Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes
© Alzate et al; licensee BioMed Central Ltd. 2009
Received: 18 May 2009
Accepted: 19 October 2009
Published: 19 October 2009
Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of α-helix 5 of the Bacillus thuringiensis Cry1Ab δ-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane.
Amino acid residue leucine 157, located in the N-terminal portion of α-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the α-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the α-helix 5 is shown to be involved in ion transport activity.
Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the α-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire α-helix becomes inserted in the insect midgut membrane.
Identification of the amino acid residues involved in the ion transport activity of Bacillus thuringiensis (Bt) δ-endotoxins remains an interesting challenge. The δ-endotoxins are insecticidal crystal proteins (ICPs) produced by the bacterium during the sporulation stage . The ICPs are composed of several proteins (also known as Cry toxins) with insecticidal activity against insects of the orders Lepidoptera, Diptera and Coleoptera. The ingested crystals are dissolved in the insect midgut to release the protoxin form of the proteins. The action of proteases in the insect midgut cleaves the protoxin at specific locations, releasing the toxic fragment of the protein. In the case of the lepidopteran-specific Cry1A δ-endotoxins, the protoxins have a molecular weight of approximately 140 kDa, while the molecular weight of the toxic fragment is approximately 65 kDa .
The toxic action of the protein is initiated by the binding of the active fragment to receptors located on the membrane lining the insect midgut. It has been suggested that after binding to the receptors, the toxin induces ion pores capable of disrupting the membrane potential [2, 3]. The molecular events occurring between binding and intoxication remain unclear. Some authors proposed that the binding to these receptors triggers conformational changes that allow the protein to translocate into the membrane, becoming inserted into the lipid moiety [4, 5]; such conformational changes, and the membrane bound state of the toxin have not yet been elucidated.
Cry1Ab δ-endotoxin is one of the many insecticidal toxins expressed by Bacillus thuringiensis. The three dimensional structure of Cry1Aa (a protein with 89% sequence homology to Cry1Ab), is composed of three structural domains . Domain I is formed by an α-helix bundle formed by seven helices with the α-helix 5 located in the core of the domain. Domains II and III are mainly composed of β-sheets. There is evidence that at least α-helix 7, located in Domain I, is involved in the ion transport activity [6, 7]. Domains II and III are involved in receptor recognition in the insect midgut [8, 9].
The insertion of the toxin into lipid membranes has been studied using artificial phospholipid vesicles, lipid bilayers [10, 11], modified protein toxins in artificial membranes , and artificial peptides resembling structural motifs of the protein [12, 13]. The protection of δ-endotoxins against proteinase K (Pk) digestion in the presence of brush border membrane vesicles (BBMV) has been used to study the insertion process of the toxin into systems resembling the midgut environment [14–17].
Expression of proteins in E. coli, purification and bioassays
Toxicity assays and voltage clamping parameters
-13.42 ± 0.70
-10.93 ± 0.27d
4.00 ± 0.14
4.22 ± 0.12d
-7.03 ± 0.22
7.58 ± 0.60
-4.27 ± 0.80
8.04 ± 0.20
-11.85 ± 0.60
3.93 ± 0.60
-10.73 ± 0.20
3.08 ± 0.60
-5.56 ± 0.10
6.84 ± 0.15
-3.39 ± 0.12
14.00 ± 0.10
The changes seen in the insertion time and in the ion transport activity are likely to occur as a result of capping the cysteine residue with the spin-label since such changes were not found in the other two mutant proteins. The mutant toxin S170C showed the same ion transport activity either with or without capping of the cysteine residue (see Table 1), and these parameters (slope = - 11.85 ± 0.60 μA/min, T0 = 3.93 ± 0.60 min) are similar to those of the wild type toxin (slope = -13.42 ± 0.70 μA/min, T0 = 4.00 ± 0.14 min). On the other hand, mutant toxin L157C showed a small decrease in ion transport that reflects the decrease observed in LC50 with no observable change in the insertion time (see Table 1). The parameters obtained for L157C mutant protein suggest that this toxin has a reduced ability to transport ions (slope = -7.03 ± 0.22 with no MTS-SL and slope = - 4.27 ± 0.80 with MTS-SL, with no change in the insertion time). The results obtained from the voltage clamp experiments are in agreement with the relative toxicities calculated from the toxicity assays (see inset in Figure 3A). These results suggest that the change in slope and insertion time (i.e., toxicity) observed for S176C is due to the particular location of the S176 residue, which is therefore critical for ion transport or for partitioning of the toxin in the membrane.
Circular dichroism spectroscopy and thermal analysis
EPR spectroscopy in solution and in BBMV
Studies on the mode of action of the Bt δ-endotoxins have shown that this is a rather complex mechanism that involves the identification of the target tissue by the activated toxin, followed by association to membrane receptors and internalization into the membrane. The regions of the toxin responsible for membrane internalization and ion transport, and the active conformation of the toxin in the membrane, have not yet been determined. In the present work, we used a combination of site-directed spin-labeling and voltage clamping to address these issues.
Three amino acid residues located in the postulated ion-transport region were individually mutated to cysteine. Each resulting toxin was tested for structural stability and biological activity. Amino acid residue L157 is located at the N-terminal end of the centrally-located α-helix 5, S170 is located at the middle, and S176 at the C-terminal end of the helix (Figure 1). The three mutated proteins produced stable protoxins with the same size as the wild type toxin (Figure 2A); after tryptic digestion, each protoxin released the stable toxin of the same size as the wild type Cry1Ab (Figure 2B). The biological effect of each introduced mutation was first analyzed by bioassays on M. sexta larva. Results show that S170C has the same toxicity as the wild type toxin while L157C and S176C show a small reduction (about 2-fold decrease). Although the LC50values for L157C and S176C clearly show that these two proteins are less toxic than the wild type, the overlapping confidence limits indicate that these bioassays are not accurate enough to determine toxicity differences within such a close range.
To determine more accurately the effects of the mutations on the toxicity, we analyzed the ion transport activity of the toxins by voltage clamping. Our results showed that mutation S170C had no effect at all in ion transport, while mutations L157C and S176C decreased the ion transport activity of the toxins (Figure 3A). Calculated values from the voltage clamping curves are shown in the table. The toxins were compared by analyzing their slopes and insertion times [6, 14]. Both the slope and the insertion time for S170C were almost identical to those of the wild type, while these parameters indicated a reduction in ion transport and insertion time for L157C and S176C. The accuracy of the voltage clamp was reflected in the correlation between the LC50 values and ion transport, as shown in the insert on Figure 3A, in which a plot of LC50 vs. slope shows a linear correlation.
To assess the effects of the spin-label on the biological properties of the toxins, each mutant toxin was reacted with MTS-SL, non-attached MTS was removed, and the bioassay and voltage clamping experiments were carried out. The S170R1 mutant toxin showed no change either in toxicity or ion transport. The L157R1 mutant toxin showed a slight decrease in toxicity, which correlates with extent of loss of ion transport activity. On the other hand, the S176R1 mutant showed a large decrease in both the toxicity and the ion transport that are statistically significant. One possible reason why this mutant was less active when the cysteine residue was modified with the spin-label could have been the introduction of large conformational changes due to the bulky side chain. The longer insertion time of the S176R1 mutant could also be due to steric limitations introduced by the modified side chain. Once the protein has been inserted, the ion transport should resemble the activity of S176C provided that the amino acid is not involved in this function. However, the slope of a plot of Isc vs. time, obtained from voltage clamp experiments, became even less steep suggesting that the amino acid residue at position 176 was involved in ion transport.
CD was used to analyze structural changes in toxin structure. The CD spectra showed that there were no changes in any of the structures, with or without the spin-label. The particular situation of S176R1, which showed a considerable decrease in ion transport, indicated that neither the mutation, nor the presence of the spin-label had any effect on the secondary structure (Figure 4). CD thermal melting was used to determine whether the spin-label had introduced any structural instability in the protein. As shown in Figure 5, the thermal melting curve of the S176R1 mutant overlaps with the thermal melting curve of the S176C mutant, with a Tm = 76°C, the same as the wild-type toxin.
By analyzing the conformation of the protein in solution, as shown in Figure 1, it is clear that some conformational changes must accompany the transfer of the protein from solution into the lipid moiety, so the S170 residue becomes located in a more viscous environment that restricts the tumbling seen in solution. One possible explanation, in agreement with two previous publications in which α-helix 5 is proposed to anchor to the membrane moiety [24, 25], would be that, after α-helix 1 is cut out of the structure , α-helices 2 and 3 open up exposing the hydrophobic face of α-helix 5 to the hydrophobic moiety of the membrane. This would explain why the motion of the modified 170 residue becomes more restricted in the membrane-bound state. It is also possible, however, that location of the spin-label in a hydrophobic environment dominated by hydrophobic amino acids would produce similar observations.
It is clear that the combination of site-directed spin-labeling and voltage clamping allows the determination of functional amino acid residues involved in ion transport activity of the Bt δ-endotoxins, and gives information about the membrane-bound conformation of these important insecticidal toxins. Two very important aspects of this combination of methodologies are, first, that voltage clamping allows for experiments on the midgut tissue containing functional receptors in their native environment and, second, that EPR gives information on the spin-labeled proteins inserted in BBMV. This is in contrast to results obtained from artificial phospholipid vesicles in which the receptors and the structure of the membrane are not necessarily the same as in the biological organism. Along with these advantages, the EPR experiments can also be carried out in artificial phospholipid vesicles. These experiments would allow one to determine the effects of membrane proteins on the EPR spectrum of the spin-labeled toxin.
Site-directed mutagenesis, toxin preparation and analysis
Selection of the amino acid residues to be targeted for mutagenesis was based on the Cry1Ab protein structure modeled from Cry1Aa (Pdb code 1CIY), whose crystal structure is available . The coordinates for Cry1Aa were used as template to introduce the changes needed to transform Cry1Aa into Cry1Ab with the protein tools included in the program QUANTA (Molecular Simulations, Inc.). Each mutation was energy minimized to Root Mean Square (R.M.S.) of 0.1 using a Polak-Ribiere algorithm with the force field MM+ (Hypercube, Inc.). Mutant toxins L157C, S170C and S176C were constructed by site-directed mutagenesis as described . The plasmid containing the target mutation was transformed into E. coli MV1190 (BioRad) and purified as described . The protoxin form of the proteins was extracted from the inclusion bodies by dissolving the crystals in 50mM carbonate buffer, pH 10.5, at 37°C with constant agitation for 2.0 hours. The presence of the protein was confirmed with SDS (10%)-PAGE gels. Toxins were obtained by digesting the protoxins in 1/20 protoxin/trypsin (w/w) ratio at 37°C for 2.0 hours. The digestion step was repeated under the same conditions. The toxins were purified by size-exclusion chromatography using an Akta Explorer workstation (GE Healthcare, Piscataway, NJ) with a GE Healthcare HiLoad 16/60 Superdex200 column.
Bioassays and other experimental procedures were performed following guidelines approved by the Institutional Animal Care and Use Committee of the Ohio State University. Toxicity levels were determined with the diet surface contamination assay. For this assay, 24 well sterile dishes (Falcon) were half filled with artificial diet (Bioserv, Frenchtown, NJ). Five dilutions of the toxin were prepared to final concentrations ranging from 1 to 300 ng/cm2, and 50 μL of each dilution was poured on the surface of the food. Two (2-3 days old) M. sexta larvae were placed in each well and the lethality was scored after five days. The lethal concentration killing 50% of the larvae (LC50) was calculated with the program SoftTox (WindowChem Software, Inc.). Each assay was repeated three times.
where Isc is the short circuit current as function of time, Isc0 is the Isc value at the time when toxin is injected in the chamber, t0 is the time needed for the current to drop 50% from the initial value and A and b are fitting parameters. The time needed for the current to drop 10% from the initial value, as calculated from equation (1), was considered to be proportional to the insertion time, T0. Experiments for each toxin were repeated at least three times. The slope corresponding to the Isc between 20% and 80% was calculated with the program Sigma-Plot (Jandel Scientific Corporation, Inc.) and was taken to be proportional to the ion-transport activity of the toxin. To test the effect of the introduced cysteine mutations the residues were reacted with MTS as explained below, using EPR spectroscopy.
Circular dichroism spectroscopy and thermal analysis
Proteins for CD analysis were purified as explained above using 40 mM phosphate buffer, pH 7.4 as mobile phase. Thirty μg of toxin was analyzed in a 1.0 cm path length Hellma quartz cuvette, in the range λ = 200-250 nm at room temperature in an Aviv 62DS spectrometer. The thermal melting analysis was done by increasing the temperature from 30 to 90°C in 3°C increments. The equilibration point was determined by the program IGOR, which controls the CD spectrometer. The CD spectrum for melting analysis was taken at λ = 223 nm.
EPR Spectroscopy and continuous wave (CW) power saturation
[1-Oxyl-2,2,5,5-tetramethyl-D-pyrroline-3-methyl] methanethiosulfonate (MTS) was from Toronto Research Chemicals (Toronto, Canada). Samples for EPR spectra were purified as described above. After purification the samples were dialyzed against 4,000 volumes of 20 mM Tris-HCl, 1.0 mM EDTA, 150 mM NaCl, pH 7.4 for 4 days with 4 to 5 changes of buffer. After the first day of dialysis, the samples formed a white precipitate that could be easily removed from solution by centrifugation. To spin-label the protein, each sample was mixed with 5.0 molar excess MTS spin-label reagent and allowed to react for 12 hours at room temperature. Excess reagent was removed by dialysis under the same conditions as above, followed by centrifugation at 12,000 RPM for 15 minutes. HPLC analysis, as described, was used to determine that the samples used in all of the different assays were in a monomeric form. Samples were concentrated to 0.1 mM, and a 200 μL aliquot was loaded into a flat cell. Spectra were recorded using a Bruker ESP300 EPR spectrometer at room temperature. The resulting spectra were the average of 20 scans.
where P is the incident microwave power, P1/2 is the power at which H is half of the unsaturated value, ε is a factor related with the homogeneity of the line, and I is a scaling factor . P1/2 was determined in the absence of paramagnetic species, in the presence of oxygen and in the presence of 40 mM NiEDDA and the change in P1/2, ΔP1/2, was determined from these measurements as described by Altenbach et al. .
List of Abbreviations
- Bt :
brush border membrane vesicles
Ni (II) ethylenediaminediacetate
electron paramagnetic resonance
We thank Dr. D. L. Dahlman (U. of Kentucky) for providing us with M. sexta eggs, and Drs. C. Felix and J. Feix from the Biophysics Research Institute (Medical College of Wisconsin) for training O. A. in the SDSL technique.
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