Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.7 No.2, Issue of August 15, 2004 © 2004 by Pontificia Universidad Católica de Valparaíso -- Chile Received November 6, 2003 / Accepted March 29, 2004 REVIEW ARTICLE Protein engineering of δ-endotoxins of Bacillus thuringiensis Nachimuthu Saraswathy National Research Centre for Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012 India E-mail: [email protected] Polumetla Ananda Kumar* National Research Centre for Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012 India Tel: 91 11 25841787 Fax: 91 11 25766420 E-mail: [email protected] Keywords: brush border membrane vesicles, cry proteins/δ-endotoxin, ion channel conductance, planar lipid bilayers, protein engineering. Abbreviations: BBMV: Brush Border Membrane Vesicles; Bt: Bacillus thuringiensis; PLB: Planar Lipid Bilayers. Bacillus thuringiensis (Bt) is a valuable environment- proteins of Bacillus thuringiensis (Bt) assume considerable friendly biopesticide, which occupies 90% of the world significance in the production of insect resistant crops such biopesticide market. Its insecticidal properties are as Maize, Cotton, Potato, Rice, etc (Kumar, 2002). Bt is a attributed to the presence of δ-endotoxins which are Gram positive spore forming aerobic bacterium isolated synthesized during the sporulation phase of the from a wide variety of environments like soil, insect bacterium. δ-endotoxin or crystal toxin is a multi- cadavers, stored grain products and phylloplane (Martin domain protein molecule comprising of three distinct and Travers, 1989). The importance of Bt is that it domains. Domain I is made of seven α-helices, domain accumulates certain proteins in crystalline form during II comprises three antiparallel β sheets, which are sporulation phase (Kumar and Bambawale, 2002). These folded into loops and domain III is made of a β proteins are known to be toxic towards larvae of different sandwich of two antiparallel β strands. Molecular orders of insect pests (Lepidoptera, Diptera, Coleoptera, studies on the structure and functional properties of Hymenoptera, and Homoptera) with different efficacies (Schnepf et al. 1998). The toxicity of a Bt toxin is highly different δ-endotoxins revealed that the domain I by specific and it is non-toxic to mammals and beneficial virtue of its membrane spanning hydrophobic and insects. amphipathic α-helices is capable of forming pores in the cell membranes of the larval midgut. Domain II being hyper variable in nature determines the insecticidal More than 150 different Cry toxins have been cloned and specificity of a toxin and domain III is involved in tested for their toxicity on various insect species till date. In varied functions like structural stability, ion channel an attempt to accommodate the growing list of new toxin gating, binding to Brush Border Membrane Vesicles genes/proteins, a new nomenclature has been formulated, and insecticidal specificity. Recent studies on toxin wherein each toxin gene/protein will be having four-letter aggregation and interaction revealed that the three code, according to their amino acid sequence identity domains interact closely to bring about the insecticidal among them (Crickmore et al. 1998). Updated list of the Bt activity of Bt. In this review we describe the protein toxin genes can be accessed at engineering studies conducted on different δ-endotoxins http://www.biols.susx.ac.uk/Home/Neil_Crickmore/BT/. Bt which led to an understanding of their molecular mode and its δ-endotoxins have been extensively studied for their of action and construction of novel toxins with enhanced molecular mechanism of action and toxin structure-function insecticidal activity and specificity. relationships. In this review we focus on how protein engineering of different Domains of δ-endotoxins helped in In the present era of transgenic technology, insecticidal understanding the structure-function relationships and. *Corresponding author This paper is available on line at http://www.ejbiotechnology.info/content/vol7/issue2/full/3 Saraswathy, N. and Kumar, P. A. Figure 2. Flow chart showing engineering of novel insecticidal proteins of Bacillus thuringiensis. Structure-function analysis of δ−endotoxins In addition to traditional bioassays on larvae of insect Figure 1. Structure of Bt δ-endotoxin Cry3Aa depicting species, advancements in molecular biology, protein the three domains (Based on Li et al. 1991) chemistry and biophysics paved the way for a thorough understanding of structure-function relationships of δ- Structure of Bt δ−endotoxins endotoxins. Protein engineering studies are based on designing of novel toxins with wider host spectrum and methods for introducing mutations in the genes that encode high potency in a rational and directed way Bt δ-endotoxins proteins of interest and for producing the proteins in large are globular protein molecules, which accumulate as amount in bacteria for further analysis. In the last decade protoxins in crystalline form during late stage of the numerous modifications were created in δ-endotoxins using sporulation. Protoxins are liberated in the midgut after various protein engineering techniques such as (a) single or solubilization and is cleaved off at C-terminal part to multiple amino acid change in variable and conserved release ~66 kDa active N-terminal toxic molecule. The regions through site directed mutageneis (b) restriction protoxin contains well-conserved cysteine residues (as fragment exchange between closely related cry toxin genes many as 16 in Cry1Ac), which helps in bridging the or with other bacterial toxin genes and (c) exchange of protoxin molecules through intermolecular disulphide domains between toxin genes through PCR mediated bonds and thereby crystal formation. Currently, 3- cloning or in vivo recombination in recA+ (recombinant dimensional protein structures have been determined for proficient) Eschericha coli Strains. The effects of mutations three Bt toxins through X-ray crystallography. Among them or modifications in δ-endotoxins have been studied on two are crystals forming (Cry) proteins or δ-endotoxins viz. various aspects like: Cry1Aa (Lepidoptera-specific; Grochulski et al. 1995) and Cry3A (Coleoptera-specific; Li et al. 1991). Since primary 1) Pore forming capacity in artificial membranes and amino acid composition determines the final structure of a measuring ion channel conductance using voltage patch protein, closely related proteins, Cry1Aa and Cry3A, with clamping, light scattering and light quenching techniques. 36% amino acid sequence identity showed superimposable structure with similar mode of action, whereas Cyt2A 2) Binding of toxins to Brush Border Membrane vesicles protein, which shares less than 20% amino acid sequence (BBMV) isolated from susceptible larval midguts using identity, is made of single domain with different functional labeled toxins. properties (Schnepf et al. 1998). The tertiary structure of δ- 3) Stability of proteins using proteases like trypsin and endotoxins is comprised of three distinct functional larval midgut juice. domains connected by a short conserved sequence. Each domain of δ-endotoxin has independent and inter-related 4) In vivo toxicity analysis on larvae. functions in the larval midgut, which brings out colloid osmotic lysis (Knowles, 1994). The nature of each domain 5) In vitro toxicity analysis in insect cell lines – lawn assay. predicted from X-ray crystallography is given in Figure 1 and Table 1 (Li et al. 1991; Grochulski et al. 1995). 6) Prediction of secondary structural changes using CD Phylogenetic analysis on the domains of δ-endotoxins spectrometry. revealed that domain I is the most conserved and domain II is hyper variable among all δ-endotoxins. (Bravo, 1997). Role of Domain I in ion channel formation 181 Saraswathy, N. and Kumar, P. A. N-terminal part of the toxic fragment comprising of six formation (Aronson et al. 1995). Synthetic peptide of amphipathic helices (a- 1, 2, 3, 4, 6, 7) with a central Cry1Aa-α-5 helix showed its ability to form ion channels hydrophobic helix (a -5) makes the domain I of δ- and toxin aggregation in PLB (Gazit and Shai, 1993). endotoxins (Li et al. 1991; Grochulski et al. 1995). Mutations in a5 helix and α4-α5 helix loops resulted in loss Although domain I of δ-endotoxins shares little sequence of toxin aggregation and loss of toxicity. Recently it has homology with other bacterial toxins like Colicin A, been found that oligomerization of toxin is important step Diphtheria B subunit and Pseudomonas exotoxin, it works and correlated with activity of δ–endotoxin (Aronson et al. similar to other bacterial toxins by forming pores in the cell 1999). It is consistent with possible oligomerisation and co- membranes (Parker and Pattus, 1993). pH dependent ion operative gating of toxin in ion channel formation. channel formation is a common feature of bacterial toxins such as colicin, botulinum toxin and diphtheria toxin (Slatin Mutations in the helix region along with synthetic peptide et al. 1990). pH dependent conformational change in δ- mimicking studies gave clear evidence for the ion channel endotoxins provided evidence for assuming a similar mode formation by domain I. Studies on orientation of the of action for Bt toxins (Convents et al. 1990). Recently it membrane-bound state of the seven a helices comprising of has been observed that pore forming capacity of the δ- the pore forming domain of Cry3A δ-endotoxin showed endotoxin is affected