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All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes.
Manoj S. Nair 1 and Donald H. Dean
1, 2
1Biophysics Program and
2Department of Biochemistry, The Ohio State University, Columbus, OH 43210
Running Title: “Entire Cry Toxin Inserts into BBMV.”
Corresponding author: 772 Biosciences building, 484 W 12
th Ave, Columbus, OH 43210,
Fax: 614-292-6773; email: [email protected]
A critical step in understanding the mode
of action of insecticidal crystal (Cry)
toxins from Bacillus thuringiensis is their
partitioning into membranes, and the
insertion of the toxin into insect brush
border membranes, in particular. The
Umbrella and Penknife models predict
that only α-helix 5 of Domain I along with
adjacent helices, α-4 or α-6 insert into the
brush border membranes because of their
hydrophobic nature. By employing
fluorescent-labeled cysteine mutations, we
observe that all 3 domains of the toxin
insert into the insect membrane. Using
proteinase K protection assays, steady
state fluorescence quenching
measurements and blue shift analysis of
acrylodan-labeled cysteine mutants, we
show that regions beyond those proposed
by the 2 models insert into the membrane.
Based on our studies, the only extended
region that does not partition into the
membrane is that of α-helix 1. Bioassays
and voltage clamping studies show that
all mutations examined, except certain
domain II mutations in loop 2 (e.g.,
F371C and G374C), which disrupt
membrane partitioning, retain their
ability to form ion channels and toxicity
in Manduca sexta larvae. This study
confirms our earlier hypothesis that
insertion of Cry toxin does not occur as
separate helices alone, but virtually the
entire molecule inserts as one or more
units of the whole molecule.
Insecticidal crystal proteins produced
by Bacillus thuringiensis are of great
commercial potential in the field of
agriculture and health (1) by targeting a
wide spectrum of crop pests and vectors of
human diseases. Cry1A toxins are active
against lepidopteran insects, which include
agricultural pests. The toxins are produced
by the bacterium in the stationary phase as
inactive crystal protoxins (1). Activation of
the 130 kDa protoxin to a 65 kDa active
toxin occurs in the alkaline environment of
the lepidopteran midgut. Crystal structures
of the active toxin show that the toxin has 3
domains that are conserved through all the
Cry toxins (2-7). Domain I is an -helical
bundle made of 7 antiparallel α-helices.
Domain II is a globin-like, wedge-shaped
prism made of antiparallel β sheets ending in
predominant -loops and Domain III is a
lectin-like β sandwich. The protease-
activated form of the toxin binds to
receptors on the surface of the insect brush
border membrane. Several receptors
implicated in binding to the toxin include
cadherins (8,9), alkaline phosphatase and
one or more forms of aminopeptidases
(10,11), glycolipids (12,13) and
glycoproteins (14). The receptor bound toxin
has been proposed to undergo
conformational changes (15,16) before or
after inserting into the membrane to form
ion channels.
Studies focused on insertion of
Cry1A toxins into the insect membrane have
limited their studies to two -helices of
http://www.jbc.org/cgi/doi/10.1074/jbc.M802895200The latest version is at JBC Papers in Press. Published on July 17, 2008 as Manuscript M802895200
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
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Domain I of Cry1A toxins, α-helix 4 and 5
based on the theoretical Umbrella Model of
insertion proposed early in the 1980s (17).
There has been little analysis of the other
regions of the toxin. Several studies using
non-specific proteases to determine the
presence of the toxin in the membrane
(18,19) have shown that almost the entire
toxin is protected from the protease but the
extent of partitioning of the toxin into the
membrane remains unmeasured.
Our current study spans the 3
domains of the toxin to identify specific
regions that may be embedded into the
insect membrane including the regions of
the toxin proposed by the Umbrella model
and beyond. Biochemical protease
protection assay and steady state
fluorescence measurements using cysteine
mutations in regions of 3 domains of
Cry1Aa or Cry1Ab toxin show that all
regions of the toxin studied except α-helix 1
are embedded into the membrane, although
to different extents. Cry1Aa and Cry1Ab
were both used as they show 89% identity in
their sequence (20) and target similar
insects.
EXPERIMENTAL PROCEDURES
Site directed mutagenesis and expression:
The cell culture containing the Bacillus
thuringiensis cry1Ab9-033 (21) was
obtained from T. Yamamoto (Sandoz Agro
Inc., Palo Alto, CA) and that for Cry1Aa
was obtained from American Type Culture
Collection (22). Uracil containing template
for Cry1Ab was obtained as described (23).
Primers for site directed mutagenesis were
obtained from either Integrated DNA
technologies Inc. or Bioneer Inc. Site
directed mutagenesis was carried out using
Mutagene M13 In vitro mutagenesis kit as
described in the manufacturer‟s manual
(BioRad). Mutations were confirmed by
double stranded DNA sequencing performed
at the Plant Microbe Genomics Facility,
Ohio State University, Columbus, Ohio.
Expression and purification of the toxin
mutants: Expression and preparation of the
toxin was carried out as described earlier
(24). Crystals were solubilized in 50 mM
Na2CO3 pH 10.5 buffer to extract the
protoxin and digested with 1/50 w/w of
trypsin/crystal protein to yield the activated
toxin. The toxins were purified using
Sepharose Q ion exchange column,
Sephacryl S300 and Superdex S200 gel
filtration columns in series.
Preparation of Small Unilamelar Vesicles
(SUV): Artificial phospholipids, 1-
Palmitoyl-2-oleyl-sn-glycerol-3-
phophatidylcholine (POPC), 1- Palmitoyl-2-
oleyl-sn-glycerol-3-
phosphatidylethanolamine (POPE) and
cholesterol (Avanti Polar lipids Inc.) were
used in the molar ratio of 7:2:1, similar in
composition to lipids commonly in bilayer
and vesicle formation of Cry toxins
(2,25,26), were used to form SUV. Small
Unilamelar Vesicles were prepared from a
preparation of Large Multilamellar Vesicles
(LMV) by using a Branson 2200 bath
sonicator. The LMV mixture free of any
choloroform was sonicated in the water bath
for 10 min intervals during which the
solution turned less opaque. The size class
of the SUV was measured on a DynaPro
light scattering instrument (Wyatt
Technologies.). SUV gave an average size
range of 25-30 nm which was reproducible
from batch to batch analysis. Typical size
ranges of SUV vary from 15-50 nm when
obtained using this protocol (27).
Preparation of Brush Border Membrane
Vesicles (BBMV): Fourth instar larvae of
Manduca sexta (Carolina Biologicals Supply
Company) were dissected using procedures
described elsewhere (28). BBMV were
prepared using modified differential
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magnesium precipitation method (29). The
final BBMV pellet was resuspended in a
binding buffer (10 mM HEPES, 150 mM
NaCl pH 7.4). Protein concentrations were
estimated using Coomassie Protein Assay
Reagent (Pierce Biotechnologies, Inc.)
Proteinase K protection assays: Pure toxin
was mixed with 10 fold excess of BBMV
and incubated at 25oC for 30 min after
which proteinase K at 10 fold excess
concentration of the toxin was added and
incubated at 37oC for 30 min. PMSF was
added to stop the reaction. The mixture was
centrifuged at 15,000 g for 10 min. The
pellet was washed with 10 mM HEPES, 150
mM NaCl, pH 7.4 and then solubilized into
1% n-octyl β-D-glucopyranoside (Sigma)
and boiled for 3-5 min before loading onto
an 8% SDS-PAGE gel. Proteins were
transferred onto a PVDF membrane and
blotted using polyclonal anti Cry1A rabbit
antisera at a dilution of 1 in 10,000 and
HRP-tagged goat anti-rabbit antisera at a
dilution of 1 in 50,000 (BioRad). Blots were
visualized using chemiluminescent HRP
substrate (BioRad).The blots exposed to X-
ray films for 10s are depicted in the Fig. 1
and for 30s for Fig. 2.
Labeling of purified cysteine mutants with
probes: 5-({2-
[(iodoacetyl)amino]ethyl}amino)-
napthalene-1-sulfonic acid (1,5-IAEDANS)
and 6-acryloyl-2-dimethyl-aminonapthalene
(Acrylodan) were purchased from Invitrogen
Inc. Purified cysteine mutants were
incubated with 10 fold molar excess of the
probes and incubated in the dark overnight.
Unbound label was removed using
Sephadex G50 gel filtration column (GE
Healthcare). Purity of the protein was
checked on 8% SDS-PAGE gel and the
efficiency of labeling was measured using
the molar extinction coefficient of each
probe.
Steady state fluorescence quenching
measurements: 50 µg of 1, 5- IAEDANS
labeled Cry1A toxin was mixed with 5mgs
of SUV and incubated for 60 min. The
bound toxin was separated from the
unbound labeled toxin by passing the sample
through a Sephadex G100 column (GE
Healthcare). Concentration of the SUV
bound protein was measured using the BCA
Protein Assay Kit (Pierce Biotechnologies
Inc.) after delipidation of the proteins using
clean up kit (Pierce Biotechnologies Inc.).
Steady state fluorescence measurements of
equal amounts of free and bound toxin were
carried out in a Fluoromax-3 fluorimeter (JY
Horiba). The sample was excited at a
wavelength of 380 nm and emission scan
showed maximum fluorescence around
460nm. The SUV bound labeled toxin was
treated with increasing aliquots of potassium
iodide (KI) in thiosulfate (final
concentration of 0.83M) to test if the label
on the bound toxin was further susceptible
to collisional quenching in the aqueous
environment. The percentage of quenching
of fluorophore was calculated as the
percentage ratio of the difference in the
quantum yield before and after partitioning
of the labeled protein to the total
fluorescence of that free labeled protein in
buffer.
Fluorescence “blue shift” measurements:
Acrylodan labeled Cry1A toxin (50 µg) was
treated with BBMV (500 µg) and incubated
for 60 min. The bound toxin was separated
from the unbound by centrifuging the
sample at 15,000 g. Proteinase K (500 µg)
was added to the reaction and incubated for
additional 30 min. The sample was
centrifuged again at 15,000 g and the
resultant pellet was resuspended in binding
buffer. Steady state fluorescence
measurements of the labeled toxin, bound
toxin before and after proteinase K treatment
were carried out on Fluoromax-3 fluorimeter
(JY Horiba). The samples were excited at
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360 nm and the fluorophore emission was
read from 390 nm to 650 nm. Maximal
emission wavelengths (max) of fluorescence
of labeled toxin and toxin bound to BBMV
before and after proteinase K treatment were
recorded for each mutant. Each experiment
was reproduced for 3 times and the average
value of the max was recorded.
Toxicity Bioassays: Toxicity levels were
determined by estimating the median lethal
concentration (LC50) on M. sexta larvae
using the diet surface contamination assay
(24). Sixteen first instar larvae were used for
each concentration of the toxin and a total of
6 concentrations of each toxin were used.
Mortalities were recorded after 5 days. The
LC50 for each toxin was calculated by probit
analysis using SoftTox (WindowsChem
Software, Inc.)
Voltage clamp measurements of Cry1A
mutants: Inhibition of short circuit currents
(Isc) was measured by clamping M. sexta
midguts using procedures described earlier
(28). Briefly, 100 ng of purified proteins
were added to the lumen side of the gut
stabilized in the buffer (30). Measurements
were made on a DVC 1000 voltage/current
clamp (World Precision Instruments,
Sarasota, FL) connected to MacLab -4 (AD
Instruments, Mountainview, CA). Data
analysis was done using SigmaPlot v.10
(Systat Software Inc. Richmond, CA). The
data was normalized to scale the percentage
of short circuit current remaining. The slope
of the linear region was used to measure the
rate of ion transport in case of each mutant
and the lag time (T0) was also calculated as
a measure of the rate of partitioning of the
toxins into BBMV (28,31,32).
RESULTS
Expression and purification of the toxin
mutants: Cysteine scanning mutagenesis of
several residues in the 3 domains of the
toxin was successfully performed using the
Kunkel method of mutagenesis (33) where
uracil rich single stranded templates were
annealed to primers and elongated in the
presence of T7 DNA polymerase and T4
DNA ligase. The resulting products were
transformed into DH5 cells (containing
dUTPase) and screened for mutants.
Proteins were expressed in DH5 cells
under a „leaky‟ promoter. The resulting
protoxins were run on an 8% SDS-PAGE
gels to obtain a 130 kDa band (data not
shown). Expressed proteins were digested
with trypsin to yield an active 65 kDa form
that was purified using ion exchange and gel
filtration chromatography. The secondary
structures of the mutants were compared to
the wild type using a circular dichroism
spectrophotometer. Only mutations with
good expression level and whose secondary
structure was not affected upon expression,
as measured by circular dichroism, were
used in this study. They are L40C, V171C,
S191C, L199C, L215C, S279C, S324C,
S364C, F371C from Cry1Ab and D62C,
E460C, K489C and I526C from Cry1Aa.
The mutations used span all the 3 domains
of the toxin and most of the chosen residues
are conserved across Cry1Aa and Cry1Ab.
Each purified toxin mutant (100ng) used for
the proteinase K protection assay has been
blotted using anti Cry1A antibody as shown
in Fig. 1. All Cry1A toxins in our hand
produced a doublet band on the SDS PAGE
gel (Fig. 1C, 1D) upon purification from the
crystals. Sequence analysis of the bands
have shown that the doublet was a result of
multiple trypsin sites at the C-terminus of
the active toxin close to each other resulting
from incomplete digestion of all molecules.
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We loaded 100 ng of toxin in Fig. 1 to
indicate multiple bands in the Western blot.
Toxicity of mutants: Biological activity of
each mutant toxin was compared to that of
the activity of the wild type toxins using a
surface contamination method against first
instar M. sexta larvae. The activity is
reported as an LC50 value (concentration of
the toxin required to kill 50% of the larvae)
shown in Table 1.
Proteinase K protection assays: To test if
the mutant toxin has retained the ability to
insert into M. sexta BBMV, we digested the
toxin bound to BBMV with proteinase K, a
non specific protease. Western blot analyses
show that even after treatment with 10 fold
excess of proteinase K for 30 minutes, most
of the mutants have retained an
approximately 60 kDa form of the toxin
(Fig. 2). For the domain II residue F371, its
mutation to tryptophan protected it from the
protease; however its mutation to cysteine
did not protect it. The effects of mutating
F371 on insertion of the toxin have been
published. F371 is a residue that has been
studied for its ability to mediate insertion of
the toxin into the membrane. The
partitioning rate of every mutant into the
brush border membrane and artificial
membranes are not the same. We have seen
that mutagenesis of F371 to other residues in
decreasing order of hydrophobicity have
compromised its insertion ability (31,34).
While mutation to Trp retained most of the
toxicity of the toxin in these studies, the
difference in the amount protected for this
mutant (Fig. 2B) could be due to the ability
of the toxin to partition only at a slower rate
than wild type, for a fixed amount of time
given for the toxin-membrane interaction in
this assay. For mutations other than that to
Trp we see further decrease in insertion rate
based on ion channel forming abilities like
voltage clamp studies and binding studies to
BBMV (34). F371C was the most affected
in these studies and using the thiol probe
acrylodan it was shown that this mutation
was completely ineffective in partitioning
into brush border membranes (35).
Labeling Cry1A mutants with fluorophore:
Purified cysteine mutants were labeled with
acrylodan or IAEDANS and purified off free
labels using gel filtration. The labeling
efficiency was measured with each
fluorophore and was found to be 95 ± 0.3 %
for acrylodan and 99 ± 0.5 % for IAEDANS
using their respective extinction coefficients.
Circular dichroism analyses of the labeled
mutants showed no variations in the spectra
in the region of 200-250 nm indicating that
the secondary structure has been retained
upon labeling with either fluorophore (data
not shown).
Quenching analysis of labeled mutants in
artificial vesicles: Using artificial SUV
made of POPC, POPE and cholesterol, the
percentage of quenching of each cysteine
mutant labeled to a fluorophore of the
aminonapthalene sulfonate (ANS) family,
IAEDANS, upon partitioning into the
vesicles were measured. The IAEDANS
fluorophore has a very high dipole moment
and hence increased quantum yield of
emission in an aqueous environment that
gets quenched inside the SUV. Thus upon
partitioning of the toxin into SUV, the
fluorescence emission of IAEDANS is
quenched. The percentage of quenching for
each of the mutants in our study has been
reported in Fig. 3. Results show that mutants
in Domain I (D62C, V171C, L199C,
L215C) , Domain II (S279C, S324C,
S364C) and Domain III (K489C , I526C) all
have about 50% or more of fluorescence
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quenched inside the SUV while residues
L40C, S191C in Domain I and E460C in
Domain III have less than 50% quenching.
Only F371C has almost no quenching into
SUVs. Addition of KI solution to a final
concentration of 0.83M quenched the
fluorescence of labeled toxin in buffer
completely at the volumes used, but when
mixed with the SUV bound labeled toxins
there was no further quenching of the
already quenched fluorescence of the vesicle
bound label, indicating that the hydrophilic
collisional quencher was not able to access
the label in the SUV bound form.
“Blue shift” measurements of acrylodan
labeled mutants: Acrylodan is an
environment sensitive fluorophore that
reacts with thiol groups of cysteines to form
a covalent conjugate. Depending on the
environment of the thiol group, there is a
variation in the fluorescence emission from
the molecule. Emission of the probe is low
and at longer wavelengths (around 500 nm)
in aqueous environments while in a lipid
environment like that of the BBMV, the
emission occurs at much shorter
wavelengths (around 460 nm). Emission
from acrylodan tagged mutants is dependent
on its dipole moment and is therefore
different for different mutants depending on
the exposure of the residue to aqueous
environment.
All our mutants, when tagged with
the fluorophore, showed a maximal emission
wavelength in aqueous environment around
480-500 nm. Upon binding to BBMV, the
maximal emission wavelength shifted to a
shorter wavelength for all of them, the
extent of which was different for each
mutant This “blue shift‟ was retained for all
the mutants even after treating the BBMV
bound labeled toxins with 100 fold excess of
proteinase K. Fig. 5A, B and C are a
representative spectra of mutants in Domain
I, II and III respectively indicating the
wavelength shift from longer to shorter
wavelength under different environments for
the label. The extent of blue shift was not
the same before and after proteinase K
treatment as indicated in Fig. 4. All these
mutants were accompanied by an increase in
the intensity of acrylodan fluorescence as
seen in case of the representative spectra
(Fig. 5A, 5B and 5C). The only exceptions
to this were Cry1Ab L40C, which showed
decrease in its fluorescence emission (Fig.
5D) and Cry1Ab F371C, which block the
protein from partitioning into the membrane
(35).
Voltage clamp measurements on Cry1A
mutants: To test the pore forming abilities of
each mutant, we carried out voltage
clamping of M. sexta midguts and measured
the percentage of remaining short circuit
current in the midgut after adding 100ng of
each toxin (Fig. 6). Slopes for the linear
region of the drop in the Isc were calculated
(Table 2). The voltage clamp response for
Cry1Ab V171C and F371C are already
reported earlier (35). The rate of ion
transport was measured as the slope of the
linear region of the drop in short circuit
current for each of the mutations. We found
that the rate of ion transport for mutants in
Domain I were overlapping with those of
mutants in Domain II and Domain III
indicating that pore formation was occurring
at a similar rate. In addition the time lag (T0)
values of 100 ng of all mutants ranged from
4.5 min to 6.5 min for all the mutants while
that of same amount of Cry1Aa and Cry1Ab
was 5.0 min. This time lag is an indicator of
the time the toxin takes, after adding to the
membrane, to initiate pore formation, in
other words, it is the time of partitioning of
the toxin into BBMV (28,31,32) suggesting
that all our mutants partitioned into the
membrane at a similar rate. The exception to
this is F371 when mutated to C or A, as
reported in our earlier studies (34,35), which
is restored to almost wild type with
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tryptophan substitution. We have recently
observed that G374C behaves as F371C
(data not shown).
DISCUSSION
Studies on the insertion of Cry toxins
for the past 2 decades have focused on the
mechanism by which the toxin forms ion
channels in the brush border membrane
vesicles of the insect midgut. Based on the
crystal structure of Cry3Aa, Li et.al (3)
proposed the Umbrella Model of insertion of
the toxin which predicts that only -helix 4
and 5 of Domain I of the toxin would insert
into the membrane because of the
hydrophobic nature of -helix 5. Subsequent
studies on the toxin were extensively
focused on the helices of Domain I
concluding that only those regions could
partition into the membrane (36,37) or line
the pore (38). These studies did not address
the fate of regions of the toxin other than the
-helices of Domain I once the toxin is
inserted. However proteinase K protection
studies (typically used for detecting the
regions of membrane proteins inside lipid
bilayer) have shown that a 60 kDa form (or
higher molecular weight aggregate) of the
toxin has been protected in membranes
(15,18,19,31,35). This suggests that most of
the toxin was likely to be embedded in the
membrane. We mutated several residues
across the 3 domains of the toxin to cysteine
to determine if these residues (and thereby
the region of the toxin around them) are
embedded into the membrane. Using
fluorescence quenching and/or blue shift
measurements, our results indicate that
regions in all 3 domains of the toxin
partition into the membrane.
This study used 6 mutations that
span Domain I, 4 mutations that span
Domain II and 3 mutations that span
Domain III. Toxicity data showed that none
of these mutations compromised the
biological activity of the toxin (Table 1) and
voltage clamp analysis (Table 2) further
indicates that all the mutations formed ion
channels at a similar rate to wild type toxin.
Quenching data for IAEDANS-
labeled mutants show that most of the
labeled residues in all 3 domains of the toxin
quenched their fluorescence upon
partitioning into SUVs. Certain residues
showed more quenching indicating that the
label attached to those residues were in a
relatively more hydrophobic environment as
compared to those that showed less
quenching. That the quenching was due to
the label being in SUV was confirmed by
the lack of any further quenching by the
hydrophilic quencher, KI added to the SUV-
bound labeled toxin. An alternative
possibility for the quenching of IAEDANS-
labeled toxin is that the quenching could be
due to hydrophobic environment generated
by the toxin molecules itself upon self
aggregation or self oligomerization. We
have examined several IAEDANS-labeled
toxins using in vitro self aggregation in low
ionic strength buffers (39). None of these
labeled toxins showed significant quenching
upon aggregation (data not shown).
A second possibility for the
quenching of IAEDANS-labeled toxin in
SUV is the possibility that SUVs do not
mimic the natural BBMV environment
where receptors play a role in determining
the regions of the toxin that would be buried
in the membrane. However when
IAEDANS-labeled toxin was used in natural
BBMV, all labeled positions, except F371C
were quenched in the 80-90% range (data
not shown). The result that the label was
quenched in presence of SUV in mutants
from all 3 domains suggests that more than
two helices of Domain I of the toxin are
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bound to the vesicles in the hydrophobic
environment.
To verify the above observations
with IAEDANS we choose a chromophore,
Acrylodan that undergoes enhanced
fluorescence and a spectral “blue shift” upon
entering a hydrophobic environment. Each
cysteine mutant used in the study except
L40C (on -helix 1) and F371C (which
blocks membrane partitioning of the whole
toxin into the membrane, (35)) showed a
“blue shift” in its fluorescence upon binding
to BBMV. We observed that not only
regions of Domain I insert into the brush
border membrane, as predicted by the
models in question, but that regions of
Domain II and Domain III used in the study
also inserted successfully into the
membrane. In these experiments, we were
able to circumvent any blue shift that might
have occurred due to self aggregation or
oligomerization of the toxin outside the
membrane by incorporating an additional
step by measuring the fluorescence of toxin
treated vesicles before and after treating
them with proteinase K for each mutant.
This treatment enabled us to confirm that the
region of the toxin to which the labeled
toxin was bound, was inserted into the
membrane bilayer and was not in a
hydrophobic environment outside the
membrane. The extent of “blue shift” seen
with each of the mutants before and after the
proteinase K treatment was different. The
“red” end of the spectrum (the environment
that the labeled toxin is exposed to in the
buffer) varies for each mutant depending on
the polarity of the environment for that
residue of the toxin. The extent of “blue
shift” that each toxin undergoes indicates the
change in the polarity of the environment
that the toxin is exposed to upon insertion
into the membrane. Domain I residues
D62C, V171C had the greatest shift while
residues in Domain II (S324C) and Domain
III (I526C) also underwent a significant blue
shift in its fluorescence even after proteinase
K treatment of the vesicles. Blue shifts of
fluorescence in case of all other proteinase
K treated mutants indicate that regions of
the toxin around those residues were also
buried in the membrane. The quantum yield
of fluorescence in all these mutants were
also increased when the toxin was in BBMV
compared to when they were in buffer.
Proteinase K treatment of labeled residue
L40C showed a complete loss in
fluorescence upon binding to BBMV
indicating that the region of α helix 1 is not
present in the membrane. SDS-PAGE of the
proteinase K protected mutant L40C showed
an intact 60 kDa form of the toxin indicating
that only the region around that residue (α-
helix 1) was vulnerable to the protease and
the rest of the toxin in this mutant was
protected intact inside the membrane.
Voltage clamping of the mutant also
generated a similar rate of formation of ion
channels as the wild type (Fig. 6). In case of
F371, our previous studies (35) show that
the residue is involved in post receptor
binding processing and therefore its
mutation to smaller amino acids like alanine
or cysteine prevents the insertion of the
toxin, while replacement to tryptophan
protects the toxin from proteinase K as
shown in Fig. 2 and by earlier studies (23).
We are further investigating the specific role
of F371 in mediating membrane insertion.
Our fluorescence partitioning data is
complemented by electrophysiological
analysis of all the mutants using voltage
clamping of M. sexta midguts.
Measurements of the rate of partitioning (T0)
and the rate of ion channel formation
(µA/min) for each mutant from Domains I,
II or III showed that all mutants were able to
partition and form ion channels at a similar
rate. The data suggests that the entry of the
toxin into brush border membranes may be
more likely at the same rate or together for
each domain, i.e., the entire toxin molecule
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rather than isolated regions, may partition
into the membrane.
Our observation do not support the
Umbrella Model of insertion of Cry1A toxin
into brush border membrane vesicles, since
we show that residues from Domain I,
Domain II and III insert into the membrane.
These observations, providing evidence of
specific regions from all 3 domains to the
toxin that are buried in the membrane, are
consistent with our previous study where we
show Domain II is involved in insertion
(35). In summary this work supports the
alternative model of insertion (32,35,40) that
proposes almost the entire toxin of about 60
kDa to insert into the insect brush border
membrane to mediate toxicity.
REFERENCES
1. Schnepf, E., Crickmore, N., VanRie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., and
Dean, D. H. (1998) Microbiol. Mol. Biol. Rev. 62, 775-806
2. Grochulski, P., Masson, L., Borisova, S., Pusztai-Carey, M., Schwartz, J.-L., Brousseau, R., and
Cygler, M. (1995) J. Mol. Biol. 254, 447-464
3. Li, J., Carroll, J., and Ellar, D. J. (1991) Nature 353, 815-821
4. Morse, R., Yamamoto, T., and Stroud, R. M. (2001) Structure 9, 409-417.
5. Galitsky, N., Cody, V., Wojtczak, A., Ghosh, D., Luft, J. R., Pangborn, W., and English, L.
(2001) Acta Crystallogr D Biol Crystallogr 57, 1101-1109.
6. Boonserm, P., Davis, P., Ellar, D. J., and Li, J. (2005) J. Mol. Biol. 348, 363-382
7. Boonserm, P., Mo, M., Angsuthanasombat, C., and Lescar, J. (2006) J Bacteriol 188, 3391-3401
8. Francis, B. R., and Bulla, L. A., Jr. (1997) Insect Biochem. Mol. Biol. 27, 541-550
9. Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051 - 28056
10. Knight, P. J. K., Crickmore, N., and Ellar, D. J. (1994) Mol. Microbiol. 11, 429-436
11. Sangadala, S., Walters, F. S., English, L. H., and Adang, M. J. (1994) J. Biol. Chem. 269, 10088-
10092
12. Griffitts, J. S., Whitacre, J. L., Stevens, D. E., and Aroian, R. V. (2001) Science 293, 860-864.
13. Griffitts, J. S., Haslam, S. M., Yang, T., Garczynski, S. F., Mulloy, B., Morris, H., Cremer, P. S.,
Dell, A., Adang, M. J., and Aroian, R. V. (2005) Science 307, 922-925.
14. Valaitis, A. P., Jenkins, J. L., Lee, M. K., Dean, D. H., and Garner, K. J. (2001) Archives of Insect
Biochemistry and Physiology 46, 186-200.
15. Aronson, A. I., Geng, C., and Wu, L. (1999) Appl. Environ. Microbiol. 65, 2503-2507
16. Bravo, A., Gómez, I., Conde, J., Muñoz-Garay, C., Sanchez, J., Miranda, R., Zhuang, M., Gill, S.
S., and Soberón, M. (2004) Biochem. Biophys. Acta 1667, 38-46.
17. Knowles, B. H. (1994) Adv. Insect Physiol. 24, 275-308
18. Aronson, A. (2000) Applied and Environmental Microbiology 66, 4568-4570
19. Tomimoto, K., Hayakawa, T., and Hori, H. (2006) Comp Biochem Physiol B Biochem Mol Biol
144, 413-422
20. Höfte, H., and Whiteley, H. R. (1989) Microbiol. Rev. 53, 242-255
21. Lee, M. K., You, T. H., Curtiss, A., and Dean, D. H. (1996) Biochem. Biophys. Res. Commun.
229, 139-146
22. Schnepf, H. E., and Whiteley, H. R. (1981) Proc. Natl. Acad. Sci. USA 78, 2893-2897
23. Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., Curtiss, A., and Dean, D. H. (1995) J.
Bacteriol. 177, 2276-2282
24. Lee, M. K., Milne, R. E., Ge, A. Z., and Dean, D. H. (1992) J. Biol. Chem. 267, 3115-3121
25. Peyronnet, O., Vachon, V., Schwartz, J.-L., and Laprade, R. (2001) J. Membrane Biol. 184, 45-54
by guest on May 12, 2020
http://ww
w.jbc.org/
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nloaded from
10
26. Schwartz, J.-L., Garneau, L., Savaria, D., Masson, L., Brousseau, R., and Rousseau, E. (1993) J.
Membrane Biol. 132, 53-62
27. Pitcher, W. H., 3rd, and Huestis, W. H. (2002) Biochem Biophys Res Commun 296, 1352-1355
28. Liebig, B., Stetson, D. L., and Dean, D. H. (1995) J. Insect Physiol. 41, 17-22
29. Wolfersberger, M., Lüthy, P., Maurer, A., Parenti, P., Sacchi, F. V., Giordana, B., and Hanozet,
G. M. (1987) Comp. Biochem. Physiol. 86A, 301-308
30. Chamberlin, M. E. (1994) Physiological Zoology 67, 82-94
31. Arnold, S., Curtiss, A., Dean, D. H., and Alzate, O. (2001) FEBS Letts. 490, 70-74
32. Alzate, O., You, T., Claybon, M., Osorio, C., Curtiss, A., and Dean, D. H. (2006) Biochemistry
45, 13597-13605
33. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492
34. Rajamohan, F., Cotrill, J. A., Gould, F., and Dean, D. H. (1996) J. Biol. Chem. 271, 2390-2397
35. Nair, M. S., Liu, X. S., and Dean, D. H. (2008) Biochemistry 47, 5814-5822
36. Gazit, E., and Shai, Y. (1993) Biochemistry 32, 3429-3436
37. Gazit, E., LaRocca, P., Sansom, M. S. P., and Shai, Y. (1998) Proc. Nat'l. Acad. Sci. 95, 12289-
12294.
38. Masson, L., Tabashnik, B. E., Liu, Y.-B., Brousseau, R., and Schwartz, J.-L. (1999) J. Biol.
Chem. 274, 31996-32000.
39. Masson, L., Mazza, A., Sangadala, S., Adang, M. J., and Brousseau, R. (2002) Biochim. Biophys.
Acta 1594, 266-275
40. Loseva, O. I., Tiktopulo, E. I., Vasiliev, V. D., Nikulin, A. D., Dobritsa, A. P., and Potekhin, S.
A. (2001) Biochemistry 40, 14143-14151.
FOOTNOTES
The abbreviations used are: BBMV, brush border membrane vesicles; SUV, small unilamelar vesicles;
IAEDANS, 5-((((2-iodoacetyl) amino) ethyl) amino)-napthalene-1-sulfonic acid; Acrylodan, 6-acryloyl-
2-dimethyl-aminonapthalene; max, Maximal emission wavelength;
FIGURE LEGENDS
Figure 1: A. Western blot analysis of purified Cry1A toxin used for proteinase K protection assays. Lane
1: Cry1Aa. Lane 2: Cry1Ab. Lane 3: Cry1AbL40C. Lane 4: Cry1AaD62C. Lane 5:
Cry1AbV171C. Lane 6: Cry1AbS191C. Lane 7: Cry1AbL199C. Lane 8: Cry1AbL215C.
B. Western blot analysis of purified Cry1A toxin used for proteinase K protection assays: Lane
1: Cry1AbS279C. Lane 2: Cry1AbS324C. Lane 3: Cry1AbS364C. Lane 4: Cry1AbF371W. Lane
5: Cry1AbF371C. Lane 6: Cry1AaE460C. Lane 7: Cry1AaK489C. Lane 8: Cry1AaI526C.
C. Purified Domain I mutant proteins run on 8% SDS_PAGE gel. Lane 1= Protein Standard;
Lane 2 = 1Ab L40C; Lane 3 = 1Aa D62C; Lane 4 = 1Ab V171C; Lane 5 = 1AbS191C; Lane 6 =
1Ab L199C; Lane 7 = 1Ab L215C.
D: Purified Domain II and Domain III mutants run on 8% SDS PAGE gels. Lane 1 & 9 = Protein
Standards; Lane 2 = 1Ab S279C; Lane 3 = 1Ab S324C; Lane 4 = 1Ab S364C; Lane 5 = 1Ab
F371C; Lane 6 = 1AaE460C; Lane 7 = 1Aa K489C and Lane 8 = 1Aa I526C.
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Figure 2: A. Western blot analysis of 8% SDS-PAGE run of proteinase K protection assay of Cry1A
mutants bound to BBMV. The mutants used are as follows: Lane 1: Cry1Ab. Lane 2. 1AbL40C.
Lane 3: 1AaD62C. Lane 4: 1AbV171C. Lane 5: 1AbS191C. Lane 6: 1AbL199C. Lane 7:
1AbL215C.
B. Western blot analysis of 8% SDS-PAGE run of proteinase K protection assay of Cry1A
mutants bound to BBMV. The mutants used are as follows: Lane 1: Cry1Aa. Lane 2: 1AbS279C.
Lane 3: 1AbS324C. Lane 4: 1AbS364C. Lane 5: 1AbF371W. Lane 6: 1AbF371C. Lane 7:
1AaE460C. Lane 8: 1AaK489C. Lane 9: 1AaI526C.
Figure 3: Percentage of quenching of fluorescence of 1, 5-IAEDANS tagged cysteine mutants calculated
as (Iaq– ISUV)/Iaq where Iaq is the quantum yield of fluorescence of the labeled mutants in aqueous
buffer and ISUV is the quantum yield of fluorescence of the labeled mutants in SUV.
Figure 4: Blue shift in the maximal emission wavelength of each acrylodan labeled mutant. The X axis
indicates the name of each labeled mutant studied and the Y axis shows the maximal emission
wavelength. Maximal emission wavelength of each mutant in aqueous carbonate buffer is
indicated by (●), in BBMV without any protease treatment is indicated by () and in BBMV after
Proteinase K treatment is indicated by (▼)
Figure 5: Steady state fluorescence measurement for the following acrylodan labeled mutants:
A. D62C (Domain I); B.S324C (Domain II), C. I526C (Domain III) and D. L40C (Domain I).
(●) represents the fluorescence in buffer, () represents the fluorescence in BBMV before
proteinase K treatment and (▼) represents the fluorescence in BBMV after proteinase K
treatment. Y–axis represents the relative intensity of fluorescence of the labeled mutant in buffer
versus membranes and not absolute values of intensity.
Figure 6: A. Voltage clamp response of Cry1Ab (●) to those of Domain I mutants: L40C (), D62C (■),
S191C (), L199C () and L215C (▼). (V171C has been reported earlier (35).
B: Voltage clamp response of Cry1Aa (●) to those in Domain II mutants: S279C () S324C (▼)
S364C () E460C (■) K489C () and I526C (). F371C and F371W have been reported earlier
(34,35).
The arrow indicates the time at which toxin was added to the stabilized midguts.
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Table 1: Bioassay measurements of Cry 1A toxins and their mutants on first instar larvae of M.sexta using
surface contamination method. 16 larvae were measured per concentration. Results were
measured after 5 days of exposure to toxin and calculated as LC50 using probit analysis (Softox).
LC50 for mutants marked in () are cited in references (23,35).
Toxin/ Mutant
Wt/Domain I
LC50 (ng/cm2) Toxin/Mutant
Domain II& III
LC50 (ng/cm2)
Cry1Aa 16.0
(8.0-25.3)
S279C 22.0
(8.2-35.6)
Cry1Ab 20.0
(7.5-31.7)
S324C 19.8
(7.5-32.2)
L40C 20.0
(7.0-33.4)
S364C 25.6
(11.2-40.2)
D62C 12.0
(4-20.2)
F371C >2000(*)
V 171C 20.0(*)
(7.5 -31.7)
F371W 13(*)
(8 -20)
S191C 28.2
(12.4- 44.2)
E460C 14.2
(5.6-23.0)
L199C 32.4
(16.2-48.6)
K489C 12.0
(3.8-21.0)
L215C 25.4
(11.2-40.4)
I526C 12.6
(3.0-21.4)
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Table 2A: The rate of ion transport measured from the slope of the linear region of the decrease in short
circuit current remaining (Isc) in M. sexta midguts for Domain I mutants.
SAMPLE T0 (min) Slope (µA/min)
Cry1Ab 4.0-5.0 -12.0± 3.2
1AbL40C 4.0-5.0 -9.42± 2.33
1AaD62C 6.0-7.0 -10.72± 5.2
1AbS191C 4.0-5.0 -13.5± 2.92
1AbL199C 5.0-6.0 -15.1± 5.0
1AbL215C 4.0-5.0 -11.45± 0.75
Table 2B: The rate of ion transport measured from the slope of the linear region of the decrease
in short circuit current remaining (Isc) in M. sexta midguts for Domain II and III mutants.
SAMPLE T0 (min) Slope (µA/min)
Cry1Aa 4.0-5.0 -11.0± 1.7
1AbS279C 5.0-6.0 -10.3± 3.0
1AbS324C 4.0-5.0 -9.12± 0.89
1AbS364C 5.0-6.0 -11.0± 1.2
1AaE460C 4.0-5.0 -10.67± 1.67
1AaK489C 5.0-6.0 -9.58± 1.67
1AaI526C 5.0-6.0 -11.66± 3.0
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Figure 1A.
250
150
100
75
50
37
1 2 3 4 5 6 7 8
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Figure 1B.
250
150
100
75
50
37
1 2 3 4 5 6 7 8
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Figure 1D.
75
50
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Figure 2: A:
Figure 2:B.
200
150
100
75
50
37
25
1 2 3 4 5 6 7
200
150
100
75
50
37
25
1 2 3 4 5 6 7 8 9
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Figure 4:
Mutant Residue Position
D6
2C
V1
71
C
S1
91
C
L1
99
C
L2
15
C
S2
79
C
S3
24
C
S3
64
C
E4
60
C
K4
89
C
I52
6C
m
ax
of
Ac
rylo
da
n E
mis
sio
n
450
460
470
480
490
500
510
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Figure 5A & B.
Wavelength (nm)
400 500 600 700
Re
lati
ve in
ten
sit
y o
f fl
uo
rescen
ce
(A
.U)
-4
-2
0
2
4
6
8
10
12
14
A
Wavelength (nm)
350 400 450 500 550 600 650
Re
lati
ve f
luo
rescen
ce
in
ten
sit
y (
A.U
)
0
2
4
6
8
10
B
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Figure 5 C & D
Wavelength (nm)
350 400 450 500 550 600 650 700
Re
lati
ve F
luo
resce
nce In
ten
sit
y (
Arb
itra
ry U
nit
s)
-2
0
2
4
6
8
10
12
C
Wavelength (nm)
400 500 600 700
Rela
tive F
luo
rescen
ce In
ten
sit
y (
Arb
itra
ry U
nit
s)
-4
-2
0
2
4
6
D
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Figure 6A:
.
Figure 6B:
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Manoj S. Nair and Donald H. DeanAll domains of Cry1A toxins insert into insect brush border membranes
published online July 17, 2008J. Biol. Chem.
10.1074/jbc.M802895200Access the most updated version of this article at doi:
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