BACKGROUND
bacillus thuringiensis strains produce a range of toxins active against invertebrates with enormous potential for use in the control of pests of importance in agriculture and health  <cit> .
during sporulation, strains synthesize cytolytic  toxins and/or crystal  protoxins and deposit them as parasporal inclusion bodies.
of the  <dig> major subclasses of cry protoxin currently recognized   <cit> , most belong to a large family of related sequences that can be proteolytically processed to yield active toxins with a three-domain fold as first seen for cry3aa  <cit> .
however, the cry nomenclature is not limited to these proteins and includes several distinct and unrelated lineages.
while much is known of the structure and function of the three-domain toxins  <cit>  and the structure of a protoxin form has recently been published  <cit> , very little is known of the non-three-domain cry proteins.
some of these proteins share sequence homology to other known toxins; for example, cry <dig> and cry <dig> are related to the bin and cry <dig> toxins of lysinibacillus sphaericus  <cit>  and may share a proposed mechanism of action involving pore formation  <cit>  and/or apoptosis  <cit> .
the structures of the ß-sheet-rich toxins cry <dig>   <cit> , cry <dig>   <cit> , and recently cry <dig>  <cit>  and the cry34/cry <dig> binary toxin  <cit>  have been published but for many non-three-domain cry proteins, neither structural data nor information on mechanism of action are available.
one such cry protein is cry6aa  <cit> , a protein with activity against coleoptera such as the western corn rootworm diabrotica virgifera virgifera  <cit>  and a range of nematodes, including both free-living  and plant pathogenic  species  that cause large-scale losses to agriculture  <cit> .
previous studies have shown by deletion of cry6aa sequences that  <dig> amino acids can be deleted from the n-terminus and  <dig> residues can be removed from the c-terminus, leaving a core region of ~43 kda that retains activity against nematode targets  <cit> .
cry6b is a protein that is closely related to cry6a but lacks  <dig> amino acids that are seen at the c-terminus of the latter protein.
cry6b is reported to be active against the coleopteran lucerne weevil hypera postica  <cit>  but showed little or no activity against a range of nematode targets  <cit> .
the absence of further data on the structure and function of these proteins has limited our ability to understand their activity against target invertebrates.
as a result, development and exploitation of the toxins in the control of agricultural pest insects and nematodes pathogenic to plants and animals may be limited.
this, in turn, inhibits their use to supplement the current chemotherapeutic approaches to nematicidal treatments that are very toxic and are being phased out  <cit> .
in this study we applied both crystallographic techniques and state of the art ab initio modeling to probe the structure of cry6aa in protoxin and trypsin-cleaved forms.
the structures obtained are novel among invertebrate-active toxins and are consistent with cry6aa acting as a pore-forming toxin.
we demonstrated pore formation in vivo and used our predictions to construct a mutant expected to show compromised toxicity.
methods
bacterial strains
b. thuringiensis strain bmb <dig> containing the pht304-derived plasmid encoding the cry6aa <dig> protein  <cit>  was kindly provided by dr ming sun, huazhong agricultural university, wuhan, china.
this strain was grown to greater than 98 % autolysis  in embrapa medium  <cit> .
spores were harvested and crystals of cry6aa were isolated and purified by discontinuous sucrose density gradient ultracentrifugation according to previously described methods  <cit> .
cry6aa protein crystals purified by this method were solubilized at 37 °c with shaking at either alkaline ph by incubation in 50 mm naoh  or at acidic ph by the addition of 100 mm sodium citrate buffer ph  <dig> .
toxin production in escherichia coli
the cry6aa gene was amplified from the above b. thuringiensis strain by 30 cycles of pcr with a 60 °c annealing temperature, using forward  and reverse  primers, incorporating ecori restriction sites .
the amplicon was cloned into the pgem-t vector  and transformed into e. coli dh5α for the selection of a single colony, plasmid isolation, and sequencing to confirm the cloning of the gene with no mutations.
the gene was subsequently cleaved from this plasmid with ecori and re-cloned into the ecori site of the pet28b expression vector to allow production of recombinant cry6aa protein with an n-terminal his-tag.
a mutation of the cry6aa gene to change leu <dig> to asp was introduced using the phusion site-directed mutagenesis kit  according to the manufacturer’s instructions by pcr  using the pet28b clone as template and the following 5′-phosphorylated oligonucleotides: 5′-gacttaggatttgttgtttatgaaattc-3′ , 5′-tggtcctaatagaaaactatattc-3′.
following ligation of the pcr product and transformation of e. coli dh5α, a colony was picked and the plasmid sequence was verified to ensure incorporation of the desired mutation and no other changes.
for recombinant production of cry6aa proteins in e. coli, the pet28b plasmids were transformed into the shuffle t <dig> express lysy e. coli strain  and isolated colonies were selected.
transformants were grown in lysogeny broth  medium in the presence of 50 μg/ml kanamycin at 37 °c with shaking overnight before the addition of isopropyl ß-d-thiogalactopyranoside  to a final concentration of 1 mm and growth for a further 3 h. expression was verified by western blotting using monoclonal anti-polyhistidine antibody  as the primary antibody and rabbit anti-mouse alkaline phosphatase conjugate antibody  as the secondary antibody to allow visualization of the proteins using the ap conjugate substrate kit .
for in vivo pore formation assays, a distinct clone of the cry6aa gene in the bamhi and psti sites of the vector pqe <dig> was produced .
an equivalent clone in pqe <dig> containing the cry5ba gene as a positive control for in vivo pore formation has been described previously  <cit> .
cry <dig> production in pseudomonas fluorescens
for inclusion body  preparation, pseudomonas fluorescens-derived cell paste expressing full-length, wild-type cry6aa <dig>  was transferred from −80 °c storage to 4 °c and resuspended at 20 % w/v in cold lysis buffer  and mixed thoroughly with a handheld homogenizer.
the suspension was then passed through a microfluidizer  twice at  <dig>  psi then centrifuged .
the supernatant was discarded.
the ib pellet was fully resuspended by homogenization in 10 % w/v room temperature lysis buffer with  <dig>  g/l lysozyme .
the suspension was incubated at 30 °c for 30 min, with brief homogenization every 10 min.
the ibs were then centrifuged  and the supernatant was discarded.
the pellet was resuspended for a final time in 10 % w/v cold lysis buffer using the homogenizer and then centrifuged  and the supernatant discarded.
the ib pellet was resuspended in 10 % w/v cold lysis buffer without triton x- <dig>  using the homogenizer and then centrifuged  and the supernatant discarded.
this step was repeated twice.
the ibs were resuspended to 30 %  in 10 mm edta, ph  <dig> , and then separated into  <dig>  ml aliquots and frozen at −80 °c until needed.
a separate aliquot of the ib preparation was stored without suspension in 10 mm edta.
purification of full-length cry6aa
about 500 mg of pseudomonas-derived ib paste was thawed at 4 °c, and centrifuged at  <dig>  g for 25 min at 4 °c.
the supernatant was removed and the pellet was solubilized in 30 ml of 100 mm caps, ph  <dig> , and the suspension was gently rocked for 2 h at room temperature to solubilize the cry6aa.
after solubilization, the mix was centrifuged at  <dig>  g for 25 min at 4 °c.
the supernatant was dialyzed against 25 mm caps, ph  <dig> , overnight.
the buffer exchanged cry6aa sample was then filtered through a  <dig>  μm syringe filter and applied to a source 15q 16/ <dig> ion exchange column at a flow rate of 5 ml/min.
the column was pre-equilibrated with buffer a .
the protein was eluted with a step gradient of 20 %, 30 %, 40 %, and 50 % buffer b .
each elution was ~50 ml.
fractions were collected from each elution based on uv absorbance and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis .
full-length cry6aa  was in the 20 % b fractions, which were pooled and concentrated to ~30 ml using a centrifugal filter device with 10 kda molecular weight cut-off membrane .
the concentrated sample was further purified by size exclusion chromatography.
for each run,  <dig>  ml of the sample was applied over a superdex  <dig> 26/ <dig> gel filtration column pre-equilibrated in 25 mm caps, ph  <dig>  and 50 mm nacl buffer, at a flow rate of  <dig>  ml/min.
two peaks were observed .
the first peak, eluted at the void volume, contained cry6aa dimers .
the fractions from peak  <dig> contained predominantly monomer and were pooled and used in crystallization experiments.
purification of cry6aa trypsin-resistant core
eight 2 ml tubes of ib suspension of full-length cry6aa were thawed and extracted in 80 ml final volume  of 10 mm caps, ph  <dig> .
the measured ph of  <dig>  was then adjusted to  <dig>  with naoh.
freshly prepared trypsin [2 ml of 25 mg/ml trypsin solution  was added, the ph was maintained at  <dig> , and the mixture was stirred overnight at 4 °c.
the digestion was applied to a source 15q 16/ <dig> column pre-equilibrated in buffer c  at a flow rate of 10 ml/min and eluted with a gradient of buffer c + 1 m nacl over 75 min.
trypsin-processed cry6aa eluted as a single large peak, which was concentrated from 100 ml to 10 ml using 15 ml amicon  <dig>  mwco spin concentrators in a ja- <dig> rotor at 4 °c and 5000 g.
the concentrated ion exchange sample was applied to a superdex  <dig> 26/ <dig> gel filtration column pre-equilibrated in 25 mm caps, ph  <dig> , and 50 mm nacl.
the sample was eluted using a  <dig>  ml/minute flow rate.
the fractions corresponding to the large cry6aa core peak were pooled  for crystallization trials.
secondary structural analysis
circular dichroism  spectra were recorded using a jasco j- <dig> cd spectrometer equipped with an mptc-490s 6-cell peltier thermostatted cell holder.
the instrument was calibrated with --10-camphorsulfonic acid.
measurements were carried out in a 1 mm path-length quartz cuvette under a constant nitrogen stream and at room temperature .
data were collected in continuous scan mode from  <dig> to 185 nm every  <dig>  nm at 50 nm/min and with 2 s averaging per point.
each spectrum is the average of five accumulations and a bandwidth of 1 nm.
the concentration of cry6aa <dig> protein in 10 mm phosphate ph  <dig>  was  <dig>  mg/ml, in 10 mm phosphate ph  <dig>  was  <dig>  mg/ml, and in 10 mm caps ph  <dig>  was  <dig>  mg/ml.
all spectra were corrected for the buffer contributions and the raw data output in ellipticity , measured in millidegrees  was converted to mean residue ellipticity  in deg cm2/dmol using the instrument software, spectrum manager  <dig>  the cdpro package was used for secondary structure estimation using the programs selcon, continll, and cdsstr.
the programs gave results that were similar to each other and relatively independent of the reference protein set used.
the secondary structure calculations reported here were from processed cd spectra using the cdsstr algorithm and the sp37a reference set.
crystallization, data collection, and structure determination
cry6aa trypsin-resistant core
the cry6aa trypsin-resistant core was concentrated to 100 mg/ml.
initial crystals were obtained using rigaku reagents, inc. wizard classic i .
after screening multiple conditions, suitable crystals were obtained under kit condition  <dig> .
data were collected at 100 k from a single crystal on a mar ccd- <dig> detector at ls-cat .
data were indexed and processed with the hkl- <dig> software suite  <cit> .
the structure of this truncated form of cry6aa toxin was solved by the molecular replacement method using phaser  <cit>   followed by manual rebuilding and model refinement.
the poly-alanine chain of the crystal structure of hemolysin b from bacillus cereus  consisting of residues 19– <dig> was used as a search model.
the final structure of the complex was obtained by carrying out several cycles of refinement consisting of manual model building using coot  <cit> , followed by restrained refinement with refmac  <cit>  using translation, libration and screw-rotation  refinement and the reference and secondary structure restraints.
the trypsin-cleaved cry6aa structure, solved at higher resolution, was used as the reference structure in this refinement.
crystallographic data and refinement statistics are given in table  <dig> and the cry6a trypsin-resistant core structure can be found at the protein data bank .table  <dig> data collection and refinement statistics
number of non-hydrogen atomsd
pdb protein data bank, rms root mean square deviation from ideal values
astatistics for the highest-resolution shell are shown in parentheses
brmerge = 100ΣΣ|i-<i>|/ ΣΣi where i is the ith intensity measurement of reflection h, and <i> is the average intensity from multiple observations
crfactor = Σ||f
obs|-|f
calc||/ Σ|f
obs|.
where f
obs and f
calc are the structure factor amplitudes from the data and the model, respectively.
to calculate r-free values, 5 % and 10 % reflections were used for cry6aa trypsin core and full-length cry6aa structures, respectively.
dper asymmetric unit
purification of full-length cry6aa
full-length cry6aa protein was concentrated to 15 mg/ml using amicon centrifugal filters with a 10 kda molecular weight cut-off  in 10 mm hepes buffer, ph  <dig>  and 25 mm nacl.
initial crystallization screens were performed using commercially available classics, classics lite, classics ii, pegs, pegs ii, phclear, and pact screens  by the sitting drop method in 96-round-bottom-well crystallization plates  using a mosquito robotic system .
diffraction quality cry6aa protein crystals were grown at 291 k from sitting drops containing 3 μl of the protein sample and  <dig>  μl of reservoir solution .
sds-page analysis of protein samples obtained by dissolving the crystals in sds-buffer did not reveal any degradation products and confirmed the presence of only full-length cry6aa protein in the crystals used for the data collection.
for data collection, crystals were harvested with 20 %  glycerol in the reservoir solution.
diffraction data were collected at 100 k from a single crystal on a mar ccd- <dig> detector at ls-cat .
data were indexed and processed with hkl- <dig>  <cit> .
the crystals belonged to orthorhombic space group p <dig> and contained one molecule of full-length cry6aa per asymmetric unit.
the structure of full-length cry6aa was solved by molecular replacement using the structure of the truncated form of cry6aa as a search model.
the final structure of full-length cry6aa toxin was obtained by carrying out several cycles consisting of manual model building using coot  <cit> , followed by structure refinement with refmac  <cit> .
crystallographic statistics are given in table  <dig>  coordinates have been deposited in the protein data bank .
molecular modeling
the cry6aa tertiary structure was modeled ab initio using the rosetta software  <cit> .
after entering the cry6aa sequence, the first stage of the web-based process returned a secondary structure prediction with options to continue full protein structure prediction either using de novo or database structure comparison methods.
both methods were followed to produce a selection of five possible structure outcomes from both de novo and database predictions .
intact molecular weight analysis/charged state distribution
intact mass analysis was performed on an agilent  <dig> hplc/msd tof 1969a system using a michrom desalting trap heated to 50 °c.
each sample was diluted to a concentration of  <dig>  μg/μl in 10 mm caps ph  <dig> buffer.
samples were also analyzed after reduction with 50 mm tcep for 10 min.
approximately 1 μg protein was injected on column.
the sample was eluted using a gradient , where buffer a is  <dig>  % formic acid in water and buffer d is 70 % isopropanol, 20 % acetonitrile, 10 % water, and  <dig>  % formic acid.
the mass was calculated using the mass hunter qualitative analysis software and the maximum entropy de-convolution algorithm.
changes in the observed charge state distribution  of proteins analyzed by liquid chromatography–mass spectrometry , as described above, were used as an indirect probe of the overall conformational flexibility of the protein under different conditions.
maldi-tof mass spectrometry and tandem mass spectrometry
digestion of cry6aa with trypsin results in two fragment fractions, a large fragment and a small fraction containing two peptides.
the small fragment sample was analyzed by matrix-assisted laser desorption/ionization - time of flight mass spectrometry  to determine the peptide masses and was subsequently fragmented in the lift mode to confirm the sequence of the peptides.
the maldi-tof ms and tandem mass spectrometry  were conducted with a bruker ultraflextreme mass spectrometer.
the c-terminal peptide  sample was diluted with  <dig>  % trifluoroacetic acid  at 1: <dig>  and desalted using a c- <dig> ziptip .
the peptides were eluted with 60 % acetonitrile  in  <dig>  % tfa and mixed with a  <dig> -dihydroxybenzoic acid  matrix .
after spotting 1 μl of the mix on a maldi sample plate, the peptide mass was analyzed using reflection-positive mode and the peptide was fragmented using the lift mode.
the instrument was calibrated with cm  <dig> .
the mass spectrum was collected and analyzed using flex analysis software.
the sequence was verified using biotools software  and the mascot search engine .
n-terminal sequencing
amino terminal sequencing of proteins was performed on a shimadzu protein sequencer  using basic edman degradation chemistry.
the protein sample was separated with sds-page under reducing conditions, and then the proteins were blotted onto polyvinylidene difluoride  membrane by liquid transfer.
a standard mix of  <dig> phenylthiohydantoin-amino acids  was run each time.
the amino acid residues from each edman degradation cycle were determined based on their retention times from the c- <dig> column compared to standards.
nematode bioassay
c. elegans bristol n <dig> strain wild-type nematodes  were cultured using standard techniques including the use of e. coli strain op <dig>  as standard food source  <cit>  on enriched nematode growth  plates  <cit>  containing 100 μm iptg and 50 μg/ml kanamycin .
to measure intoxication by cry6aa, 30– <dig> fourth-staged larvae of c. elegans were seeded on eng-ik plates with e. coli shuffle t <dig> express lysy cells  transformed with the pet28b empty vector  or pet28b with cry6aa inserts  and incubated at 25 °c for 24 h. three replica plates were used for each strain.
three to five worms were randomly picked from each of the different treatment conditions for image collection.
worm growth was assessed by measuring the area of clearly separated, individual worms , using image j  and matlab  software  <cit> .
the experiment was independently repeated three times.
data were analyzed with jmp v.  <dig> using one-way anova and dunnett’s post-test using the vector as the control group.
in vivo pore formation assay
pore formation was assessed microscopically using the method described by los et al.
<cit> .
c. elegans bristol n <dig> strain nematodes were synchronized by hypochlorite treatment.
l <dig> animals were seeded onto op <dig> and grown at 20 °c until the l <dig> stage was reached.
e. coli jm <dig> cultures  transformed with the empty pqe <dig> vector or vectors for expression of cry5ba or cry6aa were grown overnight at 37 °c with shaking in lb medium containing ampicillin.
these cultures were diluted 1: <dig> in fresh medium and grown at 37 °c with shaking for 1 h before addition of iptg to 50 μm and further incubation at 30 °c for at least 3 h with shaking.
after this induction of protein expression, 30 μl of these cells, concentrated to an optical density of  <dig>  were spread onto 60 mm diameter eng agar plates containing ampicillin and iptg and allowed to grow overnight at 25 °c for use in assays.
worms were then washed off the op <dig> plate, washed twice to remove bacteria, and pipetted onto agar plates spread with the transformed jm <dig>  plates were incubated for 2 h at 20 °c.
worms were then washed off the plates, washed twice in m <dig> medium to remove bacteria, and rotated end-over-end at room temperature in m <dig> medium containing 5 mg/ml 5-hydroxytryptamine  for 15 min to stimulate feeding.
propidium iodide was then added to the medium and worms were rotated for an additional 30–40 min.
worms were washed twice to remove excess dye and pipetted onto agar pads on microscope slides and imaged.
pictures were taken using a spot insight ccd camera and spot software connected to an olympus bx <dig> compound microscope with an uplanfl 40×/ <dig> na objective.
at least  <dig> worms were imaged for each condition, and the entire experiment was conducted three times.
the percentages for all three experiments were combined and analyzed by one-way anova followed by dunnett’s multiple comparison test between each toxin and the no-toxin control, using graphpad prism.
RESULTS
initial analysis
the cry6aa protein is active in the gut of nematodes such as c. elegans, which is an acidic environment with a ph ranging from ~ <dig>  to  <dig>   <cit>  or coleoptera such as diabrotica virgifera virgifera with ph ranging from  <dig>  to  <dig>   <cit> .
the cry6aa crystals produced in b. thuringiensis and purified by sucrose gradient centrifugation could be solubilized at ph  <dig> ; however, cry6aa derived from this source showed a tendency to form aggregates and to precipitate after solubilization at either ph  <dig>  or  <dig> , particularly if this solubilization was followed by neutralization.
as a result, further analyses utilized recombinant cry6aa produced in p. fluorescens.
the secondary structure content of the full-length cry6aa protein from this source was estimated through cd analysis.
at ph  <dig> , results indicated an alpha helical content of ~73–74 %  with a beta sheet content of ~2– <dig>  %.
at ph  <dig> , helical content of ~70–72 % and beta sheet of ~1–2 % was indicated.
results at ph  <dig>  were impacted by the absorbance of the caps buffer below 200 nm and, thus, are less reliable but suggest that the protein remains predominantly alpha helical at this ph.
overall, and considering the inherent margins of error in cd determinations, these results suggest that the protein has a stable secondary structure across a broad ph range of  <dig>  to  <dig> .
the structure of the trypsin-resistant core
the structure of the trypsin-resistant core  exhibits a group of long alpha helices  that are amphipathic in nature, with their hydrophobic sides interacting and their hydrophilic faces showing greater exposure to solvent.
this helix bundle is around 20–25 Å in diameter with a head domain folded across the helices at one end, formed from residues close to the c-terminus of the protein.
the cry6aa trypsin-resistant core structure is unlike any previously described cry protein although it is similar to several known toxins, including hlye and hblb .fig.
<dig> crystal structure of cry6a toxin.
ribbon representation of trypsin-truncated cry6aa form showing two domain architecture: the “tail” domain consists of one helical bundle with five long α-helices, labeled αa, αc, αd, αg, and αh, and shorter helices, labeled αb, αe, αf, and αi; while several long and short loops form the “head” domain.
n- and c-termini and the putative transmembrane region are labeled.
the cys88-cys <dig> disulfide bond is shown and, in the insert box, the final 2fo-fc electron density map calculated at  <dig> σ in the region of this bond is shown in blue mesh.
side and main chains of the amino acid residues are presented as sticks and colored by the atoms
analysis of the trypsin-resistant core by sds-page following reduction and edman degradation of the major band showed the n-terminal sequence hslih, corresponding to residues 12– <dig> of the cry6aa sequence and indicating processing after arg <dig>  analysis by mass spectrometry of the trypsin-processed toxin, purified by ion exchange chromatography, without reduction, showed a dominant species with a mass of  <dig> .19 da and a minor species of  <dig> .25 da.
the deconvoluted intact average molecular weight  value of  <dig> .19 da matches the theoretical value  expected for a core protein containing polypeptide chain 12– <dig> disulfide bonded to chain 444– <dig> with a mass error of + <dig>  ppm.
the minor species with mwavg of  <dig> .
<dig> matches the theoretical value of  <dig> .
<dig> for a core protein containing polypeptide chain 12– <dig> disulfide bonded to chain 451– <dig> with a mass error of + <dig>  ppm.
the mass errors for both of these measurements are within the expected mass accuracy of the lc-tof instrument.
the two ctps, m/z  <dig>  and  <dig>  da, were sequenced by maldi ms/ms.
this confirmed that the  <dig>  peak had the sequence nsnleykcpennfmiywynnsdwynnsdwynn , which matched the ctp <dig> sequence of residues 444– <dig>  the  <dig>  peak had the sequence cpennfmiywynnsdwynnsdwynn, which matched the ctp <dig> sequence from residues 451– <dig>  these results show trypsin cleavage after arg <dig> and processing sites after arg <dig> and, at a lower frequency, after lys <dig>
the solved structure of the trypsin-activated cry6aa corresponds to the major core of the toxin with the longer ctp <dig> fragment  still linked to the main body of the toxin by a disulfide bond.
this bond can be clearly resolved in the structure .
however, several residues were not resolved in the activated toxin structure: ctp <dig> residues asn <dig> and tyr473-asn475; core region residues lys126-lys <dig> and asn388-ser389-arg <dig>  indicating that these regions are not fixed in the structure.
the structure of full-length cry6aa
the full-length structure of cry6aa  is slightly less structured than that of the trypsin-resistant core and was solved to a lower resolution .
in the ordered regions where electron density can be traced, the structures are almost identical with a root mean square deviation  for c-alpha atoms  of  <dig>  Å .fig.
<dig> comparison of cry6aa structures.
a superimposed ribbon representations of the crystal structures of the truncated  and the full-length  cry6aa forms.
b the full-length cry6aa model is shown with the following features illustrated: wing-like intra-helical loops ; putative transmembrane region  with l <dig> shown in stick representation; watigaxi repeat sequence ; ttnmtsnqy repeat sequence ; wynnsdwynnsdw repeat ; and the modeled asn388–lys <dig> in dark gray and cyan
mass spectrometric analysis of the full-length cry6aa used in crystallization revealed a deconvoluted molecular mass of  <dig> .1 da and, following reduction with dithiothreitol , a mass of  <dig> .8 da, corresponding to single full-length protein chain from met <dig> to asn <dig> and containing two disulfide bonds, as revealed by the mass difference of  <dig> between reduced and non-reduced states.
the n-terminal  <dig> amino acids, the c-terminal six residues, lys126–lys <dig>  and the region from asn <dig> to lys <dig> were not resolved, indicating significant flexibility in these regions.
the latter region contains the trypsin cut sites described above and the flexibility of this region may contribute to its accessibility to this enzyme.
the sequence from cys <dig> to asn <dig> is clearly seen in the structure and appears to be held in place by a disulfide bridge between cys <dig> and cys <dig> .
to understand the activity of cry6aa from a structural point of view, and because some regions were unresolved in the structure, modeling was carried out to determine the conformation of internal missing segments and to build a full-length structure model.
the loop modeler in moe  was used to find and select loop candidates using the pdb engine and to build model loops based on the desired loop sequence.
after selecting a set of backbone loop candidates from the pdb loop library, optimization proceeded, including addition of side chains and a multi-stage energy minimization with pre-defined refinement protocols.
the two regions lys126–lys <dig> and asn388–lys <dig> were modeled manually using coot as described above to produce a structure-based model of residues 12– <dig>  ser <dig> and tyr <dig> in the crystal structure were used as anchor residues and the region from residue  <dig> to  <dig> was used as the desired sequence to determine loop length and geometry restrictions.
the loop searching calculation resulted in several model loops.
the best model loop was chosen, based on its positioning of cys <dig> and cys <dig> in close proximity  and placing cys <dig> close to cys <dig>  considering that the mass spectroscopy study indicated another disulfide bond.
the two pairs of cys residues were manually connected, followed by an energy minimization with mmff94x force field.
the resulting cry6aa model, from residue  <dig> to  <dig>  has an alpha helical content of ~70 % and a beta sheet content of ~ <dig>  %, consistent with the cd data for this protein.
during the initial sequence analysis performed as part of this study, it was noted that the cry6aa sequence contains several internally repeated sequences that have not previously been noted.
these can be visualized in the full-length model .
the c-terminal domain contains two of these repeats: the sequence ttnmtsnqy  and the repetitive sequence wynnsdwynnsdwynn forms the extreme c-terminus of the protein .
the other repeat sequence, watigaxi, occurs at residues 44– <dig> then at 332– <dig> and contributes to different alpha helices within the active core of the toxin .
disulfide bonding and conformational rigidity
the cry6aa sequence contains five cys residues at positions  <dig> and  <dig> in the long alpha helical region, and at positions  <dig>   <dig>  and  <dig> in the c-terminal region.
the structures of the cry6aa trypsin-activated form and full-length cry6aa model show that cys <dig> forms a disulfide bond with cys <dig>  while cys <dig> appears to be joined to cys <dig>  cys <dig> is not disulfide bonded in either full-length or activated cry6aa structures and it appears to face into the core of the alpha helical bundle where it is not surface exposed.
the buried nature of this residue was confirmed by mass spectrometric analysis.
treatment of wild-type cry6aa under non-denaturing conditions with iodoacetamide resulted in no shift in deconvoluted intact average molecular weight, indicating that it is not accessible to modification.
when the same protein was pre-treated with dtt followed by iodoacetamide, a mass change consistent with reduction of two disulfide bonds and alkylation of four cysteine residues was observed, suggesting that cys <dig> remains inaccessible despite the overall increased structural flexibility caused by reduction of disulfide bonds.
the conformation of the cry6aa protein was also probed by mass spectrometry.
ionization of intact proteins under electrospray mass spectrometry conditions resulted in the detection of multiply charged species with characteristic csds unique for each protein.
the distribution of charges  on a protein under the denaturing conditions used for lc-ms is influenced by the primary sequence and the conformational flexibility of the protein, which determines the available protonation sites exposed on the denatured protein surface.
this characteristic behavior of proteins is routinely used as an indirect probe of the flexibility of protein tertiary structure  <cit> .
the role of the disulfide bonds in the overall rigidity of cry6aa was probed by measuring the csd under different conditions .
comparison of the csd for cry6aa under non-reducing conditions and after treatment with 1 mm dtt shows a profound shift in the csd , even though the deconvoluted intact average molecular weight only increased by + <dig>  under non-reducing conditions, the csd is centered around m/z  <dig> , corresponding to a + <dig> charge state.
after reduction, the csd shifts to a center around m/z  <dig> , corresponding to a + <dig> charge state.
this shift towards more highly charged species is a reflection of the decreased rigidity of the overall protein tertiary structure resulting from the reduction of the disulfides with dtt, exposing additional protein surfaces to protonation during ionization.fig.
<dig> charge state distributions  of cry6aa.
a under non-reducing conditions the csd centers around m/z  <dig> .
b following treatment with 1 mm dithiothreitol , the csd centers around m/z  <dig>
the pairing of cysteine residues and the buried nature of cys <dig> described above indicate that natural cry6aa crystals are not stabilized by intermolecular disulfide bonds.
this contrasts with many three-domain cry protoxins in which disulfide bonds between the c-terminal proparts of individual protoxin molecules are important in crystal formation  <cit> .
however, while cry6aa crystal stability does not appear to be mediated by covalent intermolecular bonding, we can speculate that the rigidity conferred on the cry6aa monomers by the intramolecular disulfide bridges could be relevant to crystal packing.
similarity to other toxins
it is interesting to note that in the related cry6ba sequence, the c-terminal domain found in cry6aa  is absent.
as a result, the cry6ba sequence will lack both the c402–c <dig> and the c88–c <dig> disulfide bonds.
there is no cys residue at the position equivalent to cys <dig> in a primary sequence alignment of cry6aa and cry6ba, where this residue is asn <dig> .
cry6ba does, however, contain two additional cysteine residues at positions  <dig> and  <dig> in helix αc but these residues are too far apart to form an intramolecular disulfide bond.
cry6ba also lacks two of the repetitive features described above  and the remaining repeat feature in cry6aa  is changed in both occurrences in cry6ba and becomes non-repetitive .
cry6ba has coleopteran activity but little or no nematode toxicity  <cit> .
this indicates that this region is not necessary for toxicity per se.
in the case of cry6aa, the c-terminal region is also not essential for toxicity since deletion of the c-terminal  <dig> residues and the first  <dig> residues from the n-terminus produces proteins that retain some, albeit reduced, toxicity against c. elegans  <cit> .
the “wing” features seen in cry6aa  are regions that vary in length when compared to the equivalent regions in cry6ba .
since in cry6aa these features form interruptions to the αc and αd helices, we predict that these variations will be well tolerated in cry6ba.
the structures derived for cry6aa are striking in their structural similarity to previously described bacterial toxins.
a dali search  <cit>  conducted with the truncated cry6aa crystal structure identified the closest structural homolog as the hblb component of the bacillus cereus hemolytic toxin  <cit>   with a significant z-score value of  <dig>  %, rmsd between  <dig> c∝-atoms of  <dig>  Å and sequence identity of 12 % .
the structural similarities to other toxins – such as the nhea component of the b. cereus non-hemolytic toxin  <cit>   and hemolysin e from e. coli  <cit>   – are also remarkable, with z-scores of 21 % and 11 %, and rmsd values of  <dig>  Å and  <dig>  Å, despite low sequence identity of 12 % and 10 %, respectively .
these are pore-forming toxins, which, like cry6aa, exhibit an elongated structure composed of an alpha helical bundle with a head domain at one end.
cry6aa also shows some similarity to smc_prok_a family proteins that are also known to form dimers  <cit> ; in the related hlye crystal structure, two monomers can be seen to pack together in a head-to-tail orientation  <cit> .
dimeric forms of cry6aa were observed in this study and have been described previously  <cit>  but the packing of the monomers is not known.
despite this, there is no evidence for dimer formation in either crystal structure.fig.
<dig> comparison of cry6aa structure with structures of other related toxins.
superposition of cry6aa  with a hbl-b , b nhea , and c hlye .
in panel d, the surface representation of hydrophobic areas of the truncated cry6aa in ribbon representation are colored green and the remaining residues are colored gray.
the putative transmembrane region is labeled .
c-terminal 463– <dig> residues are removed for simplicity
it is interesting to note that in cry6aa, two of the long alpha helices are interrupted with surface loops  bounded by gly residues :; the first of these loops is also seen in the ab initio models although the second was not predicted.
these small, wing-like intra-helical loops are a unique feature of cry6aa not seen in the related structures of hlye, hblb, or nhea and their significance is unknown at present.
they do not appear to significantly interact with the rest of the molecule and residues lys <dig> and lys <dig> are not visible in the crystal structures, suggesting that they are mobile.
despite their apparent surface accessibility and the two lysine residues found near the tips of both of these loops, trypsin does not appear to cleave at these sites .
the hlye mode of action model of mueller et al.
<cit>  and the recent work of benke et al.
<cit>  propose that upon interaction with the membrane, the putative pore-forming tongue region unfolds, causing the alpha helices to extend.
it is possible that a similar rearrangement may occur for cry6aa, and that the g121dpsikkdg <dig> and g189dqkkleg <dig> loops reconfigure into alpha helical segments.
this hypothesis is consistent with the observation that each loop is seven amino acids in length  and is bounded by glycine residues, which, due to the hydrogen r group, allow free rotation around the peptide bond, allowing a low energy secondary structure transition.
the head domain of cry6aa is folded across the helices as seen in the hblb  <cit>  and nhea  <cit>  structures .
within the head domains of these proteins is a region termed the beta tongue that may contain the transmembrane sequence that allows them to form pores in target cells  <cit> .
in hlye, this putative membrane-spanning in the beta tongue  <cit>  is proposed to insert into the membrane along with the flanking helix e  <cit>  to produce an oligomeric pore from either eight  <cit>  or  <dig>  <cit>  monomers.
a similar hairpin loop feature, well conserved in cry6ba   was identified both in the ab initio models and the structures of cry6aa ; analysis using the tmpred software  <cit>  predicts this to be a candidate transmembrane sequence.
probing the cry6aa mechanism of action
prior to the completion of crystallographic analyses and the derivation of cry6aa structures, we initiated structural modeling of the cry6aa protein.
because the structural databases contained no proteins with primary sequence homology with cry <dig>  more detailed structural predictions based on known protein structures were impossible.
we decided, therefore, to employ the rosetta modeling program to predict the possible fold of the cry <dig> protein ab initio, producing five de novo models with no reference to previous structures and five models using the database structure comparison method.
all models  exhibited a high proportion of alpha helix  and very low proportions of beta sheet .
the modeling using the database structure comparison method returned a range of distinct structural models.
in contrast, the five de novo models showed a high degree of similarity to each other, differing mainly in the disposition of the n- and c-terminal domains with respect to the central domain of the long alpha helices.
these models predicted a predominantly alpha helical structure that is highly similar [the structural alignment of the backbone residues in the central region of the protein  to the actual structures of the protein that were subsequently solved through crystallography .
based on the model, we were able to form a hypothesis that cry6aa is a pore-forming toxin and identify a region that might be involved in this function as a target for mutagenesis .
nematode assays as a test for pore-forming activity of cry6aa
given that our analyses showed structural homology to toxins such as hlye and, by analogy, suggested a similar mechanism of action, we decided to test this hypothesis.
in their work on hlye, wallace et al.
<cit>  investigated the importance of the tongue region by substituting hydrophobic residues in this part of the sequence with non-hydrophobic amino acids.
they demonstrated the loss of hemolytic activity.
in a parallel experiment, to disrupt the hydrophobicity of the putative transmembrane region of cry6aa, we replaced leu <dig> with asp.
the leu <dig> residue was chosen because it is a hydrophobic residue at the center of the putative membrane-spanning domain .
the l259d-mutant protein was modeled and the change was not predicted to cause any alteration in the overall fold of the protein .
the wild-type cry6aa and the l259d mutant, both expressed in e. coli, were used in the bioassays against c. elegans.
the expression of wild-type and mutant cry6aa in e. coli on iptg induction was verified by western blotting  and cells expressing these proteins were tested for their activity against c. elegans.
bioassays showed a striking  and significant  reduction in nematode growth following exposure to the wild-type cry6aa protein compared to those feeding on cells transformed with pet28b vector alone .
in contrast, the l259d mutant revealed no reduction in nematode size compared to the controls , indicating that this targeted mutation had eliminated cry6aa bioactivity against c. elegans.
in combination with the structural data, this result is consistent with cry <dig> acting as a pore-forming toxin, with structures and mechanisms of action similar to that of the hblb and hlye toxins that could cause membrane damage through the insertion of residues into the head region of the protein, as postulated from the de novo models.fig.
<dig> nematode bioassay: c. elegans were fed on e. coli transformed with the pet28b plasmid as a negative control ; transformed with this vector containing the wild-type cry6aa gene ; or transformed with the vector with the l259d mutant of this gene .
a images of some nematodes chosen at random are shown and b results are presented as mean worm area .
p values comparing each condition to vector control are shown.
error bars represent the standard errors of the means
to test the ability of cry6aa to form pores more directly, experiments were conducted in vivo using c. elegans fourth-stage hermaphrodites and a live animal assay for the presence of pores in the intestine  <cit> .
following exposure of the hermaphrodites to e. coli either carrying empty pqe <dig> vector or expressing cry6aa or cry5ba, the penetration of ingested propidium iodide  by intestinal cells was monitored.
under conditions used, pi gets into intestinal cells exposed to active pore-forming toxins  <cit> .
as expected, nematodes fed with empty vector e. coli  showed confinement of the pi to the lumen of the gut, whereas in cry6aa- and cry5ba -treated hermaphrodites, entry of pi into the intestinal epithelial cell cytoplasm was evident .
in these experiments the percentage of hermaphrodites showing pi entry was quantitated: for empty vector  <dig>  ±  <dig>  %, for cry5b exposure 92 ±  <dig>  %, and for cry6aa exposure  <dig>  ±  <dig>  %.
the stronger effects of cry5b on pi entry into intestinal cells could be due to factors such as the absolute toxicity of each protein, the relative numbers of receptors, or the relative quantities of the proteins expressed in the cells.
nonetheless, cry6aa showed significantly higher  pore formation than control .
therefore, based on all our data, we conclude that cry6aa is an active pore-forming toxin.fig.
<dig> in vivo pore formation.
a bright field images  and fluorescence images  of the anterior regions of c. elegans fed on e. coli transformed with pqe <dig> vector or plasmids expressing cry6aa or cry5ba.
b percentage of treated worms showing propidium iodide  uptake.
the p values for the comparison between each toxin and the no-toxin control are shown
CONCLUSIONS
the elucidated crystal structures for cry6aa support a mechanism of action by pore formation, similar to toxins such as hblb, and we have demonstrated pore-forming ability in vivo.
the hblb protein and the non-hemolytic toxin components nheb and nhec  are derived from b. cereus strains and induce pore formation and osmotic lysis of target cells  <cit> , leading to the induction of diarrhea by this organism.
b. thuringiensis is a member of the b. cereus sensu lato group of bacilli and, aside from production of invertebrate-active toxins by b. thuringiensis, the two species are indistinguishable by most tests  <cit> .
interestingly, despite the low amino acid sequence identity, cry6aa shares a fold and, we propose, a mechanism of action with the b. cereus toxins and this may indicate an adaptation of this hblb protein family within the b. cereus sensu lato group for activity against target cells in invertebrates.
the known structures of delta-endotoxins from b. thuringiensis  are structurally distinct from the cry <dig> fold , which represents a new structural family among insecticidal toxins and a new paradigm for invertebrate pathogenic toxins.
based on the present study, we can now further elucidate key features of the cry6aa toxin that are responsible for its specificity and mode of action.fig.
<dig> structural families of b. thuringiensis delta-endotoxins.
representatives of different structural classes of delta-endotoxins of b. thuringiensis are shown.
cry1aa   <cit>  is a three-domain toxin; cry6aa is an alpha helical toxin ; cry <dig> is an aegerolysin-like protein   <cit>  that acts as a binary toxin with cry <dig>  a toxin_ <dig> family protein   <cit> ; cry <dig> is a member of the etx/mtx <dig> family   <cit> ; and cyt1aa, a member of the bac_thur_toxin family   <cit> .
all structures are to scale and colored according to secondary structure
additional files
additional file 1: figure s <dig>  multiple sequence alignment of cry6aa and related toxins.
sequences of full-length cry6aa and cry6ba were aligned using the clustalx program along with the sequences from the crystal structures of hlye , hblb , and nhea .
residues identical to the cry6aa sequence are highlighted in green; residues identical to cry6ba but not in cry6aa are highlighted in blue.
repeat regions in cry6aa are highlighted in yellow .
the putative transmembrane region is highlighted in gray with l <dig> indicated by a blue asterisk.
the “wing” features in the cry6aa sequence are indicated by magenta highlight.
additional file 2: figure s <dig>  ab initio models of cry6aa.
the five models constructed with reference to database information  and five models constructed entirely de novo  are shown.
the predicted n-terminal domains are shown in light gray and marked “n,” while the c-terminal domains are shown in black and marked “c.”
additional file 3: figure s <dig>  modeled l259d mutant.
the full-length structure  is overlaid with the l259d model .
the inset illustrates the putative transmembrane loop region with residue  <dig> shown in stick display.
loops are colored as above for clarity while, for other regions, the full-length structure is dark gray and the modeled mutant is light gray.
additional file 4: figure s <dig>  western blot of wild-type and l259d mutant cry6aa.
expression of the wild-type cry6aa  and the l259d mutant confirmed by western blotting.

 bacillus thuringiensis strains produce a range of toxins active against invertebrates with enormous potential for use in the control of pests of importance in agriculture and health. during sporulation, strains synthesize cytolytic toxins and/or crystal protoxins and deposit them as parasporal inclusion bodies. of the major subclasses of cry protoxin currently recognized, most belong to a large family of related sequences that can be proteolytically processed to yield active toxins with a three-domain fold as first seen for cry3aa. however, the cry nomenclature is not limited to these proteins and includes several distinct and unrelated lineages. while much is known of the structure and function of the three-domain toxins and the structure of a protoxin form has recently been published, very little is known of the non-three-domain cry proteins. some of these proteins share sequence homology to other known toxins; for example, cry and cry are related to the bin and cry toxins of lysinibacillus sphaericus and may share a proposed mechanism of action involving pore formation and/or apoptosis. the structures of the ß-sheet-rich toxins cry, cry, and recently cry and the cry34/cry binary toxin have been published but for many non-three-domain cry proteins, neither structural data nor information on mechanism of action are available. one such cry protein is cry6aa, a protein with activity against coleoptera such as the western corn rootworm diabrotica virgifera virgifera and a range of nematodes, including both free-living and plant pathogenic species that cause large-scale losses to agriculture. previous studies have shown by deletion of cry6aa sequences that amino acids can be deleted from the n-terminus and residues can be removed from the c-terminus, leaving a core region of ~43 kda that retains activity against nematode targets. cry6b is a protein that is closely related to cry6a but lacks amino acids that are seen at the c-terminus of the latter protein. cry6b is reported to be active against the coleopteran lucerne weevil hypera postica but showed little or no activity against a range of nematode targets. the absence of further data on the structure and function of these proteins has limited our ability to understand their activity against target invertebrates. as a result, development and exploitation of the toxins in the control of agricultural pest insects and nematodes pathogenic to plants and animals may be limited. this, in turn, inhibits their use to supplement the current chemotherapeutic approaches to nematicidal treatments that are very toxic and are being phased out. in this study we applied both crystallographic techniques and state of the art ab initio modeling to probe the structure of cry6aa in protoxin and trypsin-cleaved forms. the structures obtained are novel among invertebrate-active toxins and are consistent with cry6aa acting as a pore-forming toxin. we demonstrated pore formation in vivo and used our predictions to construct a mutant expected to show compromised toxicity. methods bacterial strains b. thuringiensis strain bmb containing the pht304-derived plasmid encoding the cry6aa protein was kindly provided by dr ming sun, huazhong agricultural university, wuhan, china. this strain was grown to greater than 98 % autolysis in embrapa medium. spores were harvested and crystals of cry6aa were isolated and purified by discontinuous sucrose density gradient ultracentrifugation according to previously described methods. cry6aa protein crystals purified by this method were solubilized at 37 °c with shaking at either alkaline ph by incubation in 50 mm naoh or at acidic ph by the addition of 100 mm sodium citrate buffer ph . toxin production in escherichia coli the cry6aa gene was amplified from the above b. thuringiensis strain by 30 cycles of pcr with a 60 °c annealing temperature, using forward and reverse primers, incorporating ecori restriction sites. the amplicon was cloned into the pgem-t vector and transformed into e. coli dh5α for the selection of a single colony, plasmid isolation, and sequencing to confirm the cloning of the gene with no mutations. the gene was subsequently cleaved from this plasmid with ecori and re-cloned into the ecori site of the pet28b expression vector to allow production of recombinant cry6aa protein with an n-terminal his-tag. a mutation of the cry6aa gene to change leu to asp was introduced using the phusion site-directed mutagenesis kit according to the manufacturer’s instructions by pcr using the pet28b clone as template and the following 5′-phosphorylated oligonucleotides: 5′-gacttaggatttgttgtttatgaaattc-3′, 5′-tggtcctaatagaaaactatattc-3′. following ligation of the pcr product and transformation of e. coli dh5α, a colony was picked and the plasmid sequence was verified to ensure incorporation of the desired mutation and no other changes. for recombinant production of cry6aa proteins in e. coli, the pet28b plasmids were transformed into the shuffle t express lysy e. coli strain and isolated colonies were selected. transformants were grown in lysogeny broth medium in the presence of 50 μg/ml kanamycin at 37 °c with shaking overnight before the addition of isopropyl ß-d-thiogalactopyranoside to a final concentration of 1 mm and growth for a further 3 h. expression was verified by western blotting using monoclonal anti-polyhistidine antibody as the primary antibody and rabbit anti-mouse alkaline phosphatase conjugate antibody as the secondary antibody to allow visualization of the proteins using the ap conjugate substrate kit. for in vivo pore formation assays, a distinct clone of the cry6aa gene in the bamhi and psti sites of the vector pqe was produced. an equivalent clone in pqe containing the cry5ba gene as a positive control for in vivo pore formation has been described previously. cry production in pseudomonas fluorescens for inclusion body preparation, pseudomonas fluorescens-derived cell paste expressing full-length, wild-type cry6aa was transferred from −80 °c storage to 4 °c and resuspended at 20 % w/v in cold lysis buffer and mixed thoroughly with a handheld homogenizer. the suspension was then passed through a microfluidizer twice at  psi then centrifuged. the supernatant was discarded. the ib pellet was fully resuspended by homogenization in 10 % w/v room temperature lysis buffer with  g/l lysozyme. the suspension was incubated at 30 °c for 30 min, with brief homogenization every 10 min. the ibs were then centrifuged and the supernatant was discarded. the pellet was resuspended for a final time in 10 % w/v cold lysis buffer using the homogenizer and then centrifuged and the supernatant discarded. the ib pellet was resuspended in 10 % w/v cold lysis buffer without triton x- using the homogenizer and then centrifuged and the supernatant discarded. this step was repeated twice. the ibs were resuspended to 30 % in 10 mm edta, ph , and then separated into  ml aliquots and frozen at −80 °c until needed. a separate aliquot of the ib preparation was stored without suspension in 10 mm edta. purification of full-length cry6aa about 500 mg of pseudomonas-derived ib paste was thawed at 4 °c, and centrifuged at  g for 25 min at 4 °c. the supernatant was removed and the pellet was solubilized in 30 ml of 100 mm caps, ph , and the suspension was gently rocked for 2 h at room temperature to solubilize the cry6aa. after solubilization, the mix was centrifuged at  g for 25 min at 4 °c. the supernatant was dialyzed against 25 mm caps, ph , overnight. the buffer exchanged cry6aa sample was then filtered through a  μm syringe filter and applied to a source 15q 16/ ion exchange column at a flow rate of 5 ml/min. the column was pre-equilibrated with buffer a. the protein was eluted with a step gradient of 20 %, 30 %, 40 %, and 50 % buffer b. each elution was ~50 ml. fractions were collected from each elution based on uv absorbance and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. full-length cry6aa was in the 20 % b fractions, which were pooled and concentrated to ~30 ml using a centrifugal filter device with 10 kda molecular weight cut-off membrane. the concentrated sample was further purified by size exclusion chromatography. for each run,  ml of the sample was applied over a superdex 26/ gel filtration column pre-equilibrated in 25 mm caps, ph  and 50 mm nacl buffer, at a flow rate of  ml/min. two peaks were observed. the first peak, eluted at the void volume, contained cry6aa dimers. the fractions from peak contained predominantly monomer and were pooled and used in crystallization experiments. purification of cry6aa trypsin-resistant core eight 2 ml tubes of ib suspension of full-length cry6aa were thawed and extracted in 80 ml final volume of 10 mm caps, ph . the measured ph of was then adjusted to with naoh. freshly prepared trypsin [2 ml of 25 mg/ml trypsin solution was added, the ph was maintained at, and the mixture was stirred overnight at 4 °c. the digestion was applied to a source 15q 16/ column pre-equilibrated in buffer c at a flow rate of 10 ml/min and eluted with a gradient of buffer c + 1 m nacl over 75 min. trypsin-processed cry6aa eluted as a single large peak, which was concentrated from 100 ml to 10 ml using 15 ml amicon mwco spin concentrators in a ja- rotor at 4 °c and 5000 g. the concentrated ion exchange sample was applied to a superdex 26/ gel filtration column pre-equilibrated in 25 mm caps, ph , and 50 mm nacl. the sample was eluted using a  ml/minute flow rate. the fractions corresponding to the large cry6aa core peak were pooled for crystallization trials. secondary structural analysis circular dichroism spectra were recorded using a jasco j- cd spectrometer equipped with an mptc-490s 6-cell peltier thermostatted cell holder. the instrument was calibrated with --10-camphorsulfonic acid. measurements were carried out in a 1 mm path-length quartz cuvette under a constant nitrogen stream and at room temperature. data were collected in continuous scan mode from to 185 nm every  nm at 50 nm/min and with 2 s averaging per point. each spectrum is the average of five accumulations and a bandwidth of 1 nm. the concentration of cry6aa protein in 10 mm phosphate ph   was mg/ml, in 10 mm phosphate ph   was mg/ml, and in 10 mm caps ph  was  mg/ml. all spectra were corrected for the buffer contributions and the raw data output in ellipticity, measured in millidegrees was converted to mean residue ellipticity in deg cm2/dmol using the instrument software, spectrum manager the cdpro package was used for secondary structure estimation using the programs selcon, continll, and cdsstr. the programs gave results that were similar to each other and relatively independent of the reference protein set used. the secondary structure calculations reported here were from processed cd spectra using the cdsstr algorithm and the sp37a reference set. crystallization, data collection, and structure determination cry6aa trypsin-resistant core the cry6aa trypsin-resistant core was concentrated to 100 mg/ml. initial crystals were obtained using rigaku reagents, inc. wizard classic i. after screening multiple conditions, suitable crystals were obtained under kit condition. data were collected at 100 k from a single crystal on a mar ccd- detector at ls-cat. data were indexed and processed with the hkl- software suite. the structure of this truncated form of cry6aa toxin was solved by the molecular replacement method using phaser followed by manual rebuilding and model refinement. the poly-alanine chain of the crystal structure of hemolysin b from bacillus cereus consisting of residues 19– was used as a search model. the final structure of the complex was obtained by carrying out several cycles of refinement consisting of manual model building using coot, followed by restrained refinement with refmac using translation, libration and screw-rotation refinement and the reference and secondary structure restraints. the trypsin-cleaved cry6aa structure, solved at higher resolution, was used as the reference structure in this refinement. crystallographic data and refinement statistics are given in table  and the cry6a trypsin-resistant core structure can be found at the protein data bank.table data collection and refinement statistics    number of non-hydrogen atomsd pdb protein data bank, rms root mean square deviation from ideal values astatistics for the highest-resolution shell are shown in parentheses brmerge = 100ΣΣ|i-<i>|/ ΣΣi where i is the ith intensity measurement of reflection h, and <i> is the average intensity from multiple observations crfactor = Σ||f obs|-|f calc||/ Σ|f obs|. where f obs and f calc are the structure factor amplitudes from the data and the model, respectively. to calculate r-free values, 5 % and 10 % reflections were used for cry6aa trypsin core and full-length cry6aa structures, respectively. dper asymmetric unit purification of full-length cry6aa full-length cry6aa protein was concentrated to 15 mg/ml using amicon centrifugal filters with a 10 kda molecular weight cut-off in 10 mm hepes buffer, ph  and 25 mm nacl. initial crystallization screens were performed using commercially available classics, classics lite, classics ii, pegs, pegs ii, phclear, and pact screens by the sitting drop method in 96-round-bottom-well crystallization plates using a mosquito robotic system. diffraction quality cry6aa protein crystals were grown at 291 k from sitting drops containing 3 μl of the protein sample and  μl of reservoir solution. sds-page analysis of protein samples obtained by dissolving the crystals in sds-buffer did not reveal any degradation products and confirmed the presence of only full-length cry6aa protein in the crystals used for the data collection. for data collection, crystals were harvested with 20 % glycerol in the reservoir solution. diffraction data were collected at 100 k from a single crystal on a mar ccd- detector at ls-cat. data were indexed and processed with hkl-. the crystals belonged to orthorhombic space group p and contained one molecule of full-length cry6aa per asymmetric unit. the structure of full-length cry6aa was solved by molecular replacement using the structure of the truncated form of cry6aa as a search model. the final structure of full-length cry6aa toxin was obtained by carrying out several cycles consisting of manual model building using coot, followed by structure refinement with refmac. crystallographic statistics are given in table  coordinates have been deposited in the protein data bank. molecular modeling the cry6aa tertiary structure was modeled ab initio using the rosetta software. after entering the cry6aa sequence, the first stage of the web-based process returned a secondary structure prediction with options to continue full protein structure prediction either using de novo or database structure comparison methods. both methods were followed to produce a selection of five possible structure outcomes from both de novo and database predictions. intact molecular weight analysis/charged state distribution intact mass analysis was performed on an agilent hplc/msd tof 1969a system using a michrom desalting trap heated to 50 °c. each sample was diluted to a concentration of  μg/μl in 10 mm caps ph  buffer. samples were also analyzed after reduction with 50 mm tcep for 10 min. approximately 1 μg protein was injected on column. the sample was eluted using a gradient, where buffer a is  % formic acid in water and buffer d is 70 % isopropanol, 20 % acetonitrile, 10 % water, and  % formic acid. the mass was calculated using the mass hunter qualitative analysis software and the maximum entropy de-convolution algorithm. changes in the observed charge state distribution of proteins analyzed by liquid chromatography–mass spectrometry, as described above, were used as an indirect probe of the overall conformational flexibility of the protein under different conditions. maldi-tof mass spectrometry and tandem mass spectrometry digestion of cry6aa with trypsin results in two fragment fractions, a large fragment and a small fraction containing two peptides. the small fragment sample was analyzed by matrix-assisted laser desorption/ionization - time of flight mass spectrometry to determine the peptide masses and was subsequently fragmented in the lift mode to confirm the sequence of the peptides. the maldi-tof ms and tandem mass spectrometry were conducted with a bruker ultraflextreme mass spectrometer. the c-terminal peptide sample was diluted with  % trifluoroacetic acid at 1: and desalted using a c- ziptip. the peptides were eluted with 60 % acetonitrile in  % tfa and mixed with a -dihydroxybenzoic acid matrix. after spotting 1 μl of the mix on a maldi sample plate, the peptide mass was analyzed using reflection-positive mode and the peptide was fragmented using the lift mode. the instrument was calibrated with cm. the mass spectrum was collected and analyzed using flex analysis software. the sequence was verified using biotools software and the mascot search engine. n-terminal sequencing amino terminal sequencing of proteins was performed on a shimadzu protein sequencer using basic edman degradation chemistry. the protein sample was separated with sds-page under reducing conditions, and then the proteins were blotted onto polyvinylidene difluoride membrane by liquid transfer. a standard mix of phenylthiohydantoin-amino acids was run each time. the amino acid residues from each edman degradation cycle were determined based on their retention times from the c- column compared to standards. nematode bioassay c. elegans bristol n strain wild-type nematodes were cultured using standard techniques including the use of e. coli strain op as standard food source on enriched nematode growth plates containing 100 μm iptg and 50 μg/ml kanamycin. to measure intoxication by cry6aa, 30– fourth-staged larvae of c. elegans were seeded on eng-ik plates with e. coli shuffle t express lysy cells transformed with the pet28b empty vector or pet28b with cry6aa inserts and incubated at 25 °c for 24 h. three replica plates were used for each strain. three to five worms were randomly picked from each of the different treatment conditions for image collection. worm growth was assessed by measuring the area of clearly separated, individual worms, using image j and matlab software. the experiment was independently repeated three times. data were analyzed with jmp v. using one-way anova and dunnett’s post-test using the vector as the control group. in vivo pore formation assay pore formation was assessed microscopically using the method described by los et al.. c. elegans bristol n strain nematodes were synchronized by hypochlorite treatment. l animals were seeded onto op and grown at 20 °c until the l stage was reached. e. coli jm cultures transformed with the empty pqe vector or vectors for expression of cry5ba or cry6aa were grown overnight at 37 °c with shaking in lb medium containing ampicillin. these cultures were diluted 1: in fresh medium and grown at 37 °c with shaking for 1 h before addition of iptg to 50 μm and further incubation at 30 °c for at least 3 h with shaking. after this induction of protein expression, 30 μl of these cells, concentrated to an optical density of were spread onto 60 mm diameter eng agar plates containing ampicillin and iptg and allowed to grow overnight at 25 °c for use in assays. worms were then washed off the op plate, washed twice to remove bacteria, and pipetted onto agar plates spread with the transformed jm plates were incubated for 2 h at 20 °c. worms were then washed off the plates, washed twice in m medium to remove bacteria, and rotated end-over-end at room temperature in m medium containing 5 mg/ml 5-hydroxytryptamine for 15 min to stimulate feeding. propidium iodide was then added to the medium and worms were rotated for an additional 30–40 min. worms were washed twice to remove excess dye and pipetted onto agar pads on microscope slides and imaged. pictures were taken using a spot insight ccd camera and spot software connected to an olympus bx compound microscope with an uplanfl 40×/ na objective. at least worms were imaged for each condition, and the entire experiment was conducted three times. the percentages for all three experiments were combined and analyzed by one-way anova followed by dunnett’s multiple comparison test between each toxin and the no-toxin control, using graphpad prism.  initial analysis the cry6aa protein is active in the gut of nematodes such as c. elegans, which is an acidic environment with a ph ranging from ~ to or coleoptera such as diabrotica virgifera virgifera with ph ranging from to. the cry6aa crystals produced in b. thuringiensis and purified by sucrose gradient centrifugation could be solubilized at ph ; however, cry6aa derived from this source showed a tendency to form aggregates and to precipitate after solubilization at either ph  or, particularly if this solubilization was followed by neutralization. as a result, further analyses utilized recombinant cry6aa produced in p. fluorescens. the secondary structure content of the full-length cry6aa protein from this source was estimated through cd analysis. at ph , results indicated an alpha helical content of ~73–74 % with a beta sheet content of ~2–  %. at ph , helical content of ~70–72 % and beta sheet of ~1–2 % was indicated. results at ph  were impacted by the absorbance of the caps buffer below 200 nm and, thus, are less reliable but suggest that the protein remains predominantly alpha helical at this ph. overall, and considering the inherent margins of error in cd determinations, these results suggest that the protein has a stable secondary structure across a broad ph range of to. the structure of the trypsin-resistant core the structure of the trypsin-resistant core exhibits a group of long alpha helices that are amphipathic in nature, with their hydrophobic sides interacting and their hydrophilic faces showing greater exposure to solvent. this helix bundle is around 20–25 Å in diameter with a head domain folded across the helices at one end, formed from residues close to the c-terminus of the protein. the cry6aa trypsin-resistant core structure is unlike any previously described cry protein although it is similar to several known toxins, including hlye and hblb.fig. crystal structure of cry6a toxin. ribbon representation of trypsin-truncated cry6aa form showing two domain architecture: the “tail” domain consists of one helical bundle with five long α-helices, labeled αa, αc, αd, αg, and αh, and shorter helices, labeled αb, αe, αf, and αi; while several long and short loops form the “head” domain. n- and c-termini and the putative transmembrane region are labeled. the cys88-cys disulfide bond is shown and, in the insert box, the final 2fo-fc electron density map calculated at σ in the region of this bond is shown in blue mesh. side and main chains of the amino acid residues are presented as sticks and colored by the atoms analysis of the trypsin-resistant core by sds-page following reduction and edman degradation of the major band showed the n-terminal sequence hslih, corresponding to residues 12– of the cry6aa sequence and indicating processing after arg analysis by mass spectrometry of the trypsin-processed toxin, purified by ion exchange chromatography, without reduction, showed a dominant species with a mass of.19 da and a minor species of.25 da. the deconvoluted intact average molecular weight value of.19 da matches the theoretical value expected for a core protein containing polypeptide chain 12– disulfide bonded to chain 444– with a mass error of +  ppm. the minor species with mwavg of. matches the theoretical value of. for a core protein containing polypeptide chain 12– disulfide bonded to chain 451– with a mass error of +  ppm. the mass errors for both of these measurements are within the expected mass accuracy of the lc-tof instrument. the two ctps, m/z and  da, were sequenced by maldi ms/ms. this confirmed that the peak had the sequence nsnleykcpennfmiywynnsdwynnsdwynn, which matched the ctp sequence of residues 444– the peak had the sequence cpennfmiywynnsdwynnsdwynn, which matched the ctp sequence from residues 451– these results show trypsin cleavage after arg and processing sites after arg and, at a lower frequency, after lys the solved structure of the trypsin-activated cry6aa corresponds to the major core of the toxin with the longer ctp fragment still linked to the main body of the toxin by a disulfide bond. this bond can be clearly resolved in the structure. however, several residues were not resolved in the activated toxin structure: ctp residues asn and tyr473-asn475; core region residues lys126-lys and asn388-ser389-arg indicating that these regions are not fixed in the structure. the structure of full-length cry6aa the full-length structure of cry6aa is slightly less structured than that of the trypsin-resistant core and was solved to a lower resolution. in the ordered regions where electron density can be traced, the structures are almost identical with a root mean square deviation for c-alpha atoms of  Å.fig. comparison of cry6aa structures. a superimposed ribbon representations of the crystal structures of the truncated and the full-length cry6aa forms. b the full-length cry6aa model is shown with the following features illustrated: wing-like intra-helical loops; putative transmembrane region with l shown in stick representation; watigaxi repeat sequence; ttnmtsnqy repeat sequence; wynnsdwynnsdw repeat; and the modeled asn388–lys in dark gray and cyan mass spectrometric analysis of the full-length cry6aa used in crystallization revealed a deconvoluted molecular mass of.1 da and, following reduction with dithiothreitol, a mass of.8 da, corresponding to single full-length protein chain from met to asn and containing two disulfide bonds, as revealed by the mass difference of between reduced and non-reduced states. the n-terminal amino acids, the c-terminal six residues, lys126–lys and the region from asn to lys were not resolved, indicating significant flexibility in these regions. the latter region contains the trypsin cut sites described above and the flexibility of this region may contribute to its accessibility to this enzyme. the sequence from cys to asn is clearly seen in the structure and appears to be held in place by a disulfide bridge between cys and cys. to understand the activity of cry6aa from a structural point of view, and because some regions were unresolved in the structure, modeling was carried out to determine the conformation of internal missing segments and to build a full-length structure model. the loop modeler in moe was used to find and select loop candidates using the pdb engine and to build model loops based on the desired loop sequence. after selecting a set of backbone loop candidates from the pdb loop library, optimization proceeded, including addition of side chains and a multi-stage energy minimization with pre-defined refinement protocols. the two regions lys126–lys and asn388–lys were modeled manually using coot as described above to produce a structure-based model of residues 12– ser and tyr in the crystal structure were used as anchor residues and the region from residue to was used as the desired sequence to determine loop length and geometry restrictions. the loop searching calculation resulted in several model loops. the best model loop was chosen, based on its positioning of cys and cys in close proximity and placing cys close to cys considering that the mass spectroscopy study indicated another disulfide bond. the two pairs of cys residues were manually connected, followed by an energy minimization with mmff94x force field. the resulting cry6aa model, from residue to has an alpha helical content of ~70 % and a beta sheet content of ~  %, consistent with the cd data for this protein. during the initial sequence analysis performed as part of this study, it was noted that the cry6aa sequence contains several internally repeated sequences that have not previously been noted. these can be visualized in the full-length model. the c-terminal domain contains two of these repeats: the sequence ttnmtsnqy and the repetitive sequence wynnsdwynnsdwynn forms the extreme c-terminus of the protein. the other repeat sequence, watigaxi, occurs at residues 44– then at 332– and contributes to different alpha helices within the active core of the toxin. disulfide bonding and conformational rigidity the cry6aa sequence contains five cys residues at positions and in the long alpha helical region, and at positions and in the c-terminal region. the structures of the cry6aa trypsin-activated form and full-length cry6aa model show that cys forms a disulfide bond with cys while cys appears to be joined to cys cys is not disulfide bonded in either full-length or activated cry6aa structures and it appears to face into the core of the alpha helical bundle where it is not surface exposed. the buried nature of this residue was confirmed by mass spectrometric analysis. treatment of wild-type cry6aa under non-denaturing conditions with iodoacetamide resulted in no shift in deconvoluted intact average molecular weight, indicating that it is not accessible to modification. when the same protein was pre-treated with dtt followed by iodoacetamide, a mass change consistent with reduction of two disulfide bonds and alkylation of four cysteine residues was observed, suggesting that cys remains inaccessible despite the overall increased structural flexibility caused by reduction of disulfide bonds. the conformation of the cry6aa protein was also probed by mass spectrometry. ionization of intact proteins under electrospray mass spectrometry conditions resulted in the detection of multiply charged species with characteristic csds unique for each protein. the distribution of charges on a protein under the denaturing conditions used for lc-ms is influenced by the primary sequence and the conformational flexibility of the protein, which determines the available protonation sites exposed on the denatured protein surface. this characteristic behavior of proteins is routinely used as an indirect probe of the flexibility of protein tertiary structure. the role of the disulfide bonds in the overall rigidity of cry6aa was probed by measuring the csd under different conditions. comparison of the csd for cry6aa under non-reducing conditions and after treatment with 1 mm dtt shows a profound shift in the csd, even though the deconvoluted intact average molecular weight only increased by + under non-reducing conditions, the csd is centered around m/z, corresponding to a + charge state. after reduction, the csd shifts to a center around m/z, corresponding to a + charge state. this shift towards more highly charged species is a reflection of the decreased rigidity of the overall protein tertiary structure resulting from the reduction of the disulfides with dtt, exposing additional protein surfaces to protonation during ionization.fig. charge state distributions of cry6aa. a under non-reducing conditions the csd centers around m/z. b following treatment with 1 mm dithiothreitol, the csd centers around m/z the pairing of cysteine residues and the buried nature of cys described above indicate that natural cry6aa crystals are not stabilized by intermolecular disulfide bonds. this contrasts with many three-domain cry protoxins in which disulfide bonds between the c-terminal proparts of individual protoxin molecules are important in crystal formation. however, while cry6aa crystal stability does not appear to be mediated by covalent intermolecular bonding, we can speculate that the rigidity conferred on the cry6aa monomers by the intramolecular disulfide bridges could be relevant to crystal packing. similarity to other toxins it is interesting to note that in the related cry6ba sequence, the c-terminal domain found in cry6aa is absent. as a result, the cry6ba sequence will lack both the c402–c and the c88–c disulfide bonds. there is no cys residue at the position equivalent to cys in a primary sequence alignment of cry6aa and cry6ba, where this residue is asn. cry6ba does, however, contain two additional cysteine residues at positions and in helix αc but these residues are too far apart to form an intramolecular disulfide bond. cry6ba also lacks two of the repetitive features described above and the remaining repeat feature in cry6aa is changed in both occurrences in cry6ba and becomes non-repetitive. cry6ba has coleopteran activity but little or no nematode toxicity. this indicates that this region is not necessary for toxicity per se. in the case of cry6aa, the c-terminal region is also not essential for toxicity since deletion of the c-terminal residues and the first residues from the n-terminus produces proteins that retain some, albeit reduced, toxicity against c. elegans. the “wing” features seen in cry6aa are regions that vary in length when compared to the equivalent regions in cry6ba. since in cry6aa these features form interruptions to the αc and αd helices, we predict that these variations will be well tolerated in cry6ba. the structures derived for cry6aa are striking in their structural similarity to previously described bacterial toxins. a dali search conducted with the truncated cry6aa crystal structure identified the closest structural homolog as the hblb component of the bacillus cereus hemolytic toxin with a significant z-score value of  %, rmsd between c∝-atoms of  Å and sequence identity of 12 %. the structural similarities to other toxins – such as the nhea component of the b. cereus non-hemolytic toxin and hemolysin e from e. coli – are also remarkable, with z-scores of 21 % and 11 %, and rmsd values of  Å and  Å, despite low sequence identity of 12 % and 10 %, respectively. these are pore-forming toxins, which, like cry6aa, exhibit an elongated structure composed of an alpha helical bundle with a head domain at one end. cry6aa also shows some similarity to smc_prok_a family proteins that are also known to form dimers; in the related hlye crystal structure, two monomers can be seen to pack together in a head-to-tail orientation. dimeric forms of cry6aa were observed in this study and have been described previously but the packing of the monomers is not known. despite this, there is no evidence for dimer formation in either crystal structure.fig. comparison of cry6aa structure with structures of other related toxins. superposition of cry6aa with a hbl-b, b nhea, and c hlye. in panel d, the surface representation of hydrophobic areas of the truncated cry6aa in ribbon representation are colored green and the remaining residues are colored gray. the putative transmembrane region is labeled. c-terminal 463– residues are removed for simplicity it is interesting to note that in cry6aa, two of the long alpha helices are interrupted with surface loops bounded by gly residues:; the first of these loops is also seen in the ab initio models although the second was not predicted. these small, wing-like intra-helical loops are a unique feature of cry6aa not seen in the related structures of hlye, hblb, or nhea and their significance is unknown at present. they do not appear to significantly interact with the rest of the molecule and residues lys and lys are not visible in the crystal structures, suggesting that they are mobile. despite their apparent surface accessibility and the two lysine residues found near the tips of both of these loops, trypsin does not appear to cleave at these sites. the hlye mode of action model of mueller et al. and the recent work of benke et al. propose that upon interaction with the membrane, the putative pore-forming tongue region unfolds, causing the alpha helices to extend. it is possible that a similar rearrangement may occur for cry6aa, and that the g121dpsikkdg and g189dqkkleg loops reconfigure into alpha helical segments. this hypothesis is consistent with the observation that each loop is seven amino acids in length and is bounded by glycine residues, which, due to the hydrogen r group, allow free rotation around the peptide bond, allowing a low energy secondary structure transition. the head domain of cry6aa is folded across the helices as seen in the hblb and nhea structures. within the head domains of these proteins is a region termed the beta tongue that may contain the transmembrane sequence that allows them to form pores in target cells. in hlye, this putative membrane-spanning in the beta tongue is proposed to insert into the membrane along with the flanking helix e to produce an oligomeric pore from either eight or monomers. a similar hairpin loop feature, well conserved in cry6ba was identified both in the ab initio models and the structures of cry6aa; analysis using the tmpred software predicts this to be a candidate transmembrane sequence. probing the cry6aa mechanism of action prior to the completion of crystallographic analyses and the derivation of cry6aa structures, we initiated structural modeling of the cry6aa protein. because the structural databases contained no proteins with primary sequence homology with cry more detailed structural predictions based on known protein structures were impossible. we decided, therefore, to employ the rosetta modeling program to predict the possible fold of the cry protein ab initio, producing five de novo models with no reference to previous structures and five models using the database structure comparison method. all models exhibited a high proportion of alpha helix and very low proportions of beta sheet. the modeling using the database structure comparison method returned a range of distinct structural models. in contrast, the five de novo models showed a high degree of similarity to each other, differing mainly in the disposition of the n- and c-terminal domains with respect to the central domain of the long alpha helices. these models predicted a predominantly alpha helical structure that is highly similar [the structural alignment of the backbone residues in the central region of the protein to the actual structures of the protein that were subsequently solved through crystallography. based on the model, we were able to form a hypothesis that cry6aa is a pore-forming toxin and identify a region that might be involved in this function as a target for mutagenesis. nematode assays as a test for pore-forming activity of cry6aa given that our analyses showed structural homology to toxins such as hlye and, by analogy, suggested a similar mechanism of action, we decided to test this hypothesis. in their work on hlye, wallace et al. investigated the importance of the tongue region by substituting hydrophobic residues in this part of the sequence with non-hydrophobic amino acids. they demonstrated the loss of hemolytic activity. in a parallel experiment, to disrupt the hydrophobicity of the putative transmembrane region of cry6aa, we replaced leu with asp. the leu residue was chosen because it is a hydrophobic residue at the center of the putative membrane-spanning domain. the l259d-mutant protein was modeled and the change was not predicted to cause any alteration in the overall fold of the protein. the wild-type cry6aa and the l259d mutant, both expressed in e. coli, were used in the bioassays against c. elegans. the expression of wild-type and mutant cry6aa in e. coli on iptg induction was verified by western blotting and cells expressing these proteins were tested for their activity against c. elegans. bioassays showed a striking and significant reduction in nematode growth following exposure to the wild-type cry6aa protein compared to those feeding on cells transformed with pet28b vector alone. in contrast, the l259d mutant revealed no reduction in nematode size compared to the controls, indicating that this targeted mutation had eliminated cry6aa bioactivity against c. elegans. in combination with the structural data, this result is consistent with cry acting as a pore-forming toxin, with structures and mechanisms of action similar to that of the hblb and hlye toxins that could cause membrane damage through the insertion of residues into the head region of the protein, as postulated from the de novo models.fig. nematode bioassay: c. elegans were fed on e. coli transformed with the pet28b plasmid as a negative control; transformed with this vector containing the wild-type cry6aa gene; or transformed with the vector with the l259d mutant of this gene. a images of some nematodes chosen at random are shown and b results are presented as mean worm area. p values comparing each condition to vector control are shown. error bars represent the standard errors of the means to test the ability of cry6aa to form pores more directly, experiments were conducted in vivo using c. elegans fourth-stage hermaphrodites and a live animal assay for the presence of pores in the intestine. following exposure of the hermaphrodites to e. coli either carrying empty pqe vector or expressing cry6aa or cry5ba, the penetration of ingested propidium iodide by intestinal cells was monitored. under conditions used, pi gets into intestinal cells exposed to active pore-forming toxins. as expected, nematodes fed with empty vector e. coli showed confinement of the pi to the lumen of the gut, whereas in cry6aa- and cry5ba -treated hermaphrodites, entry of pi into the intestinal epithelial cell cytoplasm was evident. in these experiments the percentage of hermaphrodites showing pi entry was quantitated: for empty vector  ±   %, for cry5b exposure 92 ±   %, and for cry6aa exposure  ±   %. the stronger effects of cry5b on pi entry into intestinal cells could be due to factors such as the absolute toxicity of each protein, the relative numbers of receptors, or the relative quantities of the proteins expressed in the cells. nonetheless, cry6aa showed significantly higher pore formation than control. therefore, based on all our data, we conclude that cry6aa is an active pore-forming toxin.fig. in vivo pore formation. a bright field images and fluorescence images of the anterior regions of c. elegans fed on e. coli transformed with pqe vector or plasmids expressing cry6aa or cry5ba. b percentage of treated worms showing propidium iodide uptake. the p values for the comparison between each toxin and the no-toxin control are shown  the elucidated crystal structures for cry6aa support a mechanism of action by pore formation, similar to toxins such as hblb, and we have demonstrated pore-forming ability in vivo. the hblb protein and the non-hemolytic toxin components nheb and nhec are derived from b. cereus strains and induce pore formation and osmotic lysis of target cells, leading to the induction of diarrhea by this organism. b. thuringiensis is a member of the b. cereus sensu lato group of bacilli and, aside from production of invertebrate-active toxins by b. thuringiensis, the two species are indistinguishable by most tests. interestingly, despite the low amino acid sequence identity, cry6aa shares a fold and, we propose, a mechanism of action with the b. cereus toxins and this may indicate an adaptation of this hblb protein family within the b. cereus sensu lato group for activity against target cells in invertebrates. the known structures of delta-endotoxins from b. thuringiensis are structurally distinct from the cry fold, which represents a new structural family among insecticidal toxins and a new paradigm for invertebrate pathogenic toxins. based on the present study, we can now further elucidate key features of the cry6aa toxin that are responsible for its specificity and mode of action.fig. structural families of b. thuringiensis delta-endotoxins. representatives of different structural classes of delta-endotoxins of b. thuringiensis are shown. cry1aa is a three-domain toxin; cry6aa is an alpha helical toxin; cry is an aegerolysin-like protein that acts as a binary toxin with cry a toxin_ family protein; cry is a member of the etx/mtx family; and cyt1aa, a member of the bac_thur_toxin family. all structures are to scale and colored according to secondary structure additional files additional file 1: figure s multiple sequence alignment of cry6aa and related toxins. sequences of full-length cry6aa and cry6ba were aligned using the clustalx program along with the sequences from the crystal structures of hlye, hblb, and nhea. residues identical to the cry6aa sequence are highlighted in green; residues identical to cry6ba but not in cry6aa are highlighted in blue. repeat regions in cry6aa are highlighted in yellow. the putative transmembrane region is highlighted in gray with l indicated by a blue asterisk. the “wing” features in the cry6aa sequence are indicated by magenta highlight. additional file 2: figure s ab initio models of cry6aa. the five models constructed with reference to database information and five models constructed entirely de novo are shown. the predicted n-terminal domains are shown in light gray and marked “n,” while the c-terminal domains are shown in black and marked “c.” additional file 3: figure s modeled l259d mutant. the full-length structure is overlaid with the l259d model. the inset illustrates the putative transmembrane loop region with residue shown in stick display. loops are colored as above for clarity while, for other regions, the full-length structure is dark gray and the modeled mutant is light gray. additional file 4: figure s western blot of wild-type and l259d mutant cry6aa. expression of the wild-type cry6aa and the l259d mutant confirmed by western blotting. 
cry proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the cry toxins of b. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. trypsin processing was found to result in the loss of some internal sequences while the c-terminal region remains disulfide-linked to the main core of the toxin. cry6aa shows structural homology to a known class of pore-forming toxins including hemolysin e from escherichia coli and two bacillus cereus proteins: the hemolytic toxin hblb and the nhea component of the non-hemolytic toxin. these proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. based on the structural similarity of cry6aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in caenorhabditis elegans was demonstrated.
 the cry family of proteins from bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. these proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. this has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. to increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of cry6aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.  the two forms of the toxin were resolved to  Å and  Å respectively and showed very similar structures. cry6aa shows structural homology to a known class of pore-forming toxins including hemolysin e from escherichia coli and two bacillus cereus proteins: the hemolytic toxin hblb and the nhea component of the non-hemolytic toxin. cry6aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. trypsin processing was found to result in the loss of some internal sequences while the c-terminal region remains disulfide-linked to the main core of the toxin. based on the structural similarity of cry6aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in caenorhabditis elegans was demonstrated. a non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.  cry proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the cry toxins of b. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. elucidation of both the structure and the pore-forming mechanism of action of cry6aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bacillus thuringiensiscry6insecticidal toxinhemolysinhttp://dx.doi.org/ /501100000275leverhulme trustf/ 407/biberry colin http://dx.doi.org/ /100000057national institute of general medical sciencesr01gm071603r01gm071603sitaram anand aroian raffi v. http://dx.doi.org/ /501100000275leverhulme trusthttp://dx.doi.org/ /100000060national institute of allergy and infectious diseasesr01ai056189aroian raffi v. issue-copyright-statement© the author 2016 