(Received for publication, September 1, 1995)
From the
Site-directed mutagenesis was used to examine the role of domain
II, loop 2 residues, RRPFNIGI
, of Bacillus thuringiensis insecticidal protein CryIAb. Alanine
substitution of residues
RRP
, called B4,
abolished potency toward Manduca sexta and Heliothis
virescens, and the loss of toxicity was correlated directly to
substantially reduced binding affinity to brush-border membrane
vesicles (BBMV) prepared from the target insect midguts. These results
indicated that these positive charges might be essential to orient the
toxin to midgut receptor molecule(s). The role of residue Phe
of CryIAb toxin to M. sexta was investigated by
substituting a series of residues at this position. Irreversible
binding and toxicity were affected significantly by hydrophilic,
aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and
Ser but not by Tyr or Trp. A hydrophobic aromatic side-chain residue at
position 371 was therefore essential for irreversible binding of CryIAb
toxin in M. sexta. The role of residues
PFNIGI
of CryIAb toxin on H. virescens was also examined. Mutants D2 (deletion of residues
370-375), G374A (alanine substitution of Gly
), and
I375A had reduced toxicity to H. virescens. In contrast to our
findings with M. sexta, the reduction in toxicity of these
mutants was correlated directly with loss of initial binding to H.
virescens BBMV, indicating that these residues perform
functionally distinct roles in binding and toxicity to different
insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.
Bacillus thuringiensis is a Gram-positive soil
bacterium that produces one or more insecticidal crystal proteins
(-endotoxins) which are largely responsible for its pathogenicity
to agriculturally important insect pests and vectors of human diseases.
In recent years, the mechanism of action of
-endotoxins to
lepidopteran insects has been studied intensively, but several factors
still need to be examined. Upon ingestion, the
-endotoxin (120 to
140 kDa) is solubilized and activated into a toxic form by removal of
28-30 residues from the N terminus and approximately 500 residues
from the C terminus by proteolytic enzymes present in the susceptible
larval midgut(1) . Following enzymatic activation, the toxic
protease-resistant core (60 to 65 kDa) binds to specific receptor(s)
(toxin-binding proteins) located in the midgut apical brush-border
membrane of the columnar cells. Binding of toxin to a receptor molecule
is thought to trigger a conformational change in the toxin and enable
the toxin to insert into the plasma membrane, generating pores or ion
channels which lead to cellular swelling and
lysis(2, 3, 4, 5, 6) .
Intoxicated insects stop feeding and eventually die.
Binding of the
activated toxin to specific gut receptor(s) is considered one of the
key factors for insect toxicity for several reasons. Firstly, ion and
water leakage induced by toxin in phosphatidylcholine vesicles is
enhanced by the addition of brush-border membrane proteins or purified
receptors(7, 8, 9) . Secondly, insect
resistance to Cry toxins is often correlated with reduced binding
affinity or binding site concentrations for specific receptors (10, 11, 12) . Thirdly, to date there have
been no reports of insecticidal activity of Cry toxins that do not have
measurable membrane binding to insect brush-border membrane vesicles
(BBMV). ()For some toxins, however, the opposite is true, i.e. more membrane binding but relatively less insect
toxicity(13) . Also, resistance to CryI toxins in a strain of
tobacco budworm, Heliothis virescens, is not caused by altered
receptor binding(14) . Factors such as imperfect activation of
the toxin by insect gut juice(15) , defective insertion of the
toxin into the membrane, and failure to make sufficient toxin oligomers
to form a functional pore might account for a lack of toxicity in the
above-mentioned exceptions.
Recently, a two-step receptor binding process for CryIAb toxin to several lepidopteran insects has been proposed(16, 17) . Interestingly, the sum of irreversibly bound toxins to midgut BBMV is correlated directly to insect toxicity(16) . Other factors, including the aggregation of toxin monomers to form oligomers either in solution or in the membrane might aid to enhance toxicity. Recently, Walters et al.(18) have shown that CryIIIA molecules exist as dimers in solution. However, the precise role of oligomerization in toxicity needs to be investigated.
The recently solved structure of a
coleopteran active -endotoxin, CryIIIA(19) , reveals a
three-domain structure. This structure is believed to be common for
other Cry toxins since the interfaces holding the domains are highly
conserved in most of the Cry toxins. Based on the crystal structure,
domain II consists of six antiparallel sheets that include the
hypervariable region of Cry toxins. The three major surface-exposed
loops of this domain have been suggested to be involved in insect
specificity and membrane binding(19) . Moreover, the transfer
of Bombyx mori activity from a highly active toxin (CryIAa) to
a relatively non-active toxin (CryIAc) by switching residues 330 to 450
has led to the proposal that insect specificity and receptor binding of
-endotoxins might be confined to the hypervariable region of
domain II, at least for this insect(20, 21) .
In
our effort to identify the amino acid residues of Cry toxins that
participate in receptor binding, we have previously shown that residues
365 to 370 of CryIAa toxin are involved in initial membrane binding on B. mori(22) and that residues 370 to 375 are
important for the irreversible association of CryIAb toxin to M.
sexta BBMV, although this region does not directly participate in
initial binding on M. sexta(16) . In this article we
report the functional role of another putative loop 2 segment, RRP
, of CryIAb
-endotoxin in toxicity
and initial binding toward H. virescens and M. sexta larvae. We substituted residue Phe
of CryIAb toxin
with amino acids of diverse physical and chemical properties to analyze
their membrane-association function on M. sexta. Membrane
binding properties of single alanine substitution mutants among
residues 370-375 of CryIAb toxin on H. virescens were
also examined. These mutational analyses, together with
voltage-clamping and ligand-blotting experiments, provided valuable
information on the functional role of these predicted loop 2 residues
in membrane binding and toxicity to the target insects, M. sexta and H. virescens.
Figure 1: Coomassie Blue-stained SDS-10% PAGE gel comparing the yield of protoxins (A) and trypsin-activated toxins (B). Lane 1, molecular mass markers. Masses of the protein markers (in kilodaltons) are shown on the left. Lane 2, CryIAb; lane 3, F371C; lane 4, F371S; lane 5, F371V; lane 6, F371L; lane 7, F371Y; lane 8, F371W; and lane 9, B4. Each lane contained 5-7 µg of protein. C, Western blot analysis of the stability of the above toxins after digestion with M. sexta gut juice enzymes. Lane 10, M. sexta gut juice. D, Western blot analysis of the stability of wild-type and mutant proteins after digestion with H. virescens gut juice enzymes. Lane 1, molecular mass markers. Masses of the protein markers (in kilodaltons) are shown on the left. Lane 2, CryIAb; lane 3, B4; lane 4, D2; lane 5, F371A; lane 6, N372A; lane 7, G374A; lane 8, I375A; and lane 9, H. virescens gut juice.
Figure 2:
Binding of I-labeled CryIAb
toxin in the presence of increasing concentrations of nonlabeled
CryIAb, B4, F371C, F371S, F371V, F371L, F371Y, and F371W toxins to M. sexta BBMV. Binding is expressed as a percentage of the
amount bound upon incubation with labeled toxin alone. On M. sexta vesicles, the amount is 2988 ± 90 cpm for
CryIAb.
Figure 3:
Determination of stability and binding of I-labeled CryIAb, B4, F371C, and D2 toxins after
incubation with M. sexta (A) and H. virescens (B) BBMV. Lane 1, amount bound to the BBMV; lane 2, total input counts. See ``Materials and
Methods'' for details.
Figure 4:
Inhibition of I across M.
sexta midgut. A total of 50 (A) and 500 (B) ng
of CryIAb, B4, F371C, F371S, F371V, F371L, F371Y, and F371W toxins per
ml were injected in separate experiments into the lumen side of the
chamber, and the drop in I
was measured. The I
measured before the addition of toxin is considered as
100%.
Figure 5:
Binding of I-labeled CryIAb
toxin in the presence of increasing concentrations of nonlabeled
CryIAb, B4, F371A, N372A, G374A, I375A, and D2 toxins to H.
virescens BBMV. Binding is expressed as a percentage of the amount
bound upon incubation with labeled toxin alone. On H. virescens vesicles, the amount is 3011 ± 82 cpm for
CryIAb.
Our toxicity results showed that F371W
was very similar to the wild-type toxin, whereas F371C, F371V, F371S,
F371L, and F371Y were >600, 400, 40, 11, and 6 times less toxic than
wild-type toxin, respectively (Table 1). Heterologous competition
experiments were performed to evaluate whether the mutant proteins
recognize the same binding site as that of the wild-type toxin. When I-labeled CryIAb was put into competition with nonlabeled
mutant proteins, all the mutants competed for the CryIAb binding
site(s) as efficiently as did nonlabeled CryIAb (Fig. 2). This
observation was further supported by the autoradiography binding study
which showed that 40% of labeled F371C was bound to the BBMV compared
to 35% binding of the wild-type toxin (Fig. 3A).
Since all the mutant toxins showed similar binding affinities but differed in toxicity, we examined whether all the BBMV-bound toxins were irreversibly associated to the BBMV. The toxins were first allowed to bind saturably to the BBMV and were then chased with 1000-fold excess of corresponding nonlabeled toxins. Our results showed that 88% of the wild-type and mutant F371W toxins bound to the BBMV could not be displaced by the addition of nonlabeled ligands (i.e. they were irreversibly associated). In contrast, only 55%, 58%, 65%, 80%, and 82% of the mutants F371C, F371V, F371S, F371L, and F371Y, respectively, were associated irreversibly with M. sexta BBMV (Fig. 6). The continued decrease in binding in the case of some mutants (F371C and F371V) were not due to labeled toxin breakdown, since we could recover the intact labeled toxin after incubation with BBMV for more than 4 h (data not shown).
Figure 6:
Dissociation of bound I-labeled toxins from M. sexta BBMV. M.
sexta BBMVs (100 µg/ml) were incubated with 1 nM
I-labeled CryIAb, F371C, F371S, F371V, F371L, F371Y,
and F371W toxins (association reaction). At 120 min after association
reaction, 1000 nM concentrations of the corresponding
nonlabeled toxins were added to the test samples and incubation was
continued (post-binding incubation). Binding is expressed as a
percentage of the amount bound compared with the amount bound at 0 h
post-incubation.
We also examined the
inhibition of I in response to the addition of wild-type
and mutant toxin to the lumen side of an isolated M. sexta midgut under voltage clamped conditions. I
measures
the active transport of ions from the hemolymph side of the midgut to
the lumen side by the ion pumps present in the membrane. When we used a
toxin concentration of 50 ng/ml, the slope of I
inhibition
of mutant F371W and wild-type was comparable (Fig. 4A),
whereas the slope for mutant F371Y was about 4 times less than that of
the wild-type (Fig. 4A). Mutants F371C, F371V, F371L,
and F371S did not show any measurable inhibition of I
at
this toxin concentration (Fig. 4A). However, when the
toxin concentration was increased to 500 ng/ml, a measurable difference
in the slope of inhibition of I
among the mutants F371C,
F371V, F371L, and F371S was observed (Fig. 4B). The
I
slope values calculated with 500 ng of toxin/ml are
given in Table 3.
Figure 7:
Binding of I-labeled CryIAb (lane 1) and B4 (lane 2) toxins to protein blots of M. sexta (A) and H. virescens (B)
BBMV proteins. A total of 30 µg of BBMV proteins were blotted and
probed with 1
10
cpm of iodine-labeled
toxins.
The relationship between the amino acid sequences of Cry
toxins and their interaction with susceptible insect gut cell membranes
(receptors) are important for understanding the molecular mechanism of
action of the toxin. Here we evaluate the correlation between
larvicidal potency, membrane binding affinity, and ion conductivity of
alanine substitution mutants constructed at the predicted loop 2
region, RRPFNIGI
, of domain II of CryIAb
toxin toward M. sexta and H. virescens. Alanine
substitutions were considered because the replacement of side-chains
with alanine could be least disruptive to the overall structure (33) .
Firstly, we generated a mutant, B4, by substituting
alanines for residues RRP
(
AAA
) using site-directed mutagenesis
techniques. This mutant abolished (>600 times less toxic for M.
sexta than CryIAb) larvicidal activity toward the target insects, M. sexta and H. virescens. To determine whether the
loss of toxicity was caused by structural instability, B4 toxin was
treated with trypsin or target insect gut juice (Fig. 1). Our
data suggested that the reduction in toxicity was simply the result of
the amino acid substitutions rather than to gross conformational
alterations in the membrane binding domain. Competition binding studies
demonstrated that B4 toxin competed only negligibly for the binding
sites of wild-type toxin on BBMV prepared from either insect ( Fig. 2and Fig. 5). Further binding experiments showed
that while CryIAb and B4 proteins had comparable specific
radioactivity, B4 exhibited only 1% binding compared to 35-40%
binding by wild-type toxin on either of the insect BBMV (Fig. 3). This direct correlation between binding affinity and
toxicity shows that in the native toxin, residues
RRP
might help the toxin to anchor to the
cell surface exposed receptors. Cummings and Ellar (34) also
observed the involvement of arginine residues in toxicity and binding
against M. sexta when they chemically modified arginine
residues of CryIAc toxin, which has more than 90% amino acid homology
to CryIAb.
Voltage clamping experiments on M. sexta midguts
provided additional evidence that the insecticidal property, inhibition
of short-circuit current across the insect midgut, did not occur with
B4 toxin, which failed to bind to the receptor. Taking together all
these results, it is reasonable to speculate that receptor binding is a
two-step process, initial reversible binding and irreversible binding.
The positively charged residues of the loop, RR
, establish the initial contact with the
receptor (reversible binding). Since the deletion of residues
PFNIGI
did not inhibit the initial
binding(16) , Pro
is not likely to be involved in
the initial binding to M. sexta. Binding is further
strengthened (irreversibly) by the interaction of residues Phe
and Gly
either with the receptor or with the
membrane(16) . However, this study does not exclude any
possible involvement of these arginine residues (since arginine can
form large number of hydrogen bonds) with neighborhood amino acids that
participate in receptor binding.
Secondly, our previous study showed
that alanine substitution mutants of CryIAb among residues PFNIGI
had initial binding similar to
CryIAb, as measured by competition binding, but significantly decreased
irreversible binding on M. sexta BBMV. The 400-fold reduction
in potency of the mutants, especially F371A, was correlated directly to
the substantially reduced irreversible association of the toxin on M. sexta BBMV(16) . To further substantiate the
physical and chemical requirement for the amino acid at position 371,
in this study we replaced Phe
with residues such as Ser,
Cys, Val, Leu, Tyr, and Trp. This set of residues contains side-chains
that vary in volume, hydrogen bonding capability, and hydrophobicity.
Hence, the influence of each of these properties on the irreversible
membrane association of CryIAb toxin could be determined precisely. Our
bioassay data showed four categories of mutants: 1) those >400 times
less toxic (F371C, F371V); 2) 10-40 times less toxic (F371S and
F371L), 3) 6 times less toxic (F371Y), and 4) comparably toxic as to
wild-type (F371W). These data are consistent with the dissociation
binding data; that is, the greater the irreversible association of the
toxin to the BBMV, the more the toxicity to the insect (Fig. 6).
Thus, the aliphatic, small side-chain substitutions are considerably
less toxic than the aromatic bulky side-chain substitutions.
Interestingly, the loss of toxicity by the smaller side-chain
substitution (F371C) could be regained consistently with the increase
in side-chain volume. The residues which strengthen the membrane
association (irreversible binding) of the toxin ranks in the order of
Trp = Phe > Tyr > Leu > Ser > Val > Cys. The
inhibition of short-circuit current of these mutant proteins to M.
sexta midgut under voltage clamp conditions also followed the same
order. Although the inhibition of I
by the mutants F371C,
F371V, and F371S were not measurable (Fig. 4A) at lower
toxin concentrations, the differences in I
inhibition were
resolved at higher concentrations of toxin (Fig. 4B).
Only a hydrophobic aromatic-ring side-chain substitution such as Trp,
retained the potency of native residue Phe
. Even the
hydroxyl group on Tyr (sterically equivalent to Phe) results in a
measurable reduction in irreversible binding. The aromatic amino acids
in the loop region of CryIC toxin were also reported to play a critical
role in post-binding function and insect toxicity(35) .
However, the smaller side-chain residues (F371C, F371V, and F371S),
which significantly affected the toxicity and rate of irreversible
binding (insertion) to the BBMV, did not affect initial binding to M. sexta. The fact that the less toxic mutants still bound to
the membrane receptor as well as the wild-type provides further
evidence that these residues are not directly involved in initial
recognition of the receptor molecule.
These experiments suggest that an aromatic hydrophobic residues in the surface exposed loop 2 is important for the irreversible association (insertion) of CryIAb toxin to membrane. Interestingly, F371A did not affect the toxicity to another CryIAb susceptible lepidopteran insect, H. virescens. It is likely that the toxin receptors located on the cell surface are different for the two target insects. Our ligand blot data showed that CryIAb toxin binds to a major 210-kDa BBMV protein in M. sexta and a 170-kDa toxin binding protein in H. virescens BBMV (Fig. 7). This is in agreement with previously published data(36, 37) .
Thirdly, the functional role of
residues 370 to 375 (PFNIGI
) of CryIAb
toxin in receptor binding and toxicity to H. virescens was
analyzed. Our bioassay data showed that mutants D2, G374A, and I375A
have reduced the larvicidal potency (>50, 9, and 5 times,
respectively) to H. virescens. As the activation of these
mutants by H. virescens gut juice is similar to the wild-type
toxin, it can be assumed that the reduction in toxicity of D2, I375A,
and G374A was not caused by structural instability of the mutant
toxins. The reduced toxicity of these mutants were directly correlated
to the reduced (4-5 times) initial binding affinity of the toxin
on H. virescens BBMV as measured by competition binding
studies. Furthermore, it is clear from our blot experiment that 40% of
total labeled CryIAb bound to BBMV, whereas only 1% of D2 toxin bound
to the BBMV prepared from H. virescens. These data are in
contrast with our previous observation in M. sexta. In M.
sexta, these mutations did not affect the initial binding to BBMV,
but the loss of larvicidal activity of these mutants was directly
attributed to the significantly reduced irreversible binding ability to
BBMV(16) . These results imply that although these residues are
involved in toxicity and receptor binding, they perform a functionally
distinct role in different insects or receptors involved in toxicity.
Determination of the role of these receptor contact residues (RRPFNIGI) in association and dissociation binding is fundamental to our understanding of the toxin-receptor molecular recognition process. Our observation that certain mutations in a surface loop of domain II affect irreversible binding (but not reversible binding) and that binding is strengthened by a hydrophobic aromatic side chain at position 371 warrants an explanation. One possibility is that toxins become tightly (irreversibly) bound (through hydrophobic interaction) to the receptor in a manner distinct from insertion of the toxin to the apical membrane. The second possibility is that loop 2, and other parts of domain II, insert into the apical membrane. Further experimental evidence is necessary to distinguish between these two models.