thuringiensis 6endotoxin affect inhibition of short circuit

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1 Proc. Natl. Acad. Sci. USA Vol. 90, pp , October 1993 Biochemistry Site-directed mutations in a highly conserved region of Bacillus thuringiensis 6endotoxin affect inhibition of short circuit current across Bombyx mori midguts (insecticidal crystal protein/voltage clamping/receptor binding/ion channel function) XUE JUN CHEN, Mi K. LEE, AND DONALD H. DEAN* Department of Biochemistry, The Ohio State University, Columbus, OH Communicated by Leo A. Paquette, June 28, 1993 (received for review January 14, 1993) ABSTRACT BaciUus thuringiensis 6-endotoxins (Cry toxins) are insecticidal proteins of =65 kda in the proteolytically processed and active form. The structure ofone of these toxins, CrylIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, ] and contains three domains. It is believed that other S-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues ), to selected other residues. This sequence corresponds to the.&sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel. The Bacillus thuringiensis 8-endotoxins (cry gene products) are a diverse family of insecticidal proteins that are deposited as crystals in the cell during sporulation (1). The activity range of different toxins covers several orders of insects, and now separate phyla are recognized as susceptible (2). The mode of action is still a matter of investigation but, in general, consists of solubilization of the crystal under the alkaline conditions of insect midgut (3), cleavage of the protoxin monomer to the active toxin by insect midgut proteases (4), binding of the toxin to receptors on the midgut brush border (5), and insertion of the toxin into the membrane of midgut epithelial cells and formation of a potassium channel (6). Lysis of the midgut cells and perforation of the midgut lead to paralysis and death of the insect. B. thuringiensis toxin is capable of disrupting the potassium flux created by proton pumps in midgut goblet cells (7). Insecticidal specificity is associated with the specificity of binding of a toxin to midgut receptors (8-10), but the molecular basis of toxicity is the ability to form an ion channel (7). Tertiary structure of one of the toxins (CryIIIA) indicates a multidomain structure (11) that is consistent with biochemical data on other toxins (12-14). For CryIIIA, the first domain consists of seven a-helices that are believed to span the membrane upon insertion of the toxin (11). The second domain is a,b-barrel that functions as the receptor binding region (15). The function of the third domain remains obscure. Most of the 8-endotoxins share five conserved tracts of amino acids (1) that are predominately located at the core of the protein structure and are proposed to play a role in structure determination of the toxin proteins (11). One of these, conserved block 4, QRYRVRIRYAS in CryIAa, the alternating arginine region (corresponding to the p-sheet 17 of CryIIIA), is located in the carboxyl-terminal domain. This sequence is predicted to form a p-sheet with a positively charged face of arginines. For convenience, the first through fourth arginine of this sequence will be referred to as Rl, R2, R3, and R4. The conserved block 4 amino acid sequence of several Cry toxins (1) is shown in Fig. 1. As mentioned above, the role of the third domain is uncertain. To gain further understanding of the mode of action of 8-endotoxins, it is important to investigate the function of the third domain. In this study, we have focused our attention upon the amino acids that comprise the highly conserved block 4 of the third domain. To investigate the importance of the conserved alternating arginine tract in CryIAa, we used site-directed mutagenesis to substitute other amino acids for the arginines. We chose conservative replacements of the arginines with lysines and nonconservative substitution of the arginines with glutamates and also investigated the effect of substituting glycines at R2 and R3 as observed in CryIVB and CryIB. We examined the mutant proteins by protease digestions, CD spectroscopy, competition hybridization to Bombyx mori brush border membrane vesicles, insect bioassay, and voltage clamping of whole B. mori midguts. Our results indicate that some residues in this region are intimately involved in protein structure, while others affect the toxin's function as an ion channel. MATERIALS AND METHODS Construction of Mutants. The construction of pos4102 (CryIAa) and expression in Escherichia coli have been described (16). To target the alternating arginine region, we subcloned the 824-bp Sac I-Kpn I fragment from pos4102 into M13mpl9 to obtain m1316. Site-directed mutagenesis was conducted by the method of Kunkel (17) using the Bio-Rad MutaGene kit. Oligonucleotides were synthesized with an Applied Biosystems model 380 B DNA synthesizer at the Biochemical Instrumentation Center (Department of Biochemistry, The Ohio State University). The list of mutagenic oligonucleotides used to substitute lysines, glutamates, and glycines for arginines is available upon request. After mutagenesis and selection, the 824-bp Sac I-Kpn I fragment was subcloned into the expression vector, pos4102, at the Sac The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact Abbreviations: Ikc, short circuit current; LC5o, median lethal concentration. *To whom reprint requests should be addressed.

2 9042 Biochemistry: Chen et al. CryIAa CryIAb CryIAc CryIB CryIC CryIF CryI I IA CryIVA CryIVB CryIVC P S T S R Y R V RV R Y A S P L T QR Y R I G F R Y A S P I T Q R Y R L R FRY A S P Q R Y R A R I R Y A S S Y S Q KY R A R IEJY A S A F Q Q S Y F I R I R Y A S PT R S Y G L R I R Y A A N V S R Q Y Q V R I R Y A T FIG. 1. Natural variation in conserved block 4 sequence among the Cry toxin sequences (1). I-Kpn I sites. Other molecular genetic techniques were carried out according to Maniatis et al. (18). Purification and Protease Digestion of Protoxins. Mutant genes were expressed in JM103. Cells were grown for 48 hr in 400 ml of LB medium (18) containing 50 ytg of ampicillin per ml. Crystal extracts were prepared and solubilized in sodium carbonate buffer (50 mm Na2CO3/10 mm dithiothreitol, ph 9.5) as described (16). The concentration of solubilized protoxin was determined by Coomassie blue protein assay reagent (Pierce). Protoxin was digested to the active toxin with trypsin with a trypsin/protoxin ratio of 1:50 (wt/wt) for 1 hr at 37 C, followed by an equal dose of trypsin for 1 hr at 37 C. Protoxin and toxins were examined by SDS/polyacrylamide gel (12%) electrophoresis (SDS/PAGE) according to Laemmli (19). Binding Assay. The midgut was isolated from fifth instar B. mori larvae. The isolated midgut was used for preparing brush border membrane vesicles as described by Wolfersberger et al. (20). Iodination of toxins and heterologous competition binding assays were carried out according to Lee et al. (15). Binding data were analyzed by the LIGAND program (21). This program calculates the dissociation constant (Kd) and binding site concentration (Bmaj) of bound ligand as a best fit of theoretical curves to the experimental curves. CD Spectra Measurement of Toxins. Sephadex G-100 column-purified toxins were dialyzed against Na2CO3 buffer (ph 9.5) and concentrated to 0.1 mg/ml. The toxin solution was added to a 0.1-cm pathlength quartz cuvette and the spectra were recorded on a Spex CD6 spectrophotometer in the far ultraviolet region between 180 and 340 nm. At room temperature (21 C), 30 scannings were averaged for each plot and the buffer baseline was subtracted before the data were analyzed. The mean residue molar ellipticity (0) at a given wavelength was calculated by the following formula: [0] = degcm2/dmol = 0.01 x (mdeg.) x 10 liters/m. Toxicity Assay. Bioassay of B. mori was performed by surface contamination of mulberry leaf disks (diameter, 1.5 cm). One second instar larvae was added into each assay cup and a 10-,ul drop of diluted protoxin was spotted on the leaf disk (diameter, 1.2 cm). Five toxin concentrations were used to calculate the median lethal concentration (LC50) value with 100 larvae at each concentration. LC50 values and 95% fidicial limits were calculated with the PROBIT.SAS program (22). A data set was taken with solubilization buffer without toxin as a control for the bioassay. Voltage Clamp Analysis. Voltage clamp analysis was carried out mainly based on the method reported by Harvey et al. (23), with some modifications. The amplifier equipment consisted of a voltage/current clamp and an A-310 Accupulser (both from World Precision Instruments, Sarasota, Proc. Natl. Acad. Sci. USA 90 (1993) FL). These were linked to a Macintosh computer with a MacLab data acquisition system. Calomel electrodes served as voltage measure devices and KCl agar bridges (4% agar in 3 M KCl) connected the electrodes to the chamber. Silver plate electrodes were used as current-passing electrodes. The voltage clamp chambers and their accessories were modified versions of models provided by M. G. Wolfersberger (Temple University) and connected to the amplifier equipment. The Chamberlin buffer (24) was used and was vigorously bubbled with 100% oxygen for 2 hr prior to adjusting the ph to 6.7. Fifth instar B. mori larvae weighing 2.3 g were used in all experiments. The procedures of dissection of insects and assembly of midgut membrane were the same as described (23). The short circuit current (I,,) was tracked with the Kipp and Zonen recorder and data were collected with the MacLab data acquisition system on a Macintosh computer. After 15 min of stable I,,, 0.4 Ag of trypsin-activated toxin was added to the lumen side of the chamber. The lag time and slope of falling 'Sc were measured. RESULTS Stability of Mutant Toxins. The mutant toxins formed in this study are illustrated in Fig. 2. Mutations in positions R2 (R528G) and R3 (R530G and R530K) were very poorly expressed as crystals in E. coli. We did not explore further the nature of the reduced expression in this study, but other work in our laboratory indicated that poor expression of some mutant protoxins is due to intracellular protease digestion (25). A mutation in which all four arginines were replaced by lysines (4R-4K) was also poorly expressed. Other mutations in the Rl, R2, and R4 positions of CryIAa (R526K, R528K, and R532K, respectively) were expressed as protox- ALLELE CryIAa AMINO ACID SEQUENCE R526K PLS Q_Y R V RI R Y A S R528K P L S Q R Y RV R I R Y A S R530K P L S Q R YERV RI R Y A S R532K P L S Q R Y R V RI RY A S 4R-4K P L S Q Y VR I Y A S R526E P L S QWY R V R I R Y A S PROTOXIN TOXIN R8E P L S Q R Y V R I R Y A S - - R530E P L S Q R Y R V I R Y A S - - R532E P L S Q R Y R V R I YAS - - 4R-4E P L S Q Y V I JY A S - - R528G P L S Q R Y V R I R Y A S - R530G PLS QRYRVVJI RYAS - _ FIG. 2. Mutations in the conserved block 4 and their effect on protoxin stability and expression. +, Expression of protoxin or toxin; -, no detectable expression. ar528k and R526E were stable with a low level of trypsin but not with a high level of trypsin.

3 ins that were processed to toxins by trypsin (Fig. 2), as was R526E in the Rl position. We examined the stability of these mutant toxins by exposing the toxins to higher concentrations of trypsin (toxin/trypsin ratio, 1:1) and full-strength B. mori midgut juice. The R526E (position R1) and R528K (position R2) mutant toxins were degraded while the other toxins remained intact. To further examine the effect of these mutations on the structure of the toxins, we analyzed the CD spectra of the mutant proteins compared to the wild-type CryIAa toxin (Fig. 3). The CD spectra of the mutant toxins R526K and R532K were essentially identical to that of wild-type toxin. Bloassays on Mutant Toins. To determine if these mutations in the conserved block 4 affected biological activity of the toxin, bioassays were performed against B. mori. Table 1 shows the bioassay results oftwo mutant toxins, R526K and R532K, compared to wild type. The LC50 values of mutants R526K and R532K are very close (16.6 and 16.5) and much higher than the wild type's (3.9) (Table 1). These two mutants have decreased toxicity to B. mori relatively to wild type. Binding Studies. Membrane binding properties of toxins were examined to determine if the mutations affected the receptor binding ability. Binding experiments with R526K and R532K on B. mori brush border membrane vesicles revealed that the Kd values were 5.07 ± 0.95 nm and 6.74 ± 0.71 nm, respectively. The Kd value of the parental toxin CryIAa was 3.80 ± 1.15 nm (n = 4 for all toxins). The data showed that mutations of R526K and R532K do not significantly affect the receptor binding. To further examine the binding affinities of these two mutant toxins, competition experiments were conducted in parallel with the parental toxin. The competition binding curves of these mutant toxins Biochemistry: Chen et al CrylAa R526K A Proc. Natl. Acad. Sci. USA 90 (1993) 9043 Table 1. Voltage sensing and biological activity of mutant insecticidal toxins Bioassay* Loss of conductancet LCso, Lag time, Toxin ng/cm2 CLt Slope min CryIAa ± ± 2 R526K ± ± 2 R532K ± ± 1 *LC5o values from five separate repetitions of five concentrations with insects per concentration. tslopes and lag times of eight separate 'Sc responses of B. mori midgut to toxin. tcl, 95% confidence limits. appear essentially identical to that of the wild-type toxin as shown in Fig. 4. Voltage Clamp Analysis. To determine the possible role of the alternating arginine region in the mode of action of the toxins, we performed conductance measurements by voltage clamp analysis with mutant toxins on B. mori midguts. & measures the active transport of ions from the blood side of midgut membrane to the lumen side (23). When toxin is added to the lumen side of the midgut, the IS falls in response to the toxin's function of disrupting ion flux by forming an adventitious ion channel. In previous studies (B. Liebig, D. Stetson, and D.H.D., unpublished data), we observed, onb. mori midguts, that the slope of the falling ISC is linearly correlated to CryIAa toxin concentration over a range of ,ug/ml. We chose a concentration of 0.11 ug/ml for CryIAa and the mutant toxins, which gave a response (slope of falling Is) midway in the range. An average of eight voltage clamp responses for CryIAa toxin, and the mutant toxins R526K and R532K, is reported in Table 1. The slopes of falling I for R526K and R532K are -1.9 and -1.8, respectively, compared to that of wild type, The values of lag time (the time period before I begins to drop after addition of toxins) for mutant toxins (about 7 min) are longer than that ofthe wild type (about 5 min). The results demonstrate that R526K and R532K mutant toxins have a much weaker ability to disturb the Is t < 0 ~~~~~~~B s CrylAa l R532K 0. c C E.E E Wavelength, nm Comparison of CD spectra of wild-type CryIAa 8-endo- FIG. 3. toxin and mutant toxins. Trypsin-activated toxin was scanned on a Spex CD6 spectrometer Competitor, nm 10,000 FIG. 4. Heterologous competition between iodine-labeled wildtype CryIAa 8-endotoxin and unlabeled mutant toxins. Toxin was purified according to a modification ofthe method ofhofte et al. (26), and labeled as described (15). Brush border membrane vesicles (300 pg/ml) were incubated with labeled CryIAa toxin (2 nm) in the presence of increasing amounts of unlabeled proteins. CryLAa (o), R526K (o), and R532K (A) were used to compete against 125I-labeled CryIAa toxin.

4 9044 Biochemistry: Chen et al DISCUSSION Studies have shown that 5-endotoxins can disturb the normal ion gradients between the blood side and the lumen side in midgut of susceptible insects by forming ion channels after binding to the receptors in the target cell membrane and cause death of insects. Harvey and Wolfersberger (6) demonstrated that the B. thuringiensis 8-endotoxins behave as potassium channels and disrupt the potassium flux that is generated by ion pumps in the goblet cells of the midgut wall. Wolfersberger and Spaeth (27) observed a correlation between insecticidal activity and the degree of disruption of the potassium flux for a series of B-endotoxins. We have observed a correlation between toxin concentration and degree of disruption of the ion flux (B. Liebig, D. Stetson, and D.H.D., unpublished data). Several patch clamp studies confirm the toxin's behavior as an ion channel (28-30), but the molecular mechanism of the toxin's function as an ion channel function is not known, nor is the location of this function in the toxin structure. In the studies reported here, we have shown that the conserved block 4 in CryIAa is involved not only in structural stability but also in ion channel function of the toxin. In our experiments on the "arginine face" of the conserved block 4 in CryIAa, we observed that mutations in the central two arginines (R2 and R3) of the conserved sequence, QRYRVRIRYAS, to glycines, glutamates, or lysines resulted in failure to express protoxin in E. coli. We assume that this is due to alteration in protein structure and in situ proteolytic degradation (25, 31). This effect on structure was more predominant in R3 than R2. Li et al. (11) observe that an intermolecular salt bridge exists in the CryIIIA crystal structure between the first domain (D224) and the third domain (R562). The latter residue is referred to as R2 in this paper. If the structure of CryIAa is similar to that of CryIIIA, one would anticipate that the arginine at position R2 or R3 would be involved in a salt bridge and thus sensitive to mutation even to a lysine. The fact that this conservative substitution at R3 lead to instability of the entire molecule indicates the interaction of the domain III with the domain I is structurally important. In light of this, it is difficult to account for the presence of glycine in the R2 position of CryIVB (32-34) or in the R3 position of CryIB (35) (Fig. 1). We observed that glycine was not tolerated in either of these positions in CryIAa (Fig. 2). Either these Cry toxins do not form salt bridges in these positions or they do so in an alternate fashion (a salt bridge at R2 for CryIB and at R3 for CrylVB). Our second observation is that the outer two arginines, Rl and R4, affect the ion channel function of the toxin. We present evidence that mutations of R to K in these positions do not affect the global structure of the mutant proteins, as measured by CD analysis, nor their sensitivity to high levels of trypsin or full-strength B. mori midgut juice. Receptor binding studies (Fig. 4) show that mutations of R526K and R532K do not significantly affect the binding ability of the toxin. This result is consistent with other studies that indicate that domain I (36) and/or domain 11 (11, 15) are involved in receptor binding; thus, mutations in the carboxyl-terminal domains would not be expected to alter binding unless they altered the overall structure of the toxin. Furthermore, since these mutant toxins are not altered in binding, the results are consistent with studies showing that factors other than binding are essential for toxicity (37, 38). From these results we conclude that the mutations of R526K and R532K specifically reduce the toxin's inhibition ofis, not as a result of structural alteration of the toxin. Li et al. (11) proposed that domain I of the &-endotoxin forms the pore. Their hypothesis is based on the hydrophobic surfaces and their potential for spanning the membrane. Proc. Natl. Acad. Sci. USA 90 (1993) There is no suggestion as to how the pore might play a role in ion channel function in their model. We think that domain I provides the scaffolding of the ion channel; other regions in the toxin could play a gating or regulation role for the channel or assist in the insertion of domain I into the membrane. We would predict that mutations in residues that form the "active site" of the ion channel would affect toxin inhibition of ISc without necessarily affecting binding properties or protein structure. In an attempt to recognize a functional role of the alternating arginine region, we scanned the literature for similar sequences. Herpesviral deoxythymidine kinase (HdTK) and porcine adenylate kinase have conserved tracts of amino acids (39) that share sequence similarity to conserved tracts 3, 4, and 5 of Cry toxins. Site 5 of HdTK, which is similar to the Cry toxin alternating arginine region, is thought to bind substrate phosphate groups (39). A kinase function in Cry proteins has not been reported, but selected nucleotides have been observed to inhibit the cytotoxic activity of Cry toxins from B. thuringiensis var. aizawai (40). The S4 a-helix, found in all classic ion channels, also has a positively charged face of arginines that is believed to be involved in the ion gating mechanism and has been called a voltage sensor (41). Supporting evidence for this hypothesis is based on mutations that alter the charges on the argininesnamely, mutations to glutamines and lysines (41, 42). These mutations reduce the single-channel conductance as measured by patch clamping. Mutations of these arginines to glutamines clearly eliminate the positive charge, but controversy arises about the role of the S4 helix because the mutation of arginines to lysines is viewed by some as not affecting the charge (43, 44). Given the lower pka of lysine (-10 in proteins), relative to arginine (=12 in proteins), a significant charge reduction would be expected from the Henderson-Hasselbalch equation (45). By application of the S4 voltage-sensing model to the Cry toxins, we hypothesize that the alternating arginine region may play a role in the ion channel function as a voltage sensor. Since the ph of the lepidopteran insect midgut is near the pka of arginine (46), the mutations of arginines to lysines would be expected to have a significant reduction in charge. Another explanation for the observed effect of these site-specific mutations might include a structural alteration of the toxin, not in solution but as it resides in the membrane, such that the ability of the toxin to form a channel would be altered. Alternately, an unrecognized function, such as a kinase activity that might play a role in second messenger activation of the ion channel, could be affected by these mutations. The finding that the conserved block 4 in the B. thuringiensis 6-endotoxin CryIAa is involved in ion conductance may assign a functional role for the third domain. Assuming that the three-dimensional structure of CryIAa is similar to that of CryIIIA, our results imply that domain III is important not only in structural stability and integrity of B-endotoxins (11) but also in function. Our working hypothesis suggests that the conserved block 4 of the 6-endotoxin is involved in a regulatory aspect of ion channel activity, possibly as a voltage sensor or in the gating mechanism. We are grateful to Ross Milne and the staff at the Forestry Pest Management Institute (Sault Ste. Marie, ON, Canada) for providing eggs and larvae of B. mori and to Qi Feng and Wayne Becktel (Department of Biochemistry, The Ohio State University) for aid in CD spectroscopy and helpful discussions. We especially thank D. Stetson and B. Liebig for help with the voltage clamp technique. This research was supported by grants from the National Institutes of Health (ROl A129092) and the U.S. Department of Agriculture Forest Service. 1. Hofte, H. & Whiteley, H. R. (1989) Microbiol. Rev. 54,

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