BBRC

Biochemical and Biophysical Research Communications 330 (2005) 519525 www.elsevier.com/locate/ybbrc

Structural requirements of the unique disulphide bond and the proline-rich motif within the a4a5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa d-endotoxin
Satita Tapaneeyakorn, Walairat Pornwiroon, Gerd Katzenmeier, Chanan Angsuthanasombat *
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand Received 9 February 2005 Available online 18 March 2005

Abstract Both the disulphide bond (Cys192Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the a4a5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry d-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the exibility of the a4a5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations signicantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buer, pH 9.0. These ndings are the rst biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the a4a5 loop of the Cry4Aa toxin. 2005 Elsevier Inc. All rights reserved.
Keywords: Bacillus thuringiensis; Disulphide bond; d-Endotoxin; Larvicidal activity; Mutagenesis; Proline-rich motif

The highly specic insecticidal proteins produced by the Gram-positive bacterium Bacillus thuringiensis (Bt) as dierent forms of cytoplasmic crystalline inclusions during sporulation are classied into two main families of crystal (Cry) and cytolytic (Cyt) d-endotoxins, based on their deduced amino acid sequence similarity [1,2]. To date, the Bt Cry d-endotoxins have been shown to be toxic towards a wide variety of insect larvae in the orders Diptera (mosquitoes and ies), Lepidoptera (moths

Corresponding author. Fax: +662 441 9906. E-mail address: stcas@mahidol.ac.th (C. Angsuthanasombat).

and ies), Coleoptera (beetles and weevils), and Hymenoptera (wasps and bees) [1,3]. Of particular interest among biological insecticides, Bt subsp. israelensis has been widely used as an ecient and safe bacterial insecticide for the control of disease-carrying mosquitoes [4]. This bacterium produces at least four major mosquitolarvicidal proteins including the two closely related toxins of ca. 130 kDa (Cry4Aa and Cry4Ba), the 65-kDa Cry11Aa toxin, and the 27-kDa Cyt1Aa toxin [2]. Upon ingestion by susceptible larvae, the Bt toxin inclusions are solubilised in the midgut lumen that is highly alkaline in a number of dipteran and lepidopteran larvae [1,3]. The released soluble protoxins are

0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.006

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subsequently processed by larval midgut proteases to liberate the active toxins. A conformational change triggers the insertion of their pore-forming portion into the target cell membrane to form ion-leakage pores that cause a net inux of ions and water which leads to osmotic cell lysis [5], resulting in larval paralysis and eventual death [6]. Thus far, the underlying molecular basis of this toxicity process, especially the steps of membrane insertion and lytic pore-formation, is still not completely elucidated. Structural data obtained by X-ray crystallography of ve dierent Bt Cry d-endotoxins, Cry1Aa [7], Cry2Aa [8], Cry3Aa [9], Cry3Bb [10], and Cry4Ba [11], reveal a high degree of overall structural similarity, comprising three distinct domains. A homology-based 3D model of the 65-kDa activated Cry4Aa mosquito-larvicidal protein also suggests a similar three-domain organisation structure [12]. Particularly, the N-terminal sevenhelix domain in which the relatively hydrophobic helix-a5 is encircled by six other amphipathic helices has been shown to be responsible for toxin insertion into the membrane, leading to formation of the ion-leakage pore [13,14]. Currently, substantial evidence supports an umbrella-like model that has been proposed to describe the membrane-bound state of the Cry toxins. This model involves an insertion of a4 and a5 into the membrane as a helical hairpin structure, with the remaining helices spread over the membrane surface [15]. A rened model suggests that the pore-forming domain swings away from the two other domains, followed by the insertion of the a4a5 hairpin into the lipid membranes with

subsequent oligomerisation of the transmembrane hairpins to form a tetrameric ion channel with a diameter of 1020 A [16]. A number of reports have suggested that a4 is aligned to face the pore lumen and participates in ion conduction [17,18], whilst a5 interacts with the lipid membranes and is involved in toxin oligomerisation [19]. Moreover, membrane permeation studies demonstrated that the a4loopa5 segment is more active than the isolated a4 and a5 helices or their mixture, indicating that the presence of the connecting loop is a structural requirement for an ecient insertion of the toxin into the membranes [20]. In our previous studies, an aromatic structure of the highly conserved tyrosine residue (Fig. 1) within the a4 a5 loop of the 130-kDa mosquito-larvicidal proteins, Cry4Aa and Cry4Ba, was shown to play a critical role in toxin activity, conceivably being involved in an interaction with lipid head groups stabilising the oligomeric pore structure [21,22]. In addition, a homology-based Cry4Aa model revealed a disulphide bond formed between Cys192 and Cys199, and a proline-rich region (Pro193ProAsnPro196) within the loop connecting a4 and a5 [12]. These characteristics are exclusively observed in the a4a5 loop of the Cry4Aa toxin (see Fig. 1). We therefore employed a series of rened site-directed mutagenesis studies aimed at determining whether this unique disulphide bridge and the prolinerich motif in the Cry4Aa a4a5 loop are an essential prerequisite for toxin action. For the rst time, our results provide biologically relevant evidence for a structural requirement of both the disulphide bridge

Fig. 1. (A) Ribbon representations of the loop linking a4 and a5 of a homology-based Cry4Aa model and those of the known structures, Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, and Cry4Ba. The highly conserved aromatic residue is shown in ball-and-stick in all six interhelical loops. Three critical proline residues (P193, P194, and P196) and the unique disulphide bond between C192 and C199 are illustrated as ball-and-stick in the Cry4Aa a4a5 loop (B), multiple sequence alignments of the transmembrane a4-loop-a5 fragment of Cry4Aa with those of the crystal structures of Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, and Cry4Ba. Mutated residues are indicated by *. The sequences were aligned using the program Clustal X. The corresponding a4-loop-a5 is shown above the sequences.

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(C192C199) and the proline-rich motif (P193PNP196) within the a4a5 loop for larvicidal activity of the Bt Cry4Aa toxin. Materials and methods
Construction of the Cry4Aa loop-mutant plasmids. The recombinant plasmid pMEx-B4A encoding the 130-kDa Bt Cry4Aa toxin under control of the tac promoter together with the cry4Ba promoter [23] was used as a template for the introduction of single mutations. One single mutant plasmid, encoding C199A, was employed further as a template for the generation of double mutations. Subsequently, the pC199A/ P194A mutant plasmid was used for generating two triple mutations, C199A/P194A/P196A and C199A/P194A/P196G. The resultant mutant plasmids, pC199A/P194A/P196A and pC199A/P194A/P196G, were then used as templates for conducting C199A/P194G/P196A and C199A/P194G/P196G mutations, respectively. Eleven pairs of complementary mutagenic oligonucleotide primers (Table 1) designed according to the published cry4Aa gene sequence [24] were purchased from Proligo (Singapore). All mutant plasmids were generated by polymerase chain reaction (PCR) using high-delity Pfu DNA polymerase, following the procedure of the QuickChange mutagenesis kit (Stratagene). All mutant clones were rst identied by restriction endonuclease digestion of the plasmids and then veried by DNA sequencing using a BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer). Expression and purication of toxin inclusions. Escherichia coli cells strain JM109 harbouring the wild-type or mutant plasmids grown at 30 C in LuriaBertani medium containing 100 lg/ml ampicillin until

OD600 of the culture reached 0.30.5. After addition of isopropanyl-b(IPTG) to a nal concentration of 0.1 mM, incubation was continued for another 10 h. Samples containing 107 cells/ml were analysed for toxin expression by using sodium dodecyl sulphate(12% w/v) polyacrylamide gel electrophoresis (SDS PAGE). Cells expressing the toxins as inclusion bodies were harvested by centrifugation and resuspended in cold distilled water. The cell suspension was disrupted by using a French Press Cell at 10,000 psi. The crude lysate was centrifuged at 8000g, 4 C for 15 min. The pellets were washed three times in cold distilled water and resuspended by sonication. Protein concentrations of the partially puried inclusions were determined by using the Bradford-based protein microassay (BioRad), with bovine serum albumin fraction V (Sigma) as a standard. Inclusion solubilisation and proteolytic digestion. Protoxin inclusions (1 mg/ml) were solubilised in 50 mM Na2CO3, pH 9.0, and incubated at 37 C for 1 h as previously described [25]. After centrifugation at 8000. for 10 min, the supernatants and inclusion suspension were analysed by SDSPAGE. Solubilised protoxins (0.7 mg/ml) were assessed for their proteolytic stability by digestion with tolysulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin at an enzyme/toxin ratio of 1/20 (w/w) in 50 mM Na2CO3, pH 9.0, for 16 h. Mosquito-larvicidal activity assays. Bioassays were performed as previously described [26], using 2-day-old Aedes aegypti mosquito larvae (hatched from eggs supplied by the mosquito-rearing facility of the Institute of Molecular Biology and Genetics, Mahidol University, Thailand). Both rearing the larvae and bioassays were done at room temperature (25 C). The assays were carried out in 1 ml of E. coli suspension (108 cells suspended in distilled water) in a 48-well microtitre plate (11.3-mm well diameter, Costar, USA), with 10 larvae per well and a total of 100 larvae for each cloned sample. E. coli cells
D-thiogalactopyranoside

Table 1 Complementary primers for the generation of Cry4Aa mutant toxins Primer C192A-f C192A-r C199A-f C199A-r P193A-f P193A-r C199A/P193A-f C199A/P193A-r C199A/P194A-f C199A/P194A-r C199A/P196A-f C199A/P196A-r C199A/P193G-f C199A/P193G-r C199A/P194A/P196A-f C199A/P194A/P196A-r C199A/P194G/P196A-f C199A/P194G/P196A-r C199A/P194A/P196G-f C199A/P194A/P196G-r C199A/P194G/P196G-f C199A/P194G/P196G-r Sequencea 5 -CCAGAGCTTGTGAATTCTGCTCCTCCTAATCC-3 5 0 -GGATTAGGAGGAGCAGAATTCACAAGCTCTGG-3 0 5 0 -CCTCCTAACCCGAGTGATGCTGATTACTATAAC-3 0 5 0 -GTTATAGTAATCAGCATCACTCGGGTTAGGAGG-3 0 5 0 -AAACTCTTGTGCGCCTAATCCTAG-3 0 5 0 -GGATTAGGCGCACAAGAGTTTAC-3 0 5 0 -CTCTTGTGCGCCTAACCCG-3 0 5 0 -GTTAGGCGCACAAGAGTTTAC-3 0 5 0 -CTCTTGTCCGGCTAACCCGAGTG-3 0 5 0 -GGTTAGCCGGACAAGAGTTTAC-3 0 5 0 -TCCTAACGCCTCAGATGCTGATTAC-3 0 5 0 -CAGCATCTGAGGCGTTAGGAGGAC-3 0 5-TAAACTCTTGTGGCCCTAACCCG-3 0 5 0 -GGGTTAGGGCCACAAGAGTTTA-3 0 5 0 -GCTAACGCGAGTGATGC-3 0 5 0 -CACTCGCGTTAGCCGG-3 0 5 0 -CTCTTGTCCTGGTAACGCGAGT-3 0 5 0 -CTCGCGTTACCAGGACAAGAGTT-3 0 5 0 -CGGCTAACGGGAGTGATGC-3 0 5 0 -GCATCACTCCCGTTAGCCG-3 0 5 0 -CTCTTGTCCTGGTAACGGGAGT-3 0 5 0 -CTCCCGTTACCAGGACAAGAGTT- 3 0 HpaII HpaII
0 0

Restriction site EcoRI AvaI HhaI HhaI HpaII DdeI HaeIII

a Recognition sites introduced for restriction enzyme analysis are underlined. Bold letters indicate mutated nucleotide residues; f and r represent forward and reversed primers, respectively.

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containing the pMEx8 vector were used as a negative control. Mortality was recorded after 24-h incubation period. Students t test [27] was performed to determine signicance levels between mutants and the wild-type.

Results and discussion As was previously suggested by Gerber and Shai [20], the loop connecting a4 and a5 of the Bt Cry d-endotoxins is needed for ecient penetration of these two transmembrane helices into the lipid membranes to form lytic pores and subsequently cause toxicity. Recently, we have strengthened this notion by demonstrating that one highly conserved tyrosine residue in this critical loop of both the two closely related dipteran-specic toxins (Cry4Aa: Tyr202; Cry4Ba: Tyr170; see Fig. 1) is an important determinant for larvicidal activity [21,22]. As shown Fig. 1, the a4a5 loop of the homology-based Cry4Aa model is substantially longer than that of the Cry crystal structures. Of particular interest, this extraordinary long loop possesses a single disulphide bond at Cys192Cys199 as well as an irregular proline-rich region (Pro193ProAsnPro196). Even though the tertiary structure of Cry4Aa is not yet completely solved and only preliminary crystallographic data are available [28], we have earlier veried the presence of this putative disulphide bond in the 20-kDa a1a5 fragment of the trypsin-treated Cry4Aa product by assaying the electrophoretic mobility of this disulphide bond-containing fragment under reducing and non-reducing conditions on SDSPAGE [12]. In the current study, attempts were therefore made to determine whether this unique disulphide bridge and the proline-rich motif in the a4a5 loop are structurally relevant for biological activity of the Cry4Aa toxin. In the beginning, we have eliminated the disulphide bridge by replacing either Cys192 or Cys199 with alanine,

using the technique of PCR-based directed mutagenesis. The resultant Cry4Aa mutant toxins were highly produced in E. coli as inclusion bodies upon IPTG induction. When equal amounts of cell lysates were analysed using SDSPAGE, both C192A and C199A mutants yielded a 130-kDa protoxin band at levels comparable to that of the wild-type toxin (see Fig. 2). This indicated that these loop mutations most likely did not have an adverse eect on protein folding, as protein misfolding can lead to low levels of protein expression caused by proteolytic degradation [29]. When E. coli cells expressing each mutant toxin were tested for their relative toxicity against A. aegypti larvae, both C192A and C199A mutants apparently exhibited the same level of larvicidal activity as the wild-type (see Fig. 3), implying no eect on toxicity due to the elimination of this disulphide bridge. However, the possibility cannot be excluded that the disulphide bond within the a4a5 loop of Cry4Aa is a requirement for the structural integrity, as shown by numerous studies demonstrating stabilisation of protein structure by this linkage [30]. Further analysis of the a4a5 loop by substitutions at Pro193, Pro194 or Pro196 in combination with C199A mutation revealed that only E. coli cells expressing the C199A/P193A mutant exhibited an almost complete loss in toxicity against mosquito larvae, whereas cells expressing C199A/P194A or C199A/P196A mutants showed an approximately 2030% decrease in larvicidal activity (see Fig. 3). In addition, the C199A/P194A/ P196A triple mutation revealed a more adverse eect on toxicity, but only by a half reduction of the activities of its double mutants, C199A/P194A and C199A/ P196A. These results suggested that this proline-rich motif is involved in toxin activity and particularly Pro193 contributes signicantly to the larvicidal activity when compared to those of the two other proline residues. The P193A single substitution was also performed

Fig. 2. SDSPAGE (Coomassie brilliant blue-stained 12% gel) analysis of lysates extracted from E. coli (107 cells) expressing 130-kDa protoxins of Cry4Aa wild-type (wt) or its mutants (C192A, C199A, P193A, C199A/P193A, C199A/P194A, C199A/P196A, C199A/P193G, C199A/P194A/ P196A, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G). E. coli cells harbouring the pMEx8 vector were used as a negative control (nc). M represents the molecular mass standards.

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Fig. 3. Larvicidal activities of E. coli cells expressing the Cry4Aa wild-type or its mutant toxins (C192A, C199A, P193A, C199A/P193A, C199A/ P194A, C199A/P196A, C199A/P193G, C199A/P194A/P196A, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G) against A. aegypti larvae at concentrations of 108 cells/ml. Error bars indicate standard errors of the mean from three independent experiments. Shaded boxes represent the larvicidal activity of the mutants that are signicantly dierent (p values < 0.001) from that of the wild-type.

to assure that the deleterious eect of the C199A/P193A double mutation resulted from both the disulphide bond reduction and the alanine substitution at Pro193. As shown in Fig. 3, the P193A mutant exhibited a signicant reduction in larvicidal activity of approximately 30% when compared to that of the wild-type, indicating that both the disulphide bridge and Pro193 play an essential role in Cry4Aa toxin activity, conceivably by maintaining the loop integrity. An increase of the loop exibility via proline-to-glycine mutations of this proline-rich motif was also investigated. The C199A/P193G double mutant and the triple mutants (C199A/P194G/P196A and C199A/P194A/ P196G) revealed a signicantly higher loss in larvicidal activity when compared to those of the C199A/P193A mutant and the C199A/P194A/P196A mutant, respectively (see Fig. 3). This indicated that all the three proline residues (Pro193, Pro194, and Pro196) play a role in contributing to the structural rigidity of the Cry4Aa a4a5 loop. Nevertheless, reduced toxicity that is observed for the C199A/P194G/P196G mutation was apparently the same as that observed for C199A/ P194G/P196A and C199A/P194A/P196G mutations (see Fig. 3). This might imply that both Pro194 and Pro196 did not contribute to the same extent as Pro193 for the loop-structure rigidity. It should be noted that all the 130-kDa mutant protoxins were expressed in E. coli cells with yields similar to that of the wild-type toxin as demonstrated by SDSPAGE (see Fig. 2). The larvae tested in the toxicity assays were therefore believed to receive a similar amount of protoxin doses. Solubility of the mutant inclusions was assessed in comparison to that of the wild-type protein using carbonate buer, pH 9.0. The amounts of the 130-kDa soluble proteins in the supernatant were compared with those of the proteins initially used in order to determine

the percentage of protein solubilisation. All of the mutant inclusions were found to be soluble in this buer, giving approximately 70% solubility, which closely resembles the solubility of wild-type inclusions under similar conditions (data not shown). When the 130kDa solubilised mutant protoxins were subsequently examined for their proteolytic stability by digestion with trypsin, all of them were found to produce trypsin-resistant fragments of ca. 47 and ca. 18 kDa, similar to those obtained with the wild-type protoxin (see Fig. 4), indicating that all the loop mutant toxins were relatively as stable as the wild-type Cry4Aa toxin. This also suggested that the mutations in the a4a5 loop residues of each mutant toxin had no apparent eect on protein folding or proteolytic processing. Therefore, the detrimental eects on larvicidal activity seen for each mutant

Fig. 4. SDSPAGE (Coomassie brilliant blue-stained 12% gel) analysis of trypsin-treated products of 130-kDa protoxins solubilised from toxin inclusions of E. coli cells expressing the Cry4Aa wild-type (lane 1) or its mutant toxins-C192A, C199A, P193A, C199A/P193A, C199A/P194A, C199A/P196A, C199A/P193G, C199A/P194A/ P196A, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/ P194G/P196G (lanes 212, respectively). U represents the 130-kDa trypsin-untreated products.

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Fig. 5. (A) Space-lling representation of a homology-based Cry4Aa model, showing the three-domain organisations (IIII). The loop linking helices 4 and 5 is also shown in the stick model. (B) Close-up view of interaction surfaces between the pyrrolidine ring of Pro193 and the aromatic phenol ring of Tyr202 within the a4a5 loop.

tion [33,34]. Hence, a disruption of this putative crossstrand interaction between Pro193 and Tyr202 by either P193A or C199A/P193A mutation could impair the structural integrity of the a4a5 loop, and thus cause a decrease in Cry4Aa toxicity. In conclusion, we have provided evidence that both the unique disulphide bridge (C192C199) and the proline-rich motif (P193PNP196), especially Pro193, in the a4a5 loop are essentially involved in Cry4Aa-larvicidal activity. Conceivably, the structural rigidity of this loop is needed for an ecient insertion of the a4a5 transmembrane hairpin into the lipid membranes to form a lytic pore. However, additional experiments are needed to verify denitively among the possible eects of these loop mutations, particularly on the steps of membrane insertion and lytic pore-formation.

are not likely a result from incomplete solubilisation or proteolytic processing. Although a role of the proline-rich motif and the single disulphide bond within the a4a5 loop in Cry4Aa toxicity could be rst have demonstrated here, it is still not clear how these critical residues, in particular Pro193, contribute to toxin activity. Previous studies demonstrated that replacements with Ala or Leu for the proline hinge in buforin II, a 21-amino acid histone H2A-derived antimicrobial peptide, revealed a signicant decrease in antimicrobial activity, resulting from the reduction of cell-penetrating eciency [31]. Also, a drastic decrease in toxicity caused by mutations at one critical proline residue (Pro345) located at the end of the nine helices (TH8) composing the diphtheria toxin translocation domain was shown to be caused by reduced translocation ability [32]. Notwithstanding the lack of membrane insertion and pore formation studies, both the proline-rich motif and the disulphide bond which are conceivably required for structural integrity of the a4a5 loop may indeed play an important role in the membrane insertion step. As was suggested earlier, Pro193 has a greater inuence on the conformational rigidity of the a4a5 loop than the two other proline residues (Pro194 and Pro196). The critical positioning of Pro193 and its neighbouring region were therefore analysed as illustrated in Fig. 5. Interestingly, the pyrrolidine ring of Pro193 that positions within the loop has a cross-strand interaction with the aromatic phenol ring of Tyr202 that could likely provide signicant stability to the a4a5 helical hairpin. The aromatic structure of this critical tyrosine residue was proposed to act as a determinant of protein orientation or confer structural rigidity to the periphery of the transmembrane segments [21]. In addition, cross-strand interactions via hydrophobic and/or aromatic interactions were shown to strongly contribute to the stability of b-hairpin forma-

Acknowledgments We are grateful to Professor Sakol Panyim, Professor Prapon Wilairat, and Dr. Panadda Boonserm for critical comments. We also thank Ms. Somsri Sakdee and Ms. Chaweewan Shimwai for technical assistance. This work was supported in part by Grant No. BRG4780004 from the Thailand Research Fund. An M.Sc. scholarship of the Development and Promotion of Science and Technology Talents Project to S.T. is gratefully acknowledged.

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