguts isolated from
Lymantria dispar (38).
With the Cry1Aa mutant D222A at pH 7.5 and all three Cry1Aa active
mutants at pH 10.5, membrane permeability increased faster upon
exposure to the mutants than in the presence of the parental toxin.
This result suggests that the removal of one of the salt bridges
facilitates the insertion of the toxin into the membrane, at least
under in vitro conditions. Interestingly, at pH 10.5 the
rate at which vesicle permeability increased in the presence of these
mutant toxins correlated with toxicity and, as mentioned above,
with the position of the missing salt bridge relative to the hinge
region. A faster rate of membrane insertion may increase the toxicity
of the mutants by reducing the time they are exposed to proteases in
the midgut. Cry1Aa was nevertheless considerably more toxic than any of
its mutants, and removal of a salt bridge is very likely to have
increased the susceptibility of the mutants to midgut proteases.
Cases of toxins with a strong capacity to form pores in
vitro despite a relatively poor toxicity have been reported
previously (25, 55, 56). As was discussed extensively (55), this may
result from a poorer stability or a stronger sensitivity to proteolysis
under the harsh conditions encountered in the insect midgut than in the
somewhat idealized in vitro conditions. Among the intestinal
factors that could affect toxin activity, the strongly alkaline pH
characteristic of the larval midgut of lepidopteran insects (40, 41)
had only minor effects on the in vitro activity of Cry1Aa
and Cry1Ac. A slightly higher permeability to sucrose and raffinose was
nevertheless observed at pH 10.5 than at pH 7.5, consistent with a
slight increase in pore size with increasing pH as was previously
reported for Cry1Ac (57). A similar effect of pH was observed for all
three Cry1Aa mutants, but it was not detected with either of the Cry1Ac mutants.
In conclusion, our results along with those of other laboratories (37,
38) clearly show that the interdomain salt bridges linking domains I
and II play an important role in toxin stability and toxicity.
Presumably, these salt bridges are necessary to maintain the toxin in a
tightly packed conformation in the midgut until, after binding to its
membrane receptor, the toxin undergoes a conformational change leading
to insertion into the membrane and pore formation. In the absence of
proteases or other intestinal factors yet to be identified, removal of
either one of these salt bridges does not necessarily alter the
toxin's capacity to form a pore in the midgut membrane. On the other
hand, under in vitro conditions removal of a salt bridge can
even increase the rate of pore formation by Cry1Aa.
*
This work was supported by a research grant from the Natural
Sciences and Engineering Research Council of Canada (to R. L. and
J.-L. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a Graduate Student Fellowship from the
Ministère de l'Éducation et de la Recherche of France.
**
To whom correspondence should be addressed: Groupe de recherche en
transport membranaire, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, H3C 3J7, Canada. Tel.:
514-343-7960; Fax: 514-343-7146. E-mail:
raynald.laprade@umontreal.ca.
Published, JBC Papers in Press, July 20, 2001, DOI 10.1074/jbc.M101887200
1
The abbreviation used is CAPS,
3-(cyclohexylamino)propanesulfonic acid.
Role of Interdomain Salt Bridges in the Pore-forming Ability of
the Bacillus thuringiensis Toxins Cry1Aa and Cry1Ac*
§¶,,
,,,,,**, and Groupe de recherche en transport
membranaire, Université de Montréal, Montreal, Quebec, H3C
3J7, Canada, § IGEPAM, CIRAD, Montpellier, France and
the Biotechnology Research Institute, National Research Council,
Montreal, Quebec, H4P 2R2, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix 7 mutants
D222A, R233A, R234A, and D242A. Two additional mutants targeting the
fourth salt bridge (R265A) and the double mutant (D242A/R265A) were
rapidly degraded during trypsin activation. Mutations were also
introduced in the corresponding Cry1Ac salt bridge (D242E, D242K,
D242N, and D242P), but only D242N and D242P could be produced. All
toxins tested, except D242A, were shown by light-scattering experiments
to permeabilize Manduca sexta larval midgut brush border
membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well
as D222A at pH 7.5, demonstrated a faster rate of pore formation than
Cry1Aa, suggesting that increases in molecular flexibility due to the
removal of a salt bridge facilitated toxin insertion into the membrane.
However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting
that increased flexibility made the toxins more susceptible to
proteolysis in the insect midgut. Interdomain salt bridges, especially
the Asp242-Arg265 bridge, therefore
contribute greatly to the stability of the protein in the larval
midgut, whereas their role in intrinsic pore-forming ability is
relatively less important.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices, is thought to be involved in membrane
insertion and pore formation (8-15). Domain II, composed of three
-sheets and two short -helices, is involved in the binding of the
toxin to its receptor on the epithelial cell surface (16-23). Domain
III, composed of two -sheets forming a face-to-face -sandwich,
appears to be involved in the stability (6), specificity (24-26), and
binding (27-34) of the toxin.
View larger version (30K):
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Fig. 1.
Salt bridges between domains I and II in
Cry1Aa. The three-dimensional rendering of the activated protein
crystal (7) was obtained using SPDV software (version 3.51) (35). The
region shown extends from Thr213, in the C-terminal region
of -helix 6, to Arg292 in domain II. It comprises the
loop between -helices 6 and 7 (Val218-Asp222), -helix 7 (Ser223-Tyr250), the hinge region between
domains I and II (Asp251-Arg265), and the
domain II region that contacts domain I, up to Arg292,
which is located between a small -helix
(Ala284-Gln289) and the second -strand
(Asp298-His310) of domain II. Conserved block
2, which starts at Tyr203 and ends at Thr269
just before the small Pro271-Glu274 helix, is
therefore almost totally shown in the figure. Salt bridges, indicated
by dashed lines, have the following atomic distances (from
bottom to top):
Asp222-Arg281, 3.92 Å;
Arg233-Glu288, 2.86 Å;
Arg234-Glu274, 3.01 Å;
Asp242-Arg265, 3.20 Å and 3.18 Å. Note that
only the last one is entirely located within block 2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B as described (43).
Cry1Ac mutants were created in the expression plasmid pMP39 (44) using
the CLONTECH Transformer kit
(CLONTECH, Palo Alto, CA). All mutants were
sequenced using an Applied Biosystems (Foster City, CA) model 370A
automated fluorescent sequencer.
80 °C until use. Brush border membrane vesicles were prepared
by Mg2+ precipitation and differential centrifugation (50).
Vesicles were resuspended at 0.44 mg of protein/ml in 10 mM
Hepes/KOH, pH 7.5 or
CAPS/KOH,1 pH 10.5 and
allowed to equilibrate overnight at 4 °C.
It), where It is the relative scattered
light intensity at time t. For rate assays, percent volume
recovery was calculated for every data point, and values obtained for
control vesicles assayed without toxin were subtracted from the
experimental values measured in the presence of toxin. Data are
reported as means ± S.E. of at least three experiments, each
performed in quintuplicate with different vesicle preparations.
Statistical significance (p < 0.05) was determined
with the two-tailed unpaired student's t test using the
Instat version 1.13 program (GraphPAD Software for Science, San Diego, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Toxicity of Cry1Aa and Cry1Ac toward M. sexta larvae
Toxicity of interdomain salt bridge mutants toward M. sexta larvae
View larger version (20K):
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Fig. 2.
Effect of Cry1Aa interdomain salt bridge
mutants on the osmotic swelling of Manduca sexta brush
border membrane vesicles. Midgut brush border membrane vesicles
isolated from 5th-instar M. sexta larvae and equilibrated in
10 mM Hepes/KOH, pH 7.5 were preincubated 60 min with the
indicated concentrations (in pmol of toxin/mg of membrane protein) of
D222A (A) or D242A (B). Vesicles were mixed
rapidly with an equal volume of 150 mM KCl, 10 mM Hepes/KOH, pH 7.5 directly in a cuvette using a
stopped-flow apparatus. Each trace corresponds to the
average of five experiments.
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Fig. 3.
Effect of interdomain salt bridge mutants on
the KCl permeability of M. sexta brush border membrane
vesicles. Midgut membrane vesicles equilibrated overnight in 10 mM Hepes/KOH, pH 7.5 (A and B) or
CAPS/KOH, pH 10.5 (C and D) were preincubated 60 min with the indicated concentrations of Cry1Aa or either one of the
Cry1Aa mutants (A and C) or with Cry1Ac or one of
its mutants (B and D). Permeability to KCl was
assayed following rapid mixing with 150 mM KCl and 10 mM Hepes/KOH, pH 7.5 (A and B) or
CAPS/KOH, pH 10.5 (C and D), as described in the
Fig. 2 legend. Percent volume recovery at t = 3 s
was derived from activity curves such as those shown in Fig. 2, as
described under "Experimental Procedures."
Effect of interdomain salt bridge mutants on the permeability of
M. sexta brush border membrane vesicles to oligosaccharides
View larger version (32K):
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Fig. 4.
Effect of interdomain salt bridge
mutants on the osmotic swelling of M. sexta brush
border membrane vesicles in the presence of oligosaccharides.
Midgut membrane vesicles equilibrated overnight in 10 mM
Hepes/KOH, pH 7.5 (A, B, E, and
F) or CAPS/KOH, pH 10.5 (C, D,
G, and H) were preincubated 60 min with 150 pmol/mg of membrane protein of Cry1Aa or either one of the Cry1Aa
mutants (A, C, E, and G),
or with Cry1Ac or either one of its mutants (B,
D, F, and H). Permeability to the
disaccharide sucrose and to the trisaccharide raffinose was assayed
following rapid mixing with 300 mM sucrose
(A-D) or raffinose (E-H)
and 10 mM Hepes/KOH, pH 7.5 (A, B,
E, and F) or CAPS/KOH, pH 10.5 (C,
D, G, and H).
View larger version (32K):
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Fig. 5.
Kinetics of pore formation by Cry1Aa, Cry1Ac,
and interdomain salt bridge mutants in M. sexta brush
border membrane vesicles. Midgut membrane vesicles equilibrated in
10 mM Hepes/KOH, pH 7.5 (A and B) or
CAPS/KOH, pH 10.5 (C and D) were mixed with an
equal volume of 150 mM KCl, 10 mM Hepes/KOH, pH
7.5 (A and B) or CAPS/KOH, pH 10.5 (C
and D) and 150 pmol/mg membrane protein of Cry1Aa or either
one of the Cry1Aa mutants (A and C), or with
Cry1Ac or one of its mutants (B and D), without
preincubation with the toxin. Percent volume recovery was calculated
for each experimental point, and control values were subtracted from
those obtained with the toxins. For clarity, error bars are
only shown every 51st data point.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix 7 and the -sheet of domain II. This in
turn may prevent the formation of the other salt bridges because the
Asp242-Arg265 salt bridge is located nearest
to the hinge region linking domains I and II (Fig. 1).
-helix 7. However, when examined by mutational
analysis conducted with the Swiss PDB viewer software on the published
structure of Cry1Aa and on that of Cry1Ac obtained by Swiss Model
modeling, a D242P mutation in -helix 7 was predicted to have very
little effect on the structure of the helix and no overall effect on
the Cry1Aa or Cry1Ac toxin structures. Following energy minimization,
the single-site mutation resulted in a maximum displacement of 0.11 Å of the C
atom at amino acid position 242. It should be
noted, however, that of all the mutants tested in the present study
D242N retained the strongest toxicity, and D242P was, along with D242A
from Cry1Aa, the least toxic to M. sexta larvae.
-helix 7 were abolished, retained an
intermediate level of toxicity.
FOOTNOTES
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Höfte, H.,
and Whiteley, H. R.
(1989)
Microbiol. Rev.
53,
242-255
2.
Feitelson, J. S.,
Payne, J.,
and Kim, L.
(1992)
Bio/Technology
10,
271-275
3.
Gill, S. S.,
Cowles, E. A.,
and Pietrantonio, P. V.
(1992)
Annu. Rev. Entomol.
37,
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