(Received for publication, October 11, 1994; and in revised form, December 8, 1994)
From the
Site-directed mutagenesis was used to generate CryIAb mutants at the selected N-terminal positions to study the function of domain I. Structurally stable mutant proteins were tested for toxicity, receptor binding kinetics, and pore function. Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity appreciably, whereas replacing this tyrosine with aspartic acid (Y153D) resulted in a great loss of toxicity. Mutation of alanine at position 92 to glutamic acid (A92E) almost completely abolished toxicity. The initial receptor binding was unchanged as measured by competition binding assays among all mutant proteins. Reduced pore function, however, was observed for mutants A92E and Y153D as tested by voltage clamping. Further studies with specially designed association and dissociation binding assays showed that irreversible binding of these two mutant toxins to Manduca sexta brush border membrane vesicles was significantly reduced. The decrease in irreversible binding was correlated with the changes in toxicity and may reflect a severely disturbed membrane insertion process in these two mutant toxins, leading to reduced pore function and toxicity. The results support the model that domain I is involved in membrane integration and pore formation.
During sporulation, Bacillus thuringiensis can produce
crystal proteins known as -endotoxins that have been shown to be
toxic to a number of insect larvae in the orders Lepidoptera, Diptera,
and Coleoptera(1) . Recently, toxins with activity against
nematodes have also been reported(2) . It is believed that
after ingestion and solubilization, protoxins are converted to active
toxins that pass through the peritrophic membrane and bind to receptors
on the surface of midgut epithelium cells of susceptible insects before
insertion into the membrane. The inserted toxins disturb the osmotic
balance by generating pores in the cell membrane, leading to cell lysis
and insect death. However, the precise mode of action is not completely
understood.
The correlation of specificity with the presence of high
affinity receptors was demonstrated by Hofmann et al.(3, 4) and Van Rie et al.(5) .
These studies indicated that binding is a crucial step in toxicity.
This conclusion is supported by the isolation of a specific receptor
for CryIAc toxin from Manduca sexta BBMV()(6, 7) . However, it has been
observed that CryI toxins bind specifically to the midgut membrane
preparation from nonsusceptible insects(8, 9) .
Wolfersberger (10) has shown that toxicities of CryIAb and
CryIAc are inversely related to their binding affinity to Lymantria
dispar BBMV. Additionally, Gould et al.(11) have
observed that a CryIAc-resistant Heliothis virescens strain
binds to the toxin as well as the sensitive strain does. Toxin binding
on BBMV has been shown to become irreversible within a short incubation
time, implying a membrane insertion step following the initial
binding(5) . Thus, it appears that post-binding events, such as
membrane insertion and channel formation and function, together with
binding, contribute to the insecticidal activity of
-endotoxins.
Because of the presence of five highly conserved amino acid blocks
among B. thuringiensis toxins(1) , it is believed that
-endotoxins may adopt a similar tertiary structure composed of
three domains, as elucidated in CryIIIA by Li et
al.(12) . Several identified insect specificity regions
are located in domain II (13, 14, 15, 16) , including one
that has been proved to be the Bombyx mori-specific binding
region for CryIAa(17, 18) , indicating that domain II
is a receptor-binding domain. Domain III has been shown to be involved
in structural stability and channel function as well as
specificity(14, 15, 19, 20) . Domain
I, a seven-
-helix bundle covering the most conserved N-terminal
region, is predicted to be the membrane insertion and pore formation
domain. Ahmad and Ellar (21) have found that mutations A165P,
L167M, F50N, and V51N in the N-terminal hydrophobic regions of CryIAa
do not affect binding to tissue culture cells, but lower cytotoxicity.
They concluded that this region is involved in formation of the
transmembrane pore rather than in receptor recognition. To the
contrary, Wu and Aronson (22) have observed that the domain I
mutation A92D in CryIAc affects both binding and toxicity in M.
sexta. More studies on domain I, which could be the key factor in
post-binding events, are important to fully understand the mode of
action of
-endotoxins.
To further investigate the function of domain I and to gain more insights into events subsequent to binding, selected amino acid residues, alanine at position 92, phenylalanine at position 148, and tyrosine at position 153, in domain I of CryIAb were mutagenized. Based on the structural alignment with CryIIIA, all these amino acids should be located on the side facing the cell membrane and could directly participate in the domain I-membrane interaction(12, 23) . Mutant toxins were tested for structural stability, toxicity, receptor binding, and pore function. We found that the dramatic decrease in toxicity, resulting from mutation, is correlated with a significant decrease in irreversible binding or insertion, which may lead to poor channel formation and function. These results support the prediction that domain I is important for membrane insertion and pore function.
BBMV were
prepared from the isolated midgut following the method described by
Wolfersberger et al.(26) . The final BBMV pellet was
resuspended in binding buffer (150 mM NaCl, 8 mM NaHPO
, 2 mM KH
PO
, pH 7.4), and the concentration of
BBMV proteins was assessed by Coomassie Blue protein assay reagent
using bovine serum albumin as a standard. The final BBMV suspension
contained 0.1% bovine serum albumin.
Figure 3: Immunoblotting of M. sexta BBMV proteins with wild type and mutant toxins. 40 µg of M. sexta BBMV proteins was blotted and incubated as described under ``Materials and Methods.'' Detection of the binding of wild type or mutant toxins to BBMV-binding proteins was carried out using polyclonal antisera to CryIAb toxin. Lane1, Coomassie Blue-stained M. sexta BBMV proteins only; lane2, control (no toxin); lane3, M. sexta nonspecific CryIIIA toxin control; lane4, IAb; lane5, A92E; lane6, Y153A; lane7, Y153D; lane8, Y153R. Positions of protein markers (in kilodaltons) are shown on the left.
To measure irreversible and reversible binding at different
incubation times, the association experiment was designed as follows.
Two sample sets of a mixture of 0.5 nMI-labeled
toxin and 20 µg of BBMV in a total volume of 100 µl were
incubated at room temperature. At the selected time, the BBMV bound by
labeled toxin in the first set was quickly spun down at 16,500 rpm for
10 min to measure total binding. At the same time, 10 µl of
unlabeled toxin (1000 nM) was added to the second set.
Incubation was continued for 60 min at room temperature to chase off
the reversible binding at the selected time. The labeled toxin-bound
BBMV pellets were always washed twice with binding buffer before
counting in a
-counter. Nonspecific binding was obtained by adding
0.5 nM
I-labeled toxin and 1000 nM unlabeled toxin together into BBMV at the beginning of the
incubation. The counts of the first set were the total specific binding
plus the nonspecific binding, whereas the counts of the second set were
the irreversible binding plus the nonspecific binding. Reversible
binding counts were obtained by subtracting the counts of the second
set from the counts of the first set. Irreversible binding counts were
calculated by subtracting the nonspecific binding from the counts of
the second set.
Figure 1: Mutations in domain I of CryIAb and their effect on protoxin expression and toxin stability. Amino acid sequences between positions 96 and 145 are not shown. +, expression of protoxin or stable toxin; -, no detectable expression. WT, wild type; mut, mutant.
Figure 2:
Competition binding studies of wild type
and mutant toxins to M. sexta BBMV. Binding is expressed as
percentages of the amount bound with labeled toxin alone. Unlabeled
competing toxins are shown as follows: IAb, ; A92E,
; Y153A,
; Y153D,
; and Y153R,
. A, labeled wild
type toxins competed by increasing amounts of unlabeled mutant toxins; B, labeled mutant toxin competed by increasing amounts of
corresponding unlabeled mutant toxin; C, labeled mutant toxin
competed by increasing amounts of unlabeled wild type IAb
toxin.
All mutant toxins exhibited almost two times higher B values than the wild type did. This minor
difference could be caused by some errors in the determination of the
specific activity of
I-labeled toxins or by other unknown
reasons, but the increases in the B
for mutants
cannot account for their loss in toxicity.
Figure 4:
Typical response curves of M. sexta midgut membrane to CryIAb and mutant toxins in voltage clamping
assays. The same amounts of wild type IAb and mutant toxins in the same
volume were respectively applied to the luminal side of the chamber.
The final concentration for each toxin was 50 ng/ml. A control
experiment was carried out with the addition of the same volume of
solubilization buffer without toxin. Time 0 indicates the time of
addition of the toxins. Curves were reproduced based on the recorded
data. , IAb;
, A92E;
, Y153A;
, Y153D;
,
Y153R;
, control.
Figure 5:
Assay of dissociation binding of labeled
wild type and mutant toxins to M. sexta BBMV. Binding is
expressed as the percentage of the amount without addition of unlabeled
toxin. Nonspecific binding was obtained by incubating the labeled toxin
with BBMV in the presence of a 1000-fold excessive amount of unlabeled
toxin and was subtracted from the total binding. , IAb;
,
A92E;
, Y153A;
, Y153D;
,
Y153R.
Association studies are shown in Fig. 6and summarized
in Table 2. In this study, total specific binding, reversible
binding, and irreversible binding at different incubation times were
tested for each toxin. For wild type toxin, more than half of the total
specific binding was irreversible at all tested time points. For mutant
A92E, after 5 min of incubation, no irreversible binding was detected,
and after 80 min of incubation, only 22% of the total specific
binding was irreversible. Similarly, for Y153D toxin, irreversible
binding accounted for only
40% of the total specific binding. In
the case of mutants Y153A and Y153R, their association binding behaved
similarly to that of the wild type.
Figure 6:
Assay
of association binding of wild type and mutant toxins to M. sexta BBMV. The curves show the total binding (), reversible
binding (
), and irreversible binding (
) expressed as
counts/minute at selected incubation times. A, wild type IAb; B, A92E; C, Y153D; D, Y153R (Y153A was
similar to Y153R in this study).
Recently, there has been considerable interest in
understanding the toxic mechanism of
-endotoxins(28, 29) . The mode of action of B. thuringiensis toxins has been described as a multicomponent
process, involving solubilization, protoxin activation, receptor
binding, membrane integration, and pore formation. The resolution of
the CryIIIA tertiary structure, consisting of three domains, provides
us with more insights into the potential mode of action of
-endotoxin. Membrane integration and channel formation have been
linked to the function of domain I.
In this study, we have constructed several mutants in domain I of CryIAb to determine the region responsible for membrane insertion. Ala-92, Phe-148, and Tyr-153, carefully selected based on amino acid alignment with CryIIIA, are located on the side that could directly interact with the membrane. Substitution of phenylalanine at position 148 with aspartic acid or arginine results in no detectable expression of protoxin. We assume that phenylalanine, located in helix 4, is a buried residue, crucial in maintaining domain I structural integrity. Thus, introducing a charged aspartic acid or arginine at this position could disrupt protein structure. Mutation A92E, which is located at the beginning of helix 3 based on the alignment, almost totally abolishes toxicity. Substitution of tyrosine at position 153 in the loop region between helixes 3 and 4 with alanine, aspartic acid, or arginine causes different effects. Mutation Y153D greatly reduces toxicity, whereas mutations Y153A and Y153R slightly affect biological activity.
To understand why these mutations affect toxicity, we have performed a series of experiments to examine factors known to be involved in toxicity. Mutants A92E, Y153A, Y153D, and Y153R express protoxins at a level similar to that of wild type IAb, and their protoxins yield trypsin-resistant cores. All these mutant toxins are as stable as wild type IAb toxin in the presence of M. sexta gut juice and exhibit essentially identical CD spectrum curves (data not shown). Therefore, structural alteration is not the likely reason for the lowered toxicity of these mutants.
The
binding capacity of the mutant proteins has been tested by competition
binding analysis and immunoblotting assay. Only minor changes in K or B
values are detected
for the toxins that have the greatest loss in toxicity, A92E and Y153D (Table 1), and the changes that are observed for increases in B
cannot account for the loss of toxicity. For
Y153A and Y153R, the changes in the dissociation constant (K
) calculated by heterologous competition binding
may account for the slight loss in toxicity, but the K
calculated from homologous competition binding indicates no
difference in binding. The homologous competition assays demonstrate
similar high affinity binding to M. sexta BBMV by mutant and
wild type toxins (Fig. 2B). The heterologous
competition assays reveal that mutant and wild type toxins recognize
the identical binding site on the BBMV and compete with each other at
similar levels for binding to the M. sexta BBMV (Fig. 2, A and B). Therefore, competition
binding results indicate that these mutations do not affect initial
receptor binding of CryIAb toxin. This conclusion is supported by our
immunoblotting experimental data, which show that wild type IAb and
mutant toxins bind to the same M. sexta BBMV-binding proteins (Fig. 3). Binding detected by blotting experiments has been
demonstrated to be specific (30) and most likely represents
only the reversible binding since membrane insertion cannot take place.
Wu and Aronson (22) present data showing that mutations within
conserved block 1 (helix 5) result in a loss of toxicity. Surprisingly,
an A92D mutant of CryIAc, very similar to the A92E mutant in this work,
lost both toxicity and receptor binding capacity(22) . This
differs from our results. At this point, it is not clear what causes
the difference in results, but we have repeated our observations using
the CryIAc A92D mutant sent to us by D. Wu. Under the binding
conditions that they published, we observe no difference in
heterologous competition between A92D and CryIAc toxins (data not
shown).
The voltage clamp method was used to measure channel
function of mutant toxins. In contrast to the binding data, dramatic
differences in the inhibition of I are observed ( Fig. 4and Table 1). The differences in the inhibition of
I
to a large extent are correlated with the toxicity of
the mutant toxins. This relationship suggests that steps following
initial receptor binding, such as membrane insertion and pore
formation, have been altered in these mutant toxins.
The binding of
toxins to BBMV may include two major steps: an initial reversible
binding and an irreversible binding. The process could be simply
expressed as follows: toxin + receptor reversible binding
irreversible binding (membrane insertion). Van Rie et al.(5) have reported that toxin binding essentially becomes
irreversible. Wolfersberger et al.(31) could not
detect toxins on the outer surface or in the inner space of BBMV after
a 10-min incubation of toxins with BBMV. However, they have found the
intact toxin molecules by denaturing BBMV with detergent. They proposed
that soon after binding to cell membrane components, the toxin
molecules become integrated into the cell membrane. It is reasonable to
consider that irreversible binding corresponds to the membrane
integration process. If more toxins are inserted into the membrane,
more pores could be formed, and higher toxicity would be observed. The
method commonly used to study toxin-BBMV interaction is a competition
binding system based on the Scatchard plot, which is best known to fit
the reversible ligand binding process, such as antibody-antigen
interaction(27) . Application of the Scatchard plot and
competition binding analysis to the toxin-BBMV interaction may
oversimplify this process. There is no doubt that this type of ligand
binding assay can demonstrate the specific or nonspecific binding of a
toxin to BBMV, but the binding data cannot be clearly interpreted in
terms of both reversible binding (initial binding) and irreversible
binding (membrane insertion). English and Slatin (32) proposed
that the binding statistics not only represented the initial binding
reaction, but also included the contributions from intercalation,
oligomerization, and even stable ion channel formation. MacIntosh et al.(33) and Ferréet al.(34) seemed to assume that the competition binding data
reflected only the initial association between toxins and BBMV. Our
results support the conclusion of these two groups.
The limitations
of the competition binding method have led us to design dissociation
and association binding assays to study irreversible binding. In the
dissociation binding studies, wild type IAb and mutants Y153A and
Y153R, both with slightly lower toxicity than the wild type, display
much more stable irreversible interaction with BBMV than do the
partially toxic Y153D and the nontoxic A92E mutants (Fig. 5).
This indicates that toxicity differences among wild type and mutant
toxins are correlated with their irreversible binding. The association
of toxin with BBMV was further analyzed as a function of incubation
time. The association assays clearly demonstrate that the binding of
toxin to BBMV includes reversible and irreversible steps. As we can see
from Fig. 6, at the beginning of the incubation, the most
specific binding is reversible binding, but as the incubation
progresses, irreversible binding gradually increases. Wild type toxin
irreversibly binds to BBMV in a much faster and more efficient way than
do the nontoxic A92E and the partially toxic Y153D mutants. If
irreversible binding represents membrane insertion, mutants A92E and
Y153D have a very low efficiency of insertion. Therefore, the loss of
or large decrease in toxicity observed in these two mutants could be
caused by a severely disturbed insertion process. In contrast to A92E
and Y153D, mutations Y153A and Y153R result in a slight change in
irreversible binding, which correlates with a minor change in toxicity.
Ihara et al.(35) have demonstrated that CryIAa is
17-fold more toxic to B. mori than is CryIAb and have observed
that the two toxins exhibit similar binding affinity and binding site
concentrations with B. mori BBMV, but that CryIAa toxin shows
much higher irreversible binding than does CryIAb. They have proposed
that the difference in irreversible binding between CryIAa and CryIAb
leads to the difference in toxicity. Our results are in agreement with
theirs. It is important to point out that insertion is a necessary step
for channel formation, and if a B. thuringiensis toxin cannot
insert into the cell membrane, the toxin is unlikely to be toxic.
Theoretically, insertion may not absolutely result in channel
formation. In other words, some insertion could be ineffective because
the molecules may not form pores or may form defective pores in the
membrane. However, more research needs to be done to understand fully
the -endotoxin membrane insertion process.
Another suggestion from this work is that the membrane region where domain I first contacts may be rich in negative charges. Introduction of a negative charge (A92E or Y153D) into the domain I region, which is proposed to start initial insertion after initial receptor binding, disrupts the insertion process. Mutation A92E or Y153D may disrupt the charge balance on the initial membrane contact region of domain I, making this region more negatively charged. If the membrane region is negatively charged, these two regions will repulse instead of attract each other. Results with Y153A and Y153R mutants support this assumption. This conclusion is also in agreement with the observation of Wu and Aronson (22) that mutations at Arg-93 of CryIAc, except R93K, lower toxicity. The different effect of A92E and Y153R could be explained if the region around Ala-92 is more important in membrane insertion than the one around Tyr-153.
In studies of resistance of H. virescens to CryIA toxins, two different groups have found no clear
correlation between initial toxin binding and resistance
levels(33, 34) . These results indicate that events
other than binding could contribute to resistance, affecting the
biological activity. Our data have shown that modification of the
membrane integration capacity could dramatically change its toxicity.
These post-binding steps might be other potential mechanisms of
resistance of insects to -endotoxins besides protease activation (36) and receptor binding(8) .
In summary, our
results support the model that domain I plays a crucial role in
insertion and pore formation rather than receptor binding. The work
reported here demonstrates that reduction in irreversible binding
(insertion) correlates with reduction in toxicity, broadens our
understanding of toxin-BBMV interaction, and favors a multistep toxic
mechanism for -endotoxins.