Accepted Manuscript

Title: Bacillus thuringiensis Toxin, Cry1 C interacts with128HLHFHLP134 region of Aminopeptidase N ofAgricultural Pest, Spodoptera litura

Author: Ravinder Kaur Anil Sharma Dinesh Gupta MridulKalita Raj K. Bhatnagar

PII: S1359-5113(14)00009-9DOI: http://dx.doi.org/doi:10.1016/j.procbio.2014.01.008Reference: PRBI 10034

To appear in: Process Biochemistry

Received date: 17-7-2013Revised date: 27-12-2013Accepted date: 11-1-2014

Please cite this article as: Kaur R, Sharma A, Gupta D, Kalita M, Bhatnagar RK, Bacillusthuringiensis Toxin, Cry1<ce:hsp sp=0̈.25/̈>C interacts with 128HLHFHLP134 regionof Aminopeptidase N of Agricultural Pest, Spodoptera litura, Process Biochemistry(2014), http://dx.doi.org/10.1016/j.procbio.2014.01.008

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

Page 1 of 30

Accep

ted

Man

uscr

ipt

1

Bacillus thuringiensis Toxin, Cry1C interacts with 128HLHFHLP134 region of Aminopeptidase N of Agricultural Pest, Spodoptera litura Ravinder Kaura,c, Anil Sharmaa,c, Dinesh Guptab, Mridul Kalitab and Raj K Bhatnagar*,a

a. Insect Resistance Group, International Center for Genetic Engineering and Biotechnology, New Delhi-110067, India.

b. Bioinformatics Group, International Center for Genetic Engineering and Biotechnology, New Delhi-110067, India.

c. These authors contributed equally to the work * Corresponding Author

Raj K. Bhatnagar Insect Resistance Group International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Phone: +91-11-26741153 Fax: +91-11-26742316 Email: raj@icgeb.res.in

Page 2 of 30

Accep

ted

Man

uscr

ipt

2

ABSTRACT We modeled Cry1C toxin and its Aminopeptidase-N receptor and in silico docking

analysis was performed. Further, we utilized biopanning against Cry1C followed by

blocking assays and mutagenesis analysis to identify the binding epitope of SlAPN.

We have identified a putative SlAPN binding region, APN-CRY (128HLHFHLP134).

A derivative of SlAPN carrying the 128HLHFHLP134 region termed as binding

region of APN (BR-APN) was cloned and its involvement in Cry1C binding and

toxicity was checked. Cry1C-BR-APN binding was competed by synthetic peptides

homologous to loop2 and loop3 of domain II but not by that of loopα. Additionally,

alanines substitution of residues H128, H130, H132 and P134 affect the binding

efficiency of receptor to Cry1C toxin (upto 4-fold lower affinity).These residues are

also implicated in Cry1C toxicity as shown by the reduced ability to affect the

mortality of Cry1C on S. litura larvae when toxin was preincubated with a fragment

of the receptor.

Key Words: Agriculture pest, Spodoptera litura, Cry1C, Aminopeptidase1, Toxin-receptor interaction, Mutagenesis.

Page 3 of 30

Accep

ted

Man

uscr

ipt

3

1. Introduction The larvicidal inclusions (δ-endotoxins) of Bacillus thuringiensis (Bt) consist of one

or more insecticidal crystal proteins (Cry) proteins which are classified into 72 classes

according to their sequence similarity [1]-[4]. Upon ingestion the toxins are active

against certain Lepidopteran, Dipteran, Coleopteran and Hymenopteran insect larvae

[5]-[8].

Most of the Bt Cry toxins are synthesized as inactive protoxins which upon ingestion

by a susceptible larvae, dissolve in the alkaline and reducing environment of the

larval midgut, thereby releasing soluble proteins. The soluble protoxin is processed

proteolytically by midgut enzymes, yielding 60-70 kDa protease-resistant toxic

fragments [3]. The activated toxin then binds specific receptor molecules located in

the midgut epithelial cell brush border membranes of the host insect [9]. The toxin

molecules oligomerize to form of a pre-pore oligomeric structure. The specific

binding involves two-steps, a reversible followed by an irreversible one leading to

insertion of the toxin oligomer into the cell membrane, resulting in the formation of

pores in the membrane and eventually cell lysis [10]- [12].

The receptors of the Cry toxins have been classified into five major groups: a

cadherin like protein (CADR), a glycosyl phosphatidyl-inositol (GPI)-anchored

aminopeptidase-N (APN), a GPI-anchored alkaline phosphatase (ALP), a 270 kDa

glycoconjugate and glycolipids [13]-[17]. Since 1994, more than 60 different APNs

from different Lepidopterans have been sequenced and registered in databases

showing the high diversity in isoforms [18]. This exopeptidase is believed to be

anchored in lipid rafts and serves as a binding molecule for Cry1A, Cry1C, Cry1F and

Cry1J toxins in different Lepidopteran species [19], [20], [21]. The fact that it is

implicated in toxin insertion [22] enhances Cry1Aa pore formation when incorporated

into lipid bilayers [23]. Further, its inactivation by natural mutations [18] or by RNAi

[24] makes the insect populations resistant to Bt toxins confirms its critical role as

functional receptor in Bt toxicity. However, the details of APN-toxin interaction still

need investigation. Although some APN binding epitopes in the toxin have been

characterized, little is known about the APN domains involved in Cry toxin binding

[25].

Previous work in our lab demonstrated that a 110 kDa recombinant APN is a receptor

for Cry1C toxin in Spodoptera litura midgut and is responsible for Cry1C toxin

Page 4 of 30

Accep

ted

Man

uscr

ipt

4

sensitivity in the larvae [21]. The mapping of binding epitopes of APN is a

challenging problem since mutagenesis studies of the receptor domains have never

been performed as in case of toxins. We have earlier shown that the toxin binding and

substrate binding regions of the SlAPN are different [26] and in this paper we took a

more detailed look at the toxin binding domain of APN. In order to map the precise

regions involved in SlAPN-Cry1C interactions, we decided to use the phage-display

technique. This technology is highly successful in receptor identification [27] and

epitope mapping [28], [29]. The findings were further confirmed by alanine

substitutions in the receptor protein aminopeptidase N.

2. Materials and Methods 2.1 Modeling of Cry1C and SlAPN and Docking of the derived structures

A standalone version of BLAST 2.2.4 [30] was used to blast the target sequences

against PDB database to retrieve the top hits to select respective templates for 3D-

homology modeling. Using Clustal X 1.83 [31], an alignment was obtained between

the best hit and the target sequence. We did extensive manual inspection and curation

of alignments and used alignment.check script of MODELLER [32] to find any trivial

alignment mistakes. These alignment files (after successful run of alignment.check)

were given as input to the MODELLER 8v2, one at a time for model building. Using

WHATIF programs [33], missing side chains were added to the respective models.

Energy minimization (EM) was done in vacuo to relieve steric interactions within the

model structure. The quality of energy minimized models was checked by various

WHATIF programs. Stereochemical parameters of the model structures were

analyzed with the PROCHECK program [34]. Local environment of atoms, packing

quality and folding states of models were further studied using more advanced

methods like PROSA II [35], Verify3D [36] and ANOLEA [37]. Finally, to access the

deviation of modeled structure with their respective templates, we used STAMP

based 3d-SS [38] and CE/CL based Compare3D. Using GRAMM v1.03 [39], we

carried out the docking process in two modes, mode1: provided the HLHFHLP

sequence moiety of APN receptor protein as interface residue constraints for

consideration during docking and mode2: without any residue constraint mode. Top

10 complexes for each mode were studied for final analysis.

Page 5 of 30

Accep

ted

Man

uscr

ipt

5

2.2 Purification of active recombinant Bt Cry1C toxin and production of

polyclonal antibodies against it

The recombinant protoxin Cry1C expressing DH5α strain was obtained from the

Bacillus Genetic Center, Ohio State University. Cry1C inclusion bodies were purified,

solubilized and trypsin-activated by a modification of the procedure described by Lee

et al. [40]. Briefly, inclusion bodies were purified and solubilized in 50mM sodium

carbonate buffer, pH 10.5, containing 10mM dithiothreitol (solubilisation buffer) at

37°C. The solubilized crystal protein was activated by trypsin (United State

Biochemical) at a trypsin: protoxin ratio of 1:10 (by mass) at 37°C for 1h. The

activated toxin was purified by anion exchange liquid chromatography using Q-

sepharose anion exchanger. The purity of the toxin was checked by SDS-PAGE and

fractions containing the purified toxin were pooled and dialyzed against tris buffered

saline (TBS) for raising antibodies and binding studies.

To raise anti-Cry1C antiserum, a New Zealand White rabbit was immunized with

100μg of purified active Cry1C administered in Freud’s complete adjuvant (Sigma).

After boosting the rabbit once with the toxin (administered in Freud’s incomplete

adjuvant), the rabbit serum was collected and assessed for its reactivity.

2.3 Biopanning and Characterization of binding phage display clones

A synthetic combinatorial library of random disulphide linked hepta-peptides fused to

the minor coat protein (pIII) of M13 phage was used in this work (New England

Biolabs Inc Ph.D.-C7C phage display peptide library kit). This library size was

1.2x109 clones and was amplified once to yield approximately 200 copies of each

sequence in 10 μl of the supplied phage. Each time 10 μl (2x1011 phages) of supplied

library was used to select peptides binding to the coated active Cry1C as ligand. For

each experiment, ligand-binding phages were isolated by panning using

immunosorbent polystyrene 96-well plates (Costar NY, USA) which were coated with

5 μg of the ligand by overnight incubation at 4°C. Three rounds of panning, with

increasing concentrations of Tween20 in wash steps, were carried out according to the

manufacturer’s instructions. After each round of selection, phage titer was determined

by plaque-assay method and enrichment of the eluted phage was analyzed by ELISA.

After the final selection round DNA from individual phages was purified and

sequenced using 28 gIII sequencing primer provided in the kit.

Page 6 of 30

Accep

ted

Man

uscr

ipt

6

2.4 Cloning, Expression and Purification of SlAPN and BR-APN

SlAPN expressed in Sf21 cells was solubilized and purified as described previously

[26]. Both biopanning and in silico docking analysis identified Cry1C interacting 128HLHFHLP134 region of APN, named as APN-CRY. Thus, a 12 kDa protein

fragment (Binding region of APN (BR-APN)) ranging from amino acid 51-162 with

APN-CRY region in it (table 1), was cloned in pQE expression vector and expressed

in E.coli M15 strain.

Production of soluble BR-APN fragment was obtained by incubating the log-phase

M15 culture with 1mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 16°C for

16h. Cells were collected by centrifugation and resuspended in 20mM Tris buffer (pH

8) containing 150mM NaCl (TBS). Total proteins were obtained by sonicating the cell

suspension on ice (four 30-second pulses) and observed under microscope for

complete lysis. The supernatant obtained after centrifuging the homogenate at low

speed (5000 rpm at 4°C for 10min) was dialysed against 50mM sodium phosphate

buffer (pH 8) (binding buffer) and loaded on Talon resin (Amersham) to purify the

His-tagged BR-APN fragment. The recombinant BR-APN fragment protein bound to

the resin were eluted according to the manufacturer’s instructions.

2.5 Toxin-overlay assays

A total of 1μg purified SlAPN or BR-APN was resolved on SDS-PAGE and electro

blotted onto a nitrocellulose (NC) membrane. The blot was incubated with 2.5 μg /ml

toxin and processed as described by Agrawal et al. [21]. Following Cry1C overlaying

the membrane was washed with TBST (20 mM Tris buffer (pH 8) containing 150 mM

NaCl and 0.5% Tween20) and incubated for 1 h in rabbit anti-Cry1C serum (diluted

1:20,000 in TBS containing 0.2% BSA). For blocking assays, strips containing

transferred SlAPN or BR-APN were cut from the NC membrane and overlaid with the

toxin previously incubated with different concentrations of peptide competitors

(APN-CRY or φ-71).

2.6 Binding assays

These were basically the reverse of toxin-overlay assays. Purified Cry1C toxin (1 μg)

was heated in SDS sample buffer containing β-mercaptoethanol (β-ME), resolved by

SDS-PAGE, and electro transferred to NC membrane. After incubation in blocking

Page 7 of 30

Accep

ted

Man

uscr

ipt

7

solution (3% BSA in tris buffered saline (TBS) containing 0.2% Tween20) for 1h at

room temperature (RT), the membrane was incubated for additional 1h in 2.5 μg of

purified SlAPN or BR-APN per ml in TBS solution. This was followed by three

washes with TBST and incubation for 1h with anti-APN antibodies (diluted

1:20,000). After three washes with TBST, the blots were incubated with alkaline

phosphatase (AP) conjugated goat anti-rabbit IgG antibodies (diluted 1:5,000) and

developed using p-nitro blue tetrazolium chloride (NBT)-5-bromo-4-chloro-3-indolyl

phosphate toluidine (BCIP) substrate (Sigma). For blocking assays, the purified

receptor (SlAPN or BR-APN) was incubated with different concentrations of peptides

in TBS for 1 h at RT before incubating the receptor with toxin containing NC blot.

2.7 Construction and Expression of BR-APN (Binding region of SlAPN) mutants

SlAPN mutants were constructed by introduction of deletions and alanine

substitutions at residues 128-134 (table 2) in the region for the portion of the gene

encoding BR-APN fragment in expression plasmid pQE-31, using the QuickChange

II XL Site-Directed Mutagenesis Kit (Stratagene). Native-type and mutant proteins

were expressed and purified as described above.

2.8 Biotin labeling of toxin and receptor proteins and competition binding assays

Native-type toxin and receptor proteins were labeled with biotin using EZ-Link Sulfo-

NHS-Biotin Reagents (Pierce) according to manufacturer’s instructions. For

heterologous competition assays involving native and mutant version of BR-APN

fragment, 1 μg of purified Cry1C was transferred on NC-membrane and overlaid with

saturating concentration of labeled BR-APN fragment in the presence of increasing

concentrations of unlabeled mutant BR-APN fragment. Binding parameters (Kd and

Bmax) were calculated from homologous competition assays (involving binding

competition of labeled proteins with the corresponding unlabeled proteins), using

GraphPad Prism version 5.0 programme.

2.9 Insect Bioassays

Activity of Cry1C against S. litura larvae was determined after one day of hatching. A

total of 200 μl (each side) of toxin dilutions (diluted in PBS) was layered on a

mulberry leaf disc placed in a petri dish and allowed to dry. Ten larvae were placed

on each petri-dish and mortality was analysed after two days of incubation [21].

Page 8 of 30

Accep

ted

Man

uscr

ipt

8

Bioassays were repeated 3-5 times. For bioassays performed in the presence of

competitors the toxin was pre-incubated with the competitor for 1h at RT and then

coated on the leaf disc.

Bioassays were performed with various BR-APN receptor mutants to study the

contribution of critical receptor residues to insect mortality. Previous experiments

have shown that 80 ng/cm2 pure native toxin is sufficient to induce 100% mortality

[21]. For mortality assays in the presence of BR-APN and mutant receptors as

competitors, 80 ng/cm2 of native-type Cry1C toxin was pre-incubated with 100-fold

molar excess of purified native-type BR-APN or mutant receptor peptides for 1h at

room temperature on end-on-end shaker and then coated on leaf disc.

3. Results 3.1 Validation of Cry1C and APN models

The results of secondary structure prediction by PSIPRED coincide with the

secondary structure of the final models. Ramachandran plots suggest that >96% of the

residues of the APN and more than 98% of the residues of Cry1C are in core plus

allowed regions. The PROSA II gave a Z-score of -8.58 for Cry model (template:

1CIY= -9.47) and -7.66 for APN model (template: 1Z5H= -13.04), which suggests

their acceptable structure. 3d-SS gave an rmsd of 0.672Å for Cry model whereas

1.142Å for APN model when compared to their respective template structures. A

highly comparable result was also obtained by Compare3D.

3.2 Description of SlAPN and Cry1C structure

The BLAST of SlAPN showed 29% identity (47% similarity) with 1Z5H protein at E-

value 1.1E-50 as top hit (Figure 1 of supplement section). 1Z5H is a zinc

aminopeptidase (tricorn interacting factor F3) from Thermoplasma acidophilum [41].

An extensive refinement of alignment leads to the increase in identity to 31%. The

two aminopeptidase (F3 and SlAPN) share the conserved HEXXH zinc binding motif.

The third zinc binding ligand in the motif NEXFA is at a conserve distance of 18

amino acid residues from the second zinc binding ligand, histidine. This makes

SlAPN a member of gluzinicin family of aminopeptidase [42]. Like F3, SlAPN has a

hook like structure formed by four domains (Figure 1a). The catalytic domain,

Domain II carries the conserved HEXXH and NEXFA motifs on two separate helices

α-3 and α-4 (respectively). A SPPIDER analysis for interacting residue prediction

Page 9 of 30

Accep

ted

Man

uscr

ipt

9

identified three high probability (>0.9) regions in SlAPN – region I involving residues

123-135, region II of residues 881-889 and region III of residues 936-941. These were

also the longest stretches of amino acids predicted to be in interfacial region.

The BLAST showed 46% identity (60% similarity) with its template, 1CIY

protein (an insecticidal toxin) at E-value 2.3E-145 as closest homologue. The final

identity of 48.5% was achieved after refinement of obtained alignment. The derived

3-dimensional structure of Cry1C revealed a folding pattern similar to those of known

crystal structures of Cry toxins (Figure 1b). The structure of the toxin provided the

basis to identify the potential loop regions. A close inspection of the alignment with

respect to the loop regions revealed that besides being in low conservation, the

regions near the loops contain many mutations either in the template or the target

sequence thus implying their importance as specificity determining regions. SPPIDER

[43] analysis of Cry1C showed that all the residues of domain II loops were

interfacial while those of domain III loops were either in interfacial or buried regions.

Prediction probabilities for interacting sites revealed that loop 2 had the highest

probability (>0.9) closely followed by loop 3 (>0.8). While loop α and loop 1 had

lower probabilities of being in interaction site (0.5 and 0.3 respectively), only 4 out of

11 loop β residues had significant interacting probability (>0.55). Structural analyses

of Cry1C showed that none of the inter-domain contacts involve loop regions.

3.3 Mapping the SlAPN epitope involved in Toxin-Receptor interaction by phage

display

To identify the SlAPN region involved in Cry1C binding, Ph.D.-C7C phage display

library was used to select a population of phages that bound immobilized purified

Cry1C. Three rounds of panning were done and phages from each round were

analyzed to determine enrichment of SlAPN binding clones (Figure 2a). Ten random

peptides obtained from third panning round were sequenced and 70% phages were

found to represent the same amino acid sequence (φ71: HPSFHWK) while all other

phages encoded unique peptides. The φ71 binds Cry1C in a dose dependent manner as

determined by ELISA (suggesting specific interaction) (Figure 2b) and shares ~43%

similarity to a 7 amino acid region present near the N-terminus of SlAPN

(128HLHFHLP134), named as APN-CRY.

3.4 Docking analysis of APN and Cry1C.

Page 10 of 30

Accep

ted

Man

uscr

ipt

10

Using GRAMM v1.03, we carried out the docking process in two modes, mode 1:

provided the APN-CRY sequence (128HLHFHLP134) of APN receptor protein as

interface residue constraints for consideration during docking and mode 2: without

any residue constraint mode. Using contact map analysis (CMA) tool [44], we studied

the interface residues of each of the ten final docked complexes of both modes 1 and

2. It was found that seven complexes of mode 1 and six complexes of mode 2 were

having the complete APN-CRY region along with flanking residues conserved within

the interaction surface. Other complexes were having at least 70% of this region as

interface residues. Most importantly, using mode1, we obtained seven complexes

where the APN-CRY region was found to be interacting with loop 3 or / and loop 2 of

Cry protein. The reliability of this prediction is enhanced as four of these complexes

were also observed in mode2 outputs, where no interface residue constraints were

given. Since our aim was to identify the APN-CRY cognate binding epitope in

Cry1C, we chose one of the complexes (complex M1.8) obtained from mode 1 as the

representative model to study APN-Cry1C interaction in detail.

The docked complex (complex M1.8) showed two regions of contact between the

receptor and the toxin (Figure 1c). While region A involves interactions between

domain I of Cry1C and latter half of domain I and 3 residues of domain II of APN,

region B showed multiple interactions involving loop 2 and loop3 of Cry1C domain II

and first half of domain I of APN. CMA analysis shows that the maximum area of

contact involves loop 3 of Cry1C and a region involving APN-CRY of (128-134)

SlAPN. An analysis of the putative forces involved in the receptor toxin interaction

revealed both hydrogen bonding and Van der Waal overlaps in the region B of contact

while the region A lacked any overlaps. CMA analysis of the docked complex

revealed that each residue in the interacting regions is capable of multiple

interactions.

3.5 Involvement of BR-APN region in Cry1C binding

To determine if the APN region that shares sequence similarity with φ71 plays any

role in toxin binding, we performed blocking assays using synthetic peptides (table 3)

corresponding either to φ71 peptide sequence or to the corresponding region in APN

(APN-CRY) as competitors. Figure 3 shows that binding of Cry1C to SlAPN was

competed by both peptides suggesting that 128HLHFHLP134 epitope is responsible for

Cry1C binding.

Page 11 of 30

Accep

ted

Man

uscr

ipt

11

3.6 Cloning of a SlAPN derivative (BR-APN) and Identification of its toxin

binding region by Competition Experiments

In order to identify the Cry1C region responsible for binding with the mapped APN

epitope, we made a peptide containing the predicted Cry1C-toxin binding region from

SlAPN. The details of primers used and expressed clone are given in table 1. This

BR-APN fragment cloned and expressed in E.coli, was able to bind to Cry1C in toxin

overlay assays and the binding was competed by the φ-71 peptide (Figure 4a). BR-

APN was used as the ligand to screen for binding clones of the random phage display

library. Seventy percent of the clones sequenced after third round of panning encoded

the same amino acid sequence (φ72: PSPNIPI) while all others represented unique

peptide sequences. φ-72 shared 58% similarity (based upon charges) with a region

involving loop 3 of domain II of Cry1C (436QRSGTPF442) suggesting that loop 3

might be the Cry1C epitope involved in binding with BR-APN region. No phage

peptide homologous to any domain III region was obtained indicating that BR-APN-

Cry1C interaction might not involve domain III. To test our hypothesis, we analyzed

BR-APN-Cry1C binding in the presence of loop 2, loop 3 and loop α peptide

competitors. Figure 4b shows that while loop2 and loop3 peptides (lanes 2-7) compete

the toxin-receptor binding, loop α peptide had no effect in this interaction (lanes 8-

10).

3.7 Involvement of BR-APN region in Cry1C toxicity

To ascertain the implication of BR-APN region in Cry1C toxicity to S. litura,

bioassays were performed on first instar larvae fed on cry1C toxin either alone or in

combination with 100-fold molar excess of peptide or proteins. Table 4 shows that

incubation of the toxin with the φ-71 peptide or BR-APN reduces the toxicity of

Cry1C by 45% and 50% respectively.

3.8 Construction and Expression of BR-APN (Binding region of SlAPN) mutants

The putative toxin binding epitope of SlAPN is a region of seven amino acids

(128HLHFHLP134) in the N-terminus of the SlAPN molecule. We expanded our

analysis of receptor ligand insecticidal protein interaction by focusing on the epitope

identified by phage display and deletion mutant data. Alanine substitutions of single

amino acids in the region 128HLHFHLP134 were done and positions of mutated amino

Page 12 of 30

Accep

ted

Man

uscr

ipt

12

acids are shown in table 2. All the mutants were highly expressed as soluble proteins

in E.coli, similar to native-type BR-APN receptor.

3.9 Effect of BR-APN mutations on toxin binding

Competition binding experiments were performed to analyse the toxin binding

properties of mutant BR-APN fragments. Binding constants determined from

homologous competition experiments are reported in table 5. Mutants L129A and

L133A bound Cry1C with affinities similar to that of native-type BR-APN (Table 5

and Figure 5) while mutants H128A, H130A, H132A and P134A showed up to 4-fold

lower affinity for the Cry1C toxin. Interestingly, mutant F131A showed only slightly

reduced affinity (Kd=14 nM) for the Cry1C toxin.

3.10 Effect of mutations in BR-APN on Cry1C toxicity

We analyzed the effects of BR-APN and its mutants on S. litura mortality caused by

Cry1C. A hundred-fold excess of purified native-type or mutated receptor protein was

incubated with the bioactive Cry1C toxin before feeding the toxin to the S. litura

larvae. Incubation of toxin with the native-type BR-APN reduced insect mortality by

55% (Table 6) while mutants H128A, P134A, H130A and H132A were not able to

affect the mortality significantly (85%, 80%, 75% and 70% mortality). On the other

hand, mutants L133A, L129A and F131A reduced Cry1C mortality to 50%, 55% and

60% respectively (Table 6). Insect bioassays with purified receptor BR-APN and

Cry1C highlighted relevance of 128HLHFHLP134 region of SlAPN in the recognition

and toxicity events of insecticidal protein.

4. Discussion The toxin-receptor interaction is a complex multi step process involving multiple

epitopes on both interacting molecules. The identification of epitopes involved in Cry

toxin-receptor interaction will be important for the characterization of resistant insect

populations in nature and to develop novel toxin formulations useful in insect pest

management.

The N-terminal region of APN is known to be responsible for the binding of the cry

toxins [45], [46]. In the present study, we took a detailed look at the SlAPN toxin

binding epitope(s). Using phage display technique, we identified a putative receptor

epitope (APN-CRY) including residues 128HLHFHLP134 and demonstrated its

Page 13 of 30

Accep

ted

Man

uscr

ipt

13

involvement in toxin binding by competition experiments. Predominance of phages

corresponding to APN-CRY region might suggest that this is a high affinity binding

site of the receptor molecule. In silico docking of cry1C and APN also revealed

involvement of the APN-CRY region in the toxin receptor interaction.

Three-dimensional model of Cry 1C toxin reveal that loops of domain II and domain

III do not lie close to each other, it is speculated that domain II of Cry1C interacts

with the APN-CRY epitope of SlAPN while the other domain might be interacting

with some other epitope of the SlAPN protein. To facilitate mapping of this second

Cry1C binding site of SlAPN we used GRAMM v1.03 which performs an exhaustive

6-D search through the relative rotations and translations of the molecules. The

advantage with the docking program is that no prior information about the binding or

interacting sites is mandatory and its ability to smoothen the surface representation to

account for possible conformational changes upon binding within the rigid body

docking approach. The docking studies identified two regions of binding in both APN

and Cry molecules. APN-CRY (128HLHFHLP134) region of SlAPN interacts with

loop3 of domain II of Cry1C toxin. The second interaction site involves domain I of

Cry1C and domain I of SlAPN. Further, biopanning with BR-APN fragment that had

APN-CRY region in it picked up phage φ-72 similar to 436QRSGTPF442 region of

Loop3 domain II of Cry 1C. This is the same region to which the APN-CRY region is

found interacting in docking analysis. Moreover, mutations in the 436QRSGTPF442

interfere with the interaction of the Cry1C toxin with the APN receptor [47]. We also

found that peptide corresponding to APN-CRY region competes for the binding to the

toxin. Moreover, bioassays in the presence of the synthetic peptides corresponding to

the mapped SlAPN epitope significantly reduced the toxicity of Cry1C, suggesting a

critical function of 128HLHFHLP134 moiety of SlAPN in insect toxicity. Phage

display, in silico docking and competition experiments with the SlAPN derivative,

BR-APN, further narrowed down the Cry1C epitopes interacting with APN-CRY

region. In Bombyx mori, the Cry1Aa-APN interacting epitopes have been mapped to

residues of domain III β16 (508STLRVN513) and β22 (582VFTLSAHV589) [45], in

Manduca sexta loop 2 and loop 3 of domain II of Cry1Ab are shown to be involved in

APN recognition [48], [49].

We have earlier shown that the APN protein serves as a small functional receptor of

Cry1C whose binding to the toxin is specific and influences the mortality of the target

Page 14 of 30

Accep

ted

Man

uscr

ipt

14

insect [21]. Since BR-APN is a small portion of the APN protein having the toxin

binding region (APN-CRY: 128HLHFHLP134) and offers the ease of prokaryotic

expression and purification system, various substitution mutations were carried out in

the APN-CRY region of this protein to study the region in detail. Since the APN-CRY

region has been shown to be involved in both toxin binding and mortality of the target

insect, the significance of individual amino acid residues of this region for both these

properties was analysed. Single point alanine substitutions were performed and the

mutants studied for their Cry1C binding and toxicity affects on S. litura larvae.

Bioassays were carried out by incubating the native-type Cry1C toxin with 100-fold

molar excess of BR-APN receptor or its mutants before feeding the toxin to the

susceptible larvae. Our results show that alanine substitution of residues H128A,

H130A, H132A and P134A resulted in considerable loss of their binding affinity to

the toxin. These mutants neither competed efficiently with BR-APN for Cry1C

binding affinity to the toxin (Figure 5 and table 6) nor could affect S.litura mortality

(Table 4). However, the mutants L129A and L133A were as efficient as native-type

BR-APN in both mortality and competition binding experiments. Thus, it seems L129

and L133 residues of SlAPN are not crucial for determining the binding and toxicity

to the larvae. However, residues H128, H130, H132 and P134 of APN receptor seems

to be responsible for interaction with the Cry1C toxin to effectuate its toxicity to the

larvae.

This is the first study reporting homology modeling of an insect aminopeptidase and

we believe that this will act as a useful benchmark in further studies of identification

of receptor epitopes. Additionally, for the first time a Cry toxin-receptor docking has

been attempted and shown to correlate with the experimental data. Analysis of docked

complex suggests that binding of domain II loops to the APN-CRY epitope might be

responsible in bringing domain I of Cry1C close to the lipid raft anchored receptor

thereby facilitating its insertion into the membrane. However, extensive research is

required in this direction to elucidate the sequence of events leading to toxin insertion

and pore formation.

5. Conclusions

Different approaches viz., phage display, in silico docking and competition binding

experiments) allowed us the identification of Cry1C and SlAPN cognate epitopes

Page 15 of 30

Accep

ted

Man

uscr

ipt

15

interacting with each other. Further mutagenesis in the APN-CRY region of APN

revealed crucial residues for binding and thus, toxicity of the Cry1C toxin to

agricultural pest, Spodoptera litura. Understanding of the toxin-receptor interaction is

must for dealing for developing resistance to the biopesticides.

Acknowledgements Prof Donald H. Dean, The Bacillus Genetic Stock Center, The Ohio State University,

Columbus, Ohio is acknowledged for providing recombinant protoxin Cry1C

expressing DH5a strain.

Page 16 of 30

Accep

ted

Man

uscr

ipt

16

References [1]. Bulla LA, Kramer KJ, Davidson LI. Characterization of the entomocidal

parasporal crystal of Bacillus thuringiensis. J Bacteriol 1977; 130: 375-83. [2]. Bulla LA, Bechtel DB, Kramer KJ, Shethna YI, Aronson AI et al.

Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis. Crit Rev Microbiol 1980; 8: 147-204.

[3]. Höfte H, Whiteley HR. Insecticidal crystal proteins of Bacillus thuringiensis.

Microbiol Rev 1989; 53: 242-55. [4]. Crickmore N, Zeigler DR, Schnepf E, Van Rie J, Lereclus D et al. Bacillus

thuringiensis toxin nomenclature 2012; http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

[5]. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J et al. Bacillus

thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

[6]. de Maagd RA, Bravo A, Crickmore N. How Bacillus thuringiensis has

evolved specific toxins to colonize the insect world. Trends Genet 2001; 17: 193-9.

[7]. van Frankenhuyzen K. Insecticidal activity of Bacillus thuringiensis crystal

proteins. J Invertebr Pathol 2009; 101: 1-16. [8]. Sharma Anil, Kumar S, Bhatnagar RK. Bacillus thuringiensis protein Cry6B

(BGSC ID 1 4D8) is toxic to larvae of Hypera postica. Curr Microbiol 2011; 62, 597–605.

[9]. Rajamohan F, Lee MK, Dean DH. Bacillus thuringiensis insecticidal proteins:

molecular mode of action. Prog Nucleic Acid Res Mol Biol 1998; 60: 1-27. [10]. Knowles BH. Mode of action of Bacillus thuringiensis upon feeding on

insects. Adv. Insect Physiol 1994; 24: 275-308. [11]. Lorence A, Darszon A, Díaz C, Liévano A, Quintero R, Bravo A. Delta-

endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers. FEBS Lett 1995; 360: 217-22.

[12]. Carroll J, Ellar DJ. An analysis of Bacillus thuringiensis delta-endotoxin

action on insect-midgut-membrane permeability using a light-scattering assay. Eur J Biochem 1993; 214: 771-8.

[13]. Jurat-Fuentes JL, Adang MJ. Characterization of a Cry1Ac-receptor alkaline

phosphatase in susceptible and resistant Heliothis virescens larvae. Eur J Biochem 2004; 271: 3127-35.

Page 17 of 30

Accep

ted

Man

uscr

ipt

17

[14]. Knight PJ, Crickmore N, Ellar DJ. The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol Microbiol 1994; 11: 429-36.

[15]. Nagamatsu Y, Koike T, Sasaki K, Yoshimoto A, Furukawa Y. The cadherin-

like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin. FEBS Lett 1999; 460: 385-90.

[16]. Valaitis AP, Jenkins JL, Lee MK, Dean DH, Garner KJ. Isolation and partial

characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity. Arch Insect Biochem Physiol 2001; 46: 186-200.

[17]. Griffitts JS, Haslam SM, Yang T, Garczynski SF, Mulloy B et al. Glycolipids

as receptors for Bacillus thuringiensis crystal toxin. Science 2005; 307: 922-5. [18]. Herrero S, Gechev T, Bakker PL, Moar WJ, de Maagd RA. Bacillus

thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes. BMC Genomics 2005; 6: 96.

[19]. Jurat-Fuentes JL, Adang MJ. Importance of Cry1 delta-endotoxin domain II

loops for binding specificity in Heliothis virescens (L.). Appl Environ Microbiol 2001; 67: 323-9.

[20]. Luo K, Lu YJ, Adang MJ. A 106-kDa form of aminopeptidase is a receptor for

Bacillus thuringiensis Cry1C δ-endotoxin in the brush border membrane of Manduca sexta. Insect Biochem Mol Biol 1996; 26: 783-91.

[21]. Agrawal N, Malhotra P, Bhatnagar RK. Interaction of gene-cloned and insect

cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C. Appl Environ Microbiol 2002; 68: 4583-92.

[22]. Bravo A, Gómez I, Conde J, Muñoz-Garay C, Sánchez J et al.

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 2004; 1667: 38-46.

[23]. Schwartz JL, Lu YJ, Söhnlein P, Brousseau R, Laprade R et al. Ion channels

formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors. FEBS Lett 1997; 412: 270-6.

[24]. Rajagopal R, Sivakumar S, Agrawal N, Malhotra P, Bhatnagar RK. Silencing

of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. J Biol Chem 2002; 277: 46849-51.

[25]. Pigott CR, Ellar DJ. Role of receptors in Bacillus thuringiensis crystal toxin

activity. Microbiol Mol Biol Rev 2007; 71: 255–81.

Page 18 of 30

Accep

ted

Man

uscr

ipt

18

[26]. Kaur R, Agrawal N, Bhatnagar R. Purification and characterization of aminopeptidase N from Spodoptera litura expressed in Sf21 insect cells. Protein Expr Purif 2007; 54: 267-74.

[27]. Fernández LE, Pérez C, Segovia L, Rodríguez MH, Gill SS et al. Cry11Aa

toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II. FEBS Lett 2005; 579: 3508-14.

[28]. Demangel C, Maroun RC, Rouyre S, Bon C, Mazié JC et al. Combining phage

display and molecular modeling to map the epitope of a neutralizing antitoxin antibody. Eur J Biochem 2000; 267: 2345-53.

[29]. Gómez I, Oltean DI, Gill SS, Bravo A, Soberón M. Mapping the epitope in

cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display. J Biol Chem 2001; 276: 28906-12.

[30]. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment

search tool. J Mol Biol 1990; 215: 403-10. [31]. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The

CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997; 25: 4876-82.

[32]. Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial

restraints. J Mol Biol 1993; 234: 779-815. [33]. Vriend G. WHAT IF: a molecular modeling and drug design program. J Mol

Graph 1990; 8: 52-6. [34]. Laskowski RA, Moss DS, Thornton JM. Main-chain bond lengths and bond

angles in protein structures. J Mol Biol 1993; 231: 1049-67. [35]. Sippl MJ. Recognition of errors in three-dimensional structures of proteins.

Proteins 1993; 17: 355-62. [36]. Lüthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-

dimensional profiles. Nature 1992; 356: 83-5. [37]. Melo F, Devos D, Depiereux E, Feytmans E. ANOLEA: a www server to

assess protein structures. Proc Int Conf Intell Syst Mol Biol 1997; 97: 110-3. [38]. Sumathi K, Ananthalakshmi P, Roshan MN, Sekar K. 3dSS: 3D structural

superposition. Nucleic Acids Res 2006; 34: W128-32. [39]. Vakser IA. Protein docking for low-resolution structures. Protein Eng 1995; 8:

371-7.

Page 19 of 30

Accep

ted

Man

uscr

ipt

19

[40]. Lee MK, Milne RE, Ge AZ, Dean DH. Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin. J Biol Chem 1992; 267: 3115-21.

[41]. Kyrieleis OJ, Goettig P, Kiefersauer R, Huber R, Brandstetter H. Crystal

structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations. J Mol Biol 2005; 349: 787-800.

[42]. Hooper NM. Families of zinc metalloproteases. FEBS Lett 1994; 354: 1-6. [43]. Porollo A, Meller J. Prediction-based fingerprints of protein-protein

interactions. Proteins 2007; 66: 630-45. [44]. Vladimir S, Eran E, Sergey G, Vladimir P, Mariana B et al. SPACE: a suite of

tools for protein structure prediction and analysis based on complementarity and environment. Nucleic Acids Res 2005; 33: W39-43.

[45]. Atsumi S, Mizuno E, Hara H, Nakanishi K, Kitami M et al. Location of the

Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin. Appl Environ Microbiol 2005; 71: 3966-77.

[46]. Kitami M, Kadotani T, Nakanishi K, Atsumi S, Higurashi S et al. Bacillus

thuringiensis Cry toxins bound specifically to various proteins via Domain III, which had a galactose-binding domain like fold. Biosci Biotechnol Biochem 2011; 75: 305-12.

[47]. Kaur R, Sharma A, Chauhan VS, Bhatnagar RK. Mutagenesis of domain II

and domain III loop residues of Bacillus thuringiensis Cry1C delta endotoxin reveal their role in binding of aminopeptidase N of Spodoptera litura. Process Biochem 2013; (Accompanied Manuscript).

[48]. Pacheco S, Gómez I, Arenas I, Saab-Rincon G, Rodríguez-Almazán C, et al.

Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a "ping pong" binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors. J Biol Chem 2009; 284: 32750-7.

[49]. Jenkins JL, Dean DH. Enzyme stabilization by directed evolution. In: Setlow

JK editor. Genetic Engineering: Principles and Methods. New York: Plenum Press 2000; Vol. 22. p. 55-76.

FOOTNOTES The abbreviations used are: Bt, Bacillus thuringiensis; SlAPN, recombinantly expressed APN of Spodoptera litura; BBMV, brush border membrane vesicle

Page 20 of 30

Accep

ted

Man

uscr

ipt

20

FIGURE LEGENDS Figure 1. (a) Homology based model for SlAPN. Schematic ribbon presentation of the hook shaped structure of SlAPN, illustrating the four-domain organization of the receptor molecule. (b) Homology-based model for Cry1C. Schematic ribbon presentation of 65-kDa Cry1C toxin illustrating the three-domain organization of the protein molecule. Loops analyzed in the study are labeled. (c). Docked complex M1.8. (a) Ribbon representation. The two regions of contact (A and B) of the interacting toxin (blue) and receptor (green) molecules are shown. The interacting residues are: region A= residues E74, I76, I78, V79, V96-V101, R113, P115, D116, Y123-T143 of SlAPN and S220, T221, Q223, N273-S280, L284, P285, L369-P375, R437-F442 of Cry1C, region B= residues R198, A217-S220, T231, Y233, P234, P262, D299, M306 of SlAPN and D73, V77, E80, Q81, N84-F90, R92 of Cry1C. The amino acid residues in the binding epitopes are in sphere representation. Figure 2. Phage display assay with Cry1C as ligand. (a). Assaying the enrichment of clones for binding with Cry1C. Equal number of phages after each round of selection was assayed for toxin binding by ELISA. Tween20 concentrations used in the ELISA washing steps were same as that used during individual biopanning step. The titer output and the absorbance value of each panning round are shown. (b). Assaying the binding affinities of phages selected after third round of panning (φ-71=phage clone 71% homologous to 128HLHFHLP134 sequence of SlAPN, φ-1 to φ-3= other three phage clones obtained after third round of panning). Equal numbers of individual peptide displaying phages were allowed to bind to 2.5 μg Cry1C and relative affinities determined by standard ELISA protocol. Figure 3. Synthetic peptides homologous to φ-71 and BR- APN compete with Cry1C binding to SlAPN. Toxin overlay assays of Cry1C to SlAPN. Purified SlAPN (1 μg) was loaded on SDS- polyacrylamide gel and transferred to NC membrane which was probed for toxin binding. Pure active Cry1C toxin (2.5 μg /ml) was pre incubated with increasing concentrations of peptides for 1 h at RT and then overlaid on the receptor containing NC membrane. Binding was visualized by standard NBT-BCIP assay using anti-Cry1C antibodies. Lane1, binding of Cry1C to SlAPN; lanes 2-4, competition of Cry1C binding with a 125-, 250- and 500- fold molar excess of peptide φ-71 respectively; lanes 5-7, competition of Cry1C binding with a 125-, 250- and 500- fold molar excess of peptide APN-CRY respectively. Figure 4. Identification of Cry1C epitope for BR-APN by Competition Experiments. (a). Synthetic φ-71 peptide compete Cry1C binding to BR-APN. Lane1, binding of Cry1C to BR-APN; lanes 2-4 binding of Cry1C to BR-APN with a 75-, 125- and 250- fold molar excess of φ-71 peptide. (b). Synthetic peptides homologous to DII loops 2 & 3 compete with BR-APN binding to Cry1C while that of loop α does not. Purified BR-APN (1 μg) was loaded on SDS- polyacrylamide gel and transferred to NC membrane which was probed for toxin binding. Pure active Cry1C toxin (2.5 μg /ml) was pre incubated with increasing concentrations of peptides for 1 h at RT and then overlaid on the receptor containing NC membrane. Lane1, binding of Cry1C to BR-APN; lanes 2-4, binding of Cry1C to BR-APN with a 125-, 250- and 500- fold molar excess of loop 2 peptide; lanes 5-7, binding of Cry1C to BR-APN with a 125-, 250- and 500- fold molar excess of loop 3 peptide; lanes 8-

Page 21 of 30

Accep

ted

Man

uscr

ipt

21

10, binding of Cry1C to BR-APN with a 125-, 250- and 500- fold molar excess of loop α peptide. Figure 5. Effect of BR-APN mutations on toxin binding. Binding of biotin-labeled BR-APN receptor protein in the presence of increasing concentrations of unlabeled BR-APN, H128A, L129A, H130A, F131A, H132A, L133A, and P134A proteins to Cry1C. (a) Purified Cry1C toxin was electro blotted on NC membrane and overlaid with saturating concentration of purified biotin-labeled BR-APN in the presence of increasing concentrations (lane 1-no competitor, lane 2-0.1X, lane 3-1X, lane 4-10X and lane 5-100X fold molar excess of competitor protein) of non labeled native type receptor or mutant proteins as the competitors. Blots were developed using HRPO-conjugated streptavidin. (b) Binding is expressed as a percentage of the amount bound upon incubation with labeled receptor protein alone. Average integrated density values (IDVs) (obtained from the above ligand blots) were taken as bound toxin values and plotted against competitor concentration using graph-pad software package.

Page 22 of 30

Accep

ted

Man

uscr

ipt

22

TABLES Table 1. Summary of primers used in the study

Name Abbreviation Primers Base pairs

Amino acids

Mass (kDa)

Binding region of SlAPN

BR-APN (primer 1) TGT GTA CCC TAC TGA TGT (primer 2) CGA CGC GGC CGC ACC TCT GTA GAA GCC

336 51-162 12

Table 2 Summary of BR-APN receptor mutants generated by site-directed mutagenesis. Residue Mutated Sequence region of mutation

H128A 126LLHLHFHLPIV136 126--A--------136

L129A 126LLHLHFHLPIV136 126---A-------136

H130A 126LLHLHFHLPIV136 126----A------136

F131A 126LLHLHFHLPIV136 126-----A-----136

H132A 126LLHLHFHLPIV136 126------A----136

L133A 126LLHLHFHLPIV136 126-------A---136

P134A 126LLHLHFHLPIV136 126--------A--136

Residues substituted by alanine are underlined. Table 3. Synthetic Peptide sequences used in the study

Name Description Sequence

φ-71 Amino acid sequence of Cry1C binding phage clone HPSFHWK

APN-CRY Amino acid sequence of residues 128-134 of SlAPN HLHFHLP

Scr Scrambled peptide

Page 23 of 30

Accep

ted

Man

uscr

ipt

23

Table 4. Toxicity of Cry1C to S.litura first instar larvae in the presence or absence of competitors. Treatment Mortality a (%)

Cry1C ( 80ng/cm2) 100

Cry1C+BR-APN (100 X)b 45

Cry1C+φ-71(1012 plaque forming units) 50

Cry1C+scr (1012 plaque forming units) 1 a40 larvae per treatment. Representative results of three experiments are given. bMolar excess of competitor.

A total of 200 μl (each side) of toxin was layered on a mulberry leaf disc placed in a petri dish

and allowed to dry. Ten larvae were placed on each petri-dish and mortality was analysed after

two days of incubation. For competition assays, toxin was pre-incubated with excess of

competitor for 1h at room temperature and then fed to the larvae.

Table 5 Binding efficiency of BR-APN and mutants to Cry1C toxin.

Receptor Kd (nM) Bmax

(pmol/mg) BR-APN 9.318 138.7 H128A 24.63 95.09 L129A 9.239 140.4 H130A 39.69 105.4 F131A 14.66 122.8 H132A 25.45 74.4 L133A 10.42 135.7 P134A 25.11 92.33

Dissociation constant (Kd) and concentration of binding sites (Bmax) are estimated from homologous competition experiments performed with biotin-labeled, purified BR-APN receptor or mutant receptor proteins and Cry1C toxin electro-blotted on nitro-cellulose membrane. Each value is the mean of three experiments.

Page 24 of 30

Accep

ted

Man

uscr

ipt

24

Table 6 Effect of BR-APN and its mutants on toxicity of Cry1C protein to Spodoptera litura larvae.

Treatment Mortalitya (%)Cry1C (80 ng/cm2) 100 ± 2.2 Cry1C+BR-APN 45 ± 1.3 Cry1C+H128A 85 ± 3.4 Cry1C+L129A 55 ± 1.8 Cry1C+H130A 75 ± 2.6 Cry1C+F131A 60 ± 4.1 Cry1C+H132A 70 ± 3.1 Cry1C+L133A 50 ± 2.5 Cry1C+P134A 80 ± 3.7

a40 larvae per treatment. ± S.D. of three experiments Toxicity of Cry1C is measured in the presence of 100-fold molar excess of purified BR-APN or mutant receptor proteins as the competitors.

Page 25 of 30

Accep

ted

Man

uscr

ipt

Figure 1

Page 26 of 30

Accep

ted

Man

uscr

ipt

Figure 2

Page 27 of 30

Accep

ted

Man

uscr

ipt

Figure 3

Page 28 of 30

Accep

ted

Man

uscr

ipt

Figure 4

Page 29 of 30

Accep

ted

Man

uscr

ipt

Figure 5

Page 30 of 30

Accep

ted

Man

uscr

ipt

Highlights • In silico docking and biopanning identified a putative binding region of Cry1C 128HLHFHLP134 to APN of Spodoptera litura • Synthetic peptides homologous to domain II loop 2 and 3 of Cry 1C competed Cry1C- APN binding. • Alanines substitution of H128, H130, H132 and P134 affect the toxicity of Cry1C toxin to APN receptor of S. litura.