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: dean.10@osu.edu

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

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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

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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

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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

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20. Höfte, H., and Whiteley, H. R. (1989) Microbiol. Rev. 53, 242-255

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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.

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24. Lee, M. K., Milne, R. E., Ge, A. Z., and Dean, D. H. (1992) J. Biol. Chem. 267, 3115-3121

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G. M. (1987) Comp. Biochem. Physiol. 86A, 301-308

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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-

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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 1C.

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Figure 1D.

75

50

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Figure 2: A:

Figure 2:B.

200

150

100

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25

1 2 3 4 5 6 7

200

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Figure 3:

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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

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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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