doi:10.1016/j.jmb.2008.05.010 J. Mol. Biol. (2008) 380, 820–827
Available online at www.sciencedirect.com
High-Resolution Crystal Structure of Activated Cyt2BaMonomer from Bacillus thuringiensis subsp. israelensis
Shmuel Cohen1,2⁎, Orly Dym3, Shira Albeck3, Eitan Ben-Dov1,4,Rivka Cahan2, Michael Firer2 and Arieh Zaritsky1
1Department of Life Sciences,Ben-Gurion University of theNegev, P.O.B. 653,Be'er-Sheva 84105, Israel2Department of ChemicalEngineering and Biotechnology,Ariel University Center ofSamaria, Ariel 40700, Israel3The Israel StructuralProteomics Center (ISPC),Weizmann Institute of Science,Rehovot 76100, Israel4Achva Academic College,MP Shikmim, 78900, Israel
Received 4 March 2008;received in revised form5 May 2008;accepted 6 May 2008Available online11 May 2008
*Corresponding author. DepartmentGurion University of the Negev, P.O84105, Israel. E-mail address: shmulAbbreviations used: ICP, insectici
VVA2, volvatoxin A2; SE, size exclucell; EDTA, ethylenediaminetetraaceData Bank.
The Cyt family of proteins consists of δ-endotoxins expressed during spo-rulation of several subspecies of Bacillus thuringiensis. Its members possessinsecticidal, hemolytic, and cytolytic activities through pore formation andattract attention due to their potential use as vehicles for targetedmembranedestruction. The δ-endotoxins of subsp. israelensis include three Cyt species:a major Cyt1Aa and twominor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cytprotein that lacks the N- and C-terminal segments forms a toxic monomer.Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino andcarboxy termini by bacterial endogenous protease(s). Overall, its foldresembles that of the previously described volvatoxin A2 and the nontoxicform of Cyt2Aa. The structural similarity between these three proteins mayprovide information regarding the mechanism(s) of membrane-perforatingtoxins.
Bacillus thuringiensis is a species of Gram-positive,aerobic bacterium that produces endospores undercertain environmental conditions.1,2 Coincident withsporulation, it creates parasporal crystals, δ-endo-toxins, composed of insecticidal crystal proteins(ICPs).3 These entomopathogenic toxins are highlyspecific against larvae of Lepidoptera, Coleoptera, orDiptera1,4 and are classified into two families:Crystal (Cry) and Cytolytic (Cyt).5 There is no se-quence or structure homology between the two fa-
of Life Sciences, Ben-.B. 653, Be'[email protected] crystal protein;sion; RBC, red bloodtic acid; PDB, Protein
lsevier Ltd. All rights reserve
milies, but they share common biochemical proper-ties. The ICPs are produced and crystallized in vivo asprotoxins, solubilized in alkaline pH, and convertedinto active membrane-perforating toxins followingproteolytic cleavage, removing short segments of theN andC termini.6,7 The attachment of Cry proteins tothe larval intestinal membrane is mediated throughspecific receptors,8,9 whereas Cyt proteins attachnonspecifically8 and are mediated by nonsaturatedphospholipids such as phosphatidylcholine, phos-phatidylethanolamine, and sphingomyelin.10
B. thuringiensis subsp. israelensis exhibits the mostpotent bacteria-derived mosquito larvicidal activityknown so far. This stems from synergy between someof its major ICPs encoded by genes that are mappedat the 128-kb plasmid pBtoxis: four of the Cry family(Cry4Aa, Cry4Ba, Cry10Aa, and Cry11Aa) and threeCyts (Cyt1Aa, Cyt2Ba, and Cyt1Ca).2,11
Themosquito larvicidal activity of Cyt1Aa is lowerthan those of the Cry proteins, but it is highly syner-gistic with them.12,13 In addition, the native protoxin
d.
821Crystallization and Structure Analysis of Cyt2Ba
(27.2 kDa) is active in vitro against insect cells, ery-throcytes, and mammalian cells14 and lethal to micefollowing an intravenous injection.10,14 The hemoly-tic and cytolytic activities of Cyt1Aa are higher thanall other family members15,16 and are enhanced byNand C termini proteolysis in the larval gut, thusforming a 22- to 25-kDa activated toxin form.6,17,18
Cyt1Aa (249 amino acids) shares 39% identity and64% similarity in its amino acid sequence withCyt2Aa (259 amino acids) from subsp. kyushuensis,which is also hemolytic and cytolytic followingproteolytic processing in similar domains.19 There-fore, they are likely to have a high degree of struc-tural similarity.19,20To date, the only solved crystal structure of the Cyt
family is that of Cyt2Aa, which is distinguished fromthe Cry structure and is composed of a single α–βdomain comprising two outer layers of α-helix hair-pins and a β-sheet in between.20 In the protoxinform, Cyt2Aa is a nonhemolytic19 dimer linked bythe intertwined N-terminal strands. Proteolytic pro-cessing cleaves the N- and C-terminal segments,leading to dimer dissociation and toxin activation.Themechanism of action of Cyt toxins is still under
controversy. According to one approach, the twoouter layers of α-helix hairpins swing away from theβ-sheet uponmembrane contact, and the three lastβ-strands are allowed to insert into the membrane.Consequently, oligomerization with other mono-mers and formation of a β-barrel pore occur, re-sulting in a colloid osmotic lysis.20–24 Another modelsuggests that the hydrophilic side of the helicesinteracts with residues from othermonomers to formoligomer. This results in nonspecific aggregation ofCyt molecules on the surface of the lipid bilayer, lea-ding to a detergent-like action and membrane dis-assembly.10,25,26 The crystal structure of volvatoxinA2 (VVA2), a pore-forming cardiotoxin from themushroom Volvariella volvacea, adopts a similarstructure to that of Cyt2Aa.27 It was suggested thatthe N-terminal domain of VVA2 (residues 1–127)consisting of α-helices is responsible for oligomer-ization, and the C-terminal domain (residues 128–199) consists of three β-strands that are inserted intothe membrane.28 These strands correspond to thestrands of Cyt2Aa, which may be responsible formembrane insertion as well.Cyt2Ba is a minor component within the complex
of the crystal's proteins expressed by subsp. israe-lensis. It is composed of 263 amino acids with aboutof 41% and 67% identity to Cyt1Aa and Cyt2Aa,respectively.29,30 Despite the similarities in sequencesamong these three Cyt proteins, no antigenic cross-reaction has been detected between them.31 Cyt2Bais highly homologous (more than 99% identity) toseven Cyt2Ba variants from other B. thuringiensissubspecies, suggesting that it is responsible for animportant task within the crystal complex of thisspecies.29,30
Toxicities of recombinant solubilized crystalCyt1Aa and Cyt2Aa to mosquito larvae, as well astheir hemolytic activities, are similar but higher thanthose of Cyt2Ba. LC50 values of Cyt1Aa and of
Cyt2Aa against larvae of Aedes aegypti, Anophelesgambiae, and Culex pipiens ranged between 0.5 and2 μg ml−1 and between 0.5 and 4 μg ml−1, respect-ively, and hemolytic end-point values for undi-gested, endogenously digested, and fully digestedCyt1Aa were up to 3.7-fold lower than those ofCyt2Aa.19 In another study,15 nonsolubilized crys-tals of recombinant Cyt1Aa and Cyt2Ba displayedLC50 values of 0.4–2.7 μg ml−1 and 1.8–5.5 μg ml−1,respectively, against larvae of A. aegypti, C. pipiens,Culex quinquefasciatus, and Anopheles stephani,whereas hemolytic end-point values of solubilizedor trypsin-activated Cyt1Aa were more than 100-fold lower than those of Cyt2Ba. The reasons for thedifferences in activities between Cyt proteins are stillunknown.Here, we describe the crystal structure of the pro-
teolytically cleaved active form of Cyt2Ba to 1.8 Åresolution. This is the first structure of a toxic form ofa Cyt family member to be reported. The structurehas a striking similarity to the protoxin form ofCyt2Aa and to fungal VVA2, suggesting that thetoxic monomer of these proteins has a similar modeof activity against cell membrane.
Results and Discussion
Isolation of the Cyt2Ba monomer and itsanalysis and identification
Expression of cyt2Ba in recombinant acrystallifer-ous B. thuringiensis subsp. israelensis produces Cyt2Baas easily isolatable hexagonal crystals that ranged insize from 0.4 to 0.6 μm.32 The solubilized crystalsyielded a protein that underwent partial proteolysis(Fig. 1a). The undigested Cyt2Ba appears on SDS-PAGE with a mass of 24 kDa while the endogenouslydigested product has a mass of 22 kDa. This mixturewas purified by size exclusion (SE) chromatographyyielding three peaks, including a large peak thateluted in the void volume and contained predomi-nantly DNA (established by its 260/280 nm absor-bance ratio and by lack of protein bands on SDS-PAGE). The second peak contains Cyt2Ba migratingas a dimer, which runs on SDS-PAGE as the un-cleaved protein (upper band in Fig. 1a). The last peakcontains Cyt2Ba migrating as a monomer on the SEcolumn, which runs on SDS-PAGE as its degradationproduct (lower band in Fig. 1a). In order to obtain thefully digested product, in vivo Cyt2Ba crystals weresolubilized in the absence of protease inhibitors at37 °C. The fully digested Cyt2Ba (Fig. 1b) migratedon an SE column as a monomer and produced crys-tals for X-ray analysis, whereas the uncleaved protein(24 kDa) did not.The activity of the cleaved purified Cyt2Ba sample
was determined using hemolysis assay. Variousconcentrations of this fraction incubated with 0.2%of human red blood cells (RBCs) for 3 h yielded anHC50 value of 15.2 μg ml−1. However, when the in-cubation was extended to 24 h, 95% hemolysis of the
Fig. 1. Analysis of proteolytically activated Cyt2Ba. (a) SDS-PAGE analysis of Cyt2Ba extracted from crystals formedin vivo. This preparation contains full-length (upper band) and proteolytically digested Cyt2Ba (lower band). (b) Purified,digested Cyt2Ba, which crystallized in vitro. (c) MALDI-TOF mass spectrum analysis of the purified, proteolyticallycleaved protein. Two fragments are identified: one with a mass of 22.4 kDa corresponding to T34–P232 (22.38 kDa) andthe second fragment with a mass of 22.7 kDa corresponding to segment T34–I234 (22.6 kDa). y-axis, arbitrary intensity; x-axis, mass/charge ratio.
822 Crystallization and Structure Analysis of Cyt2Ba
RBC was induced. These results are similar to thoseobtained with Cyt2Ba treated by endogenous pro-teases of subsp. israelensis.32
MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectroscopy (Fig.1c) of this fraction revealed two peaks, one with amass of 22.7 kDa and the other with a mass of22.4 kDa. The N-terminal sequence of the proteolyticfragments of solubilized in vivo Cyt2Ba crystals thathad been treated by endogenous proteases beganwith residue T34.32 Based on the masses of the twofragments, we concluded that our two fragmentscorrespond to T34–I234 and T34–P232 (22.6 and22.38 kDa, respectively) (marked by green arrows inFig. 2). Interestingly, these two digested forms arehomologous to the proteolytically activated frag-ments of Cyt2Aa digested by proteinase K (beforeT34 and S37 and after S228 and F237).20
The overall structure of monomeric Cyt2Ba
The crystal structure of the proteolytically acti-vated, monomeric form of Cyt2Bawas solved to 1.8 Åresolution (Fig. 3). It is composed of a single domainof α/β architecture with a β-sheet surrounded by twoα-helical layers representing a cytolysin fold. Thesheet consists of six antiparallel β-strands (β1−β6)flanked by an α-helix layer composed of α1 and α2 onone side and by a second α-helix layer composed ofα3–α5 on the other. The four longest β-strands(β2–β5) of the central β-sheet have a modifiedGreek-key topology. The model consists of residuesT41–S228 (with 120 water molecules), while the two
fragments introduced into crystallization correspon-ded to T34–I234 and T34–P232 (22.6 and 22.4 kDa,respectively). The lack of observed density forresidues 34–40 and 229–234 in our structure suggeststhat these regions are disordered.
Comparison of Cyt2Ba with structurally relatedproteins
A striking similarity was observed between thestructures of the endogenously cleaved Cyt2Bamonomer (residues T41–S228) and the correspond-ing region (residues D41–S228) within the inactiveprotoxin dimer of Cyt2Aa (rmsd of 0.59 Å). Eachmonomer of Cyt2Aa consists of an extra β-strand atits N terminus and α-helix at its C terminus com-pared to the cleaved Cyt2Ba (Fig. 4a). The dimerinterface of Cyt2Aa is held together by the inter-twined N-terminal strands from both monomers.20
The cleavage of Cyt2Aa removes the N and Ctermini segments, thereby preventing dimer forma-tion and, hence, releasing a monomer active toxin.Similarly, in Cyt2Ba, the proteolysis causes theremoval of 34 amino acids at its N terminus and28 or 30 residues at its C terminus, forming thecrystallized toxic monomer.Cyt2Ba shares only 16% sequence identity to VVA2
(Fig. 2). Nevertheless, they both adopt a cytolysinfold and their structure is very similar with an rmsdof 3.59 Å (Fig. 4b). VVA2 consist of 199 amino acidsand possesses hemolytic and cytolytic proper-ties.27,28,35 Themain difference between the structureof VVA2 and that of Cyt2Ba is an insertion of a β-
Fig. 2. Sequence alignment of Cyt2Ba, Cyt2Aa, Cyt1Aa, and VVA2. Cyt2Ba secondary structure elements are labeledabove the corresponding sequence (numbering refers to the sequence of Cyt2Ba); α- and η-helices are spirals and β-strands are arrows. The residues conserved in all four proteins are in red blocks. The figure was created using ESPript.33
Green arrows designate the proteolytic fragments T34–I234 and T34–P232.
823Crystallization and Structure Analysis of Cyt2Ba
hairpin between α1 and α2 in VVA2 (indicated by anarrow in Fig. 4b). Due to the structural resemblancebetween the activated forms of Cyt2Ba, Cyt2Aa, andVVA2, it seems plausible that they could have similarpore-forming mechanism(s).VVA consists of two proteins, VVA1 and VVA2,
with molecular masses of 44.8 and 22.4 kDa,respectively. While VVA2 is toxic, VVA1 does notpossess any significant biological activity and is nottoxic.35 VVA1 is twice the length of VVA2, whichexists only as a monomer. Interestingly, sequencealignment between VVA2 to VVA1 revealed that theVVA2 monomer is homologous to each half ofVVA1 (32% identity for residues 1–189 and 36% forresidues 190–393). Therefore, it is reasonable toassume that each domain of VVA1 adopts a similarfold to that observed for VVA2 and Cyt2Ba. Thus,VVA1 most likely exists as a pseudo-dimer, whichmay explain its lack of toxicity. The most significantoutcome of this structural comparison is that thetoxicity of Cyt2Ba, Cyt2Aa, and VVA2 is aninherent property of the monomer and not theresult of secondary structure rearrangement uponcleavage. While the protoxins of the Cyt familyform dimers, VVA2 exists solely as a monomer.Interestingly, VVA2 lacks the N- and C-terminalsegments that are involved in dimer formation inthe Cyt family.Soluble VVA2 was shown to be prone to cleavage
by trypsin and chemotrypsin before K127 andW132,respectively.28 These positions are located in the
region corresponding to the loop between β3 and β4of Cyts. The aligned homologous regions in solubleCyt1Aa, Cyt2Aa, and Cyt2Ba are protected fromcleavage by these and other proteases.6,19,32 How-ever, when Cyt1Aa and Cyt2Aa are in a liposome-bound state, they were cleaved by trypsin andproteinase K not only at the N and C termini butalso between the end of α4 and β3 (before K154and N155 in Cyt1Aa and before G141 and I150 inCyt2Aa), probably by changing their spatial con-formation.21 In addition, the C-terminal region ofVVA2, Cyt1Aa, and Cyt2Aa starting with β4 hasbeen shown to be resistant to proteolysis uponassociation to the membrane.21,28 Therefore, it hasbeen suggested that β4, β5, and β6 are embeddedin the membrane lipid bilayer.A comprehensive understanding of the toxic
activities of these proteins may not only broadenour understanding as to the cytolytic machinery ofpore-forming toxins but also help to design bettermembrane-active cytotoxins. Indeed, utilization ofproteolytically active Cyt1Aa and Cyt2Aa in tar-geted drug delivery systems has recently been de-monstrated: A conjugant of Cyt1Aa–insulin has beenused against cells expressing high levels of insulinreceptors,36 and chemical binding or genetic fusionof active Cyt1Aa to a peptide (p87–99) of myelinbasic protein (MBPp) targets Cyt1Aa to malignant B-cells expressing surface anti-MBPp antibodies.37 Inyet another study, specific toxicity against breast andovarian cancer cells was obtained when the active
Fig. 3. Crystal structure of the Cyt2Ba monomer.Ribbon representation of the Cyt2Ba crystal structurecomposed of a single domain of α/β architecture with a β-sheet surrounded by two α-helical layers representing acytolysin fold. Secondary elements and N and C terminiare labeled (created with PyMOL34).
824 Crystallization and Structure Analysis of Cyt2Ba
fragment of Cyt2Aa was genetically fused to anantibody that recognizes p185HER-2.38,39
Fig. 4. Structure comparison of Cyt2Ba to Cyt2Aa andVVA2. (a) Cyt2Ba monomer (green) and Cyt2Aa dimer(monomer A, orange; monomer B, blue) (PDB accessioncode 1CBY). Note: the N-terminal intertwined strands andthe C-terminal helices of Cyt2Aa that are both involved indimer formation are shown in red dashed ellipsoid. Bothsegments do not exist in the proteolytically cleavedCyt2Ba structure. (b) Cyt2Ba (green) and VVA2 (red).(PDB accession code 1PP0). The insertion of a β-hairpinbetween α1 and α2 in the VVA2 structure is shown by ablack arrow. Ribbon diagrams were created by PyMOL.34
Experimental Procedures
Purification of Cyt2Ba
Strain IPS(cyt2Ba/p20) of acrystalliferous B. thuringiensissubsp. israelensis containing pHTcy2B–p2032 and expres-sing cyt2Ba and p20 (encoding the helper protein)40 duringsporulation was used. Cyt2Ba was assembled in vivo toeasily isolatable hexagonal crystals, which ranged between0.4 and 0.6 μm in size32 after growth in CCY sporulationmedium41 for 4 days when most cells sporulated and auto-lyzed. The culture was centrifuged, and the sedimentincluding crystals, spores, and debris was rinsed thricewith double-distilled water. The crystals were separated ona discontinuous sucrose gradient,42 and the pure Cyt2Bacrystals were rinsed thrice with water and solubilized in analkaline buffer [50 mM Na2CO3, pH 10.5, 5 mM PMSF,
10 mM DTT, and 10 mM ethylenediaminetetraacetic acid(EDTA)]. The supernatant was filtered and dialyzed threetimes against Buffer A (20 mMTris–HCl, pH 8, 5% glycerol,
Table 1.Data collection and structure refinement statistics
Data collectionResolution range (Å) 50.0–1.8 (1.83–1.8)Space group C2Unit cell dimensions
a (Å) 83.48b (Å) 43.26c (Å) 55.65β (°) 112.44
Number of molecules in the asymmetric unit 1Number of reflections measured 60,445Number of unique reflections 16,805 (767)Rsym
a 0.075 (0.26)Completeness (%) 97.4 (94.9)Redundancy 3.67 (3.2)⟨I⟩/⟨σ(I)⟩ 16.5 (4.5)
Bond lengths (Å) 0.017Bond angles (°) 1.54Torsion angles (°) 12.98
Ramachandran plot (%)Most favored 90.3
825Crystallization and Structure Analysis of Cyt2Ba
5 mM DTT, 1 mM EDTA, 10 mM ethanolamine, 0.1 mMPMSF, and 3 mM NaN3).During the preparation of soluble Cyt2Ba, there was a
massive aggregation of the protoxin at neutral pH, whichprobably hindered its crystallization in vitro. Removingamino and carboxy termini of Cyt by proteolysis seemed tolower the aggregation level considerably and allowedbinding to cell membrane.6,20 For proteolyzed Cyt2Ba, thecrystal sediment was incubated (1 h at 37 °C in shaker,200 rpm) with 50 mMNa2CO3 buffer (pH 10.5) containing10 mM DTT without any protease inhibitors. The super-natant was filtered, dialyzed three times against Buffer Aand concentrated by placing the dialysis bag on a mat ofcarboxymethylcellulose (Sigma).Proteolyzed Cyt2Ba was detected by 15% SDS-PAGE
and quantified by OD280. Absorbance of 1 unit isequivalent to 1.09 mg ml−1.43
Cyt2Ba was further purified by SE chromatography(HiLoad 16/60 Superdex 75, Amersham Biosciences) witha buffer containing 20 mM Tris–HCl, pH 8, 50 mM NaCl,5% glycerol, and 5 mM DTT. The eluted peaks wereanalyzed by 15% SDS-PAGE. Those containing proteoly-tically cleaved Cyt2Ba were pooled and concentrated byCentricon® centrifugal filter YM-10 (Millipore, Billerica,MA, USA) to a volume of 300 μl (23 mg ml−1 according toOD280) and submitted to crystallization.
Additional allowed 9.1Generously allowed 0.6Disallowed regions 0.0
Values in parentheses are for the highest-resolution shells.a Rsym=∑ |⟨Ihkl⟩− Ihkl|/Ihkl|, where ⟨Ihkl⟩ is the average
intensity over symmetry-related reflections and Ihkl is the
Hemolysis assay
Human RBCs were treated and analyzed as previouslydescribed.32
observed intensity.b R=∑||Fo|−|Fc||/∑|Fo|, where Fo denotes the observed
structure factor amplitude and Fc the structure factor calculatedfrom the model.
Crystallization, data collection, and refinement
Single crystals of the proteolytically cleaved Cyt2Ba(residues T34–I234) were obtained by the microbatchmethod under oil, using the IMPAX 1–5 robot (DouglasInstruments, East Garston, Hungerford, Berkshire, UK).The protein was crystallized in a mixture containing 1.0 MNa/K tartrate, 0.1 M Tris, pH 7.0, 0.2 M Li2SO4, and10 mM EDTA sodium salt. A complete data set wascollected from a single crystal on a Rigaku R-AXIS IV+imaging plate area detector using a Rigaku RU-H3Rrotating anode operated at 5 kW and Osmic multilayer X-ray focusing mirrors. The diffraction images were indexedand integrated by using the program HKL2000.44 Theintegrated reflections were scaled by using the programSCALEPACK.44 Crystals formed in space group C2, withcell constants a=83.472 Å, b=43.262 Å, c=55.64 Å, andβ=112.438°, contained one monomer in the asymmetricunit cell with a VM of 2.26 Å3/Da and diffracted to 1.8 Åresolution. Structure factors' amplitudes were calculatedby using TRUNCATE from the CCP4 program suite.45
The structure was solved by molecular replacement withthe program PHASER,46 using the refined structure of themosquito larvicidal δ-endotoxin Cyt2Aa from B. thurin-giensis subsp. kyushuensis [Protein Data Bank (PDB) acces-sion code 1CBY] as amodel. All steps of atomic refinementwere carried out with the program CCP4/Refmac5.47 Themodel was built to σA-weighted, 2Fobs−Fcalc, and Fobs−Fcalc maps using the program COOT.48 Refinement moveswere accepted only when they produced a decrease in theRfree value. In later rounds of refinement, water moleculeswere built into peaks greater than 3σ in Fobs−Fcalc maps.The current model contains residues 44–228 and 120 watermolecules. The Rfree value is 21.6% (for the 5% of ref-lections not used in the refinement), and the Rwork value is
18.7% for all data to 1.8 Å. The coordinates of Cyt2Ba havebeen deposited in the PDB with the accession code 2RCI.The Cyt2Ba model was evaluated with the programPROCHECK.49 Data collection and refinement statisticsare described in Table 1. The figures were created withPyMOL.34 Sequence alignment was prepared withESPript.33
PDB accession code
Coordinates for the crystal structure of the Cyt2Ba-digested protein have been assigned the PDB accessioncode 2RCI.
Acknowledgements
We thank Prof. Joel L. Sussman for helpful dis-cussions. We are grateful to Anna Branzburg andYigal Michael from the Israel Structural ProteomicsCenter for their skilled assistance. The structure wasdetermined at the Israel Structural ProteomicsCenter, supported by The Israel Ministry of Science,Culture and Sport; the Divadol Foundation; theNeuman Foundation; and the European Commis-sion Sixth Framework Research and TechnologicalDevelopment Programme ‘SPINE2-COMPLEXES’
826 Crystallization and Structure Analysis of Cyt2Ba
Project under Contract No. 031220. Partial supportwas awarded by a grant (No. 2001-042) from the US–Israel Binational Science Foundation, Jerusalem,Israel (to A.Z.).
References
1. Aronson, A. (2002). Sporulation and delta-endotoxinsynthesis by Bacillus thuringiensis. Cell. Mol. Life Sci. 59,417–425.
2. Margalith, Y. & Ben-Dov, E. (2000). Biological controlby Bacillus thuringiensis subsp. israelensis. In Insect PestManagement: Techniques for Environmental Protection(Rechcigl, J. E. & Rechcigl, N. A., eds.), pp. 243–301,CRC press LLC, Boca Raton, FL.
3. Baum, J. A. & Malvar, T. (1995). Regulation ofinsecticidal crystal protein production in Bacillusthuringiensis. Mol. Microbiol. 18, 1–12.
4. Aronson, A. I. (1993). The two faces of Bacillusthuringiensis: insecticidal proteins and post-exponen-tial survival. Mol. Microbiol. 7, 489–496.
5. Hofte, H. & Whiteley, H. R. (1989). Insecticidal crystalproteins of Bacillus thuringiensis. Microbiol. Rev. 53,242–255.
6. Al-yahyaee, S. A. S. & Ellar, D. J. (1995). Maximaltoxicity of cloned CytA δ-endotoxin from Bacillusthuringiensis subsp. israelensis requires proteolyticprocessing from both the N- and C-termini. Micro-biology, 141, 3141–3148.
7. Angsuthanasombat, C., Crickmore, N. & Ellar, D. J.(1993). Effects on toxicity of eliminating a cleavage sitein a predicted interhelical loop in Bacillus thuringiensisCryIVB delta-endotoxin. FEMS Microbiol. Lett. 111,255–261.
8. Bravo, A., Gill, S. S. & Soberón, M. (2007). Mode ofaction of Bacillus thuringiensis Cry and Cyt toxins andtheir potential for insect control. Toxicon, 49, 423–435.
9. Pigott, C. R. & Ellar, D. J. (2007). Role of receptors inBacillus thuringiensis crystal toxin activity. Microbiol.Mol. Biol. Rev. 71, 255–281.
10. Thomas, W. E. & Ellar, D. J. (1983). Mechanism ofaction of Bacillus thuringiensis var. israelensis insectici-dal delta-endotoxin. FEBS Lett. 154, 362–368.
11. Berry, C., O'Neil, S., Ben-Dov, E., Jones, A. F., Murphy,L., Quail, M. A. et al. (2002). Complete sequence andorganization of pBtoxis, the toxin-coding plasmid ofBacillus thuringiensis subsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.
12. Khasdan, V., Ben-Dov, E., Manasherob, R., Boussiba,S. & Zaritsky, A. (2001). Toxicity and synergism intransgenic Escherichia coli expressing four genes fromBacillus thuringiensis subsp. israelensis. Environ. Micro-biol. 3, 798–806.
13. Park, H. W., Bideshi, D. K. & Federici, B. A. (2003).Recombinant strain of Bacillus thuringiensis producingCyt1A, Cry11B, and the Bacillus sphaericus binarytoxin. Appl. Environ. Microbiol. 69, 1331–1334.
14. Knowles, B. H., White, P. J., Nicholls, C. N. & Ellar,D. J. (1992). A broad-spectrum cytolytic toxin fromBacillus thuringiensis var. kyushuensis. Proc. R. Soc.London, Ser. B. 248, 1–7.
15. Juarez-Perez, V., Guerchicoff, A., Rubinstein, C. &Delecluse, A. (2002). Characterization of Cyt2Bc toxinfrom Bacillus thuringiensis subsp. medellin. Appl.Environ. Microbiol. 68, 1228–1231.
16. Mizuki, E., Ohba, M., Akao, T., Yamashita, S., Saitoh,H. & Park, Y. S. (1999). Unique activity associated with
non-insecticidal Bacillus thuringiensis parasporal inclu-sions: in vitro cell-killing action on human cancer cells.J. Appl. Microbiol. 86, 477–486.
17. Butko, P., Huang, F., Pusztai-Carey, M. & Surewicz,W. K. (1996). Membrane permeabilization induced bycytolytic delta-endotoxin CytA from Bacillus thurin-giensis var. israelensis. Biochemistry, 35, 11355–11360.
18. Du, J., Knowles, B. H., Li, J. & Ellar, D. J. (1999). Bio-chemical characterization of Bacillus thuringiensis cyto-lytic toxins in association with a phospholipid bilayer.Biochem. J. 338, 185–193.
19. Koni, P. A. & Ellar, D. J. (1994). Biochemical charac-terization of Bacillus thuringiensis cytolytic delta-endotoxins. Microbiology, 140, 1869–1880.
20. Li, J., Koni, P. A. & Ellar, D. J. (1996). Structure of themosquitocidal delta-endotoxin CytB from Bacillusthuringiensis sp. kyushuensis and implications formembrane pore formation. J. Mol. Biol. 257, 129–152.
21. Du, J., Knowles, B. H., Li, J. & Ellar, D. J. (1999).Biochemical characterization of Bacillus thuringiensiscytolytic toxins in association with a phospholipidbilayer. Biochem. J. 338, 185–193.
22. Li, J., Derbyshire, D. J., Promdonkoy, B. & Ellar, D. J.(2001). Structural implications for the transformationof the Bacillus thuringiensis delta-endotoxins fromwater-soluble to membrane-inserted forms. Biochem.Soc. Trans. 29, 571–577.
23. Promdonkoy, B. & Ellar, D. J. (2000). Membrane porearchitecture of a cytolytic toxin from Bacillus thur-ingiensis. Biochem. J. 350, 275–282.
24. Promdonkoy, B. & Ellar, D. J. (2005). Structure–function relationships of a membrane pore formingtoxin revealed by reversion mutagenesis. Mol. Membr.Biol. 22, 327–337.
25. Butko, P. (2003). Cytolytic toxin Cyt1A and its mecha-nism of membrane damage: data and hypotheses.Appl. Environ. Microbiol. 69, 2415–2422.
26. Manceva, S. D., Pusztai-Carey, M., Russo, P. S. &Butko, P. (2005). A detergent-like mechanism of actionof the cytolytic toxin Cyt1A from Bacillus thuringiensisvar. israelensis. Biochemistry, 44, 589–597.
27. Lin, S. C., Lo, Y. C., Lin, J. Y. & Liaw, Y. C. (2004).Crystal structures and electron micrographs of fungalvolvatoxin A2. J. Mol. Biol. 343, 477–491.
28. Weng, Y. P., Lin, Y. P., Hsu, C. I. & Lin, J. Y. (2004).Functional domains of a pore-forming cardiotoxicprotein, volvatoxin A2. J. Biol. Chem. 279, 6805–6814.
29. Guerchicoff, A., Delecluse, A. & Rubinstein, C. P.(2001). The Bacillus thuringiensis cyt genes for hemoly-tic endotoxins constitute a gene family. Appl. Environ.Microbiol. 67, 1090–1096.
30. Guerchicoff, A., Ugalde, R. A. & Rubinstein, C. P. (1997).Identification and characterization of a previouslyundescribed cyt gene in Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 63, 2716–2721.
31. Held, G. A., Kawanishi, C. Y. & Huang, Y. S. (1990).Characterization of the parasporal inclusion of Bacillusthuringiensis subsp. kyushuensis. J. Bacteriol. 172,481–483.
32. Nisnevitch, M., Cohen, S., Ben-Dov, E., Zaritsky, A.,Sofer, Y. & Cahan, R. (2006). Cyt2Ba of Bacillus thu-ringiensis israelensis: activation by putative endogenousprotease. Biochem. Biophys. Res. Commun. 344, 99–105.
33. Gouet, P., Courcelle, E., Stuart, D. I. &Metoz, F. (1999).ESPript: analysis of multiple sequence alignments inPostScript. Bioinformatics, 15, 305–308.
34. DeLano, W. L. (2002). The PyMOL molecular graphicssystem. DeLano Scientific, San Carlos, CA, USA. http://www.pymol.org.
827Crystallization and Structure Analysis of Cyt2Ba
35. Lin, J. Y., Jeng, T. W., Chen, C. C., Shi, G. Y. & Tung,T. C. (1973). Isolation of a new cardiotoxic proteinfrom the edible mushroom, Volvariella volvacea.Nature,246, 524–525.
36. Al-yahyaee, S. A. & Ellar, D. J. (1996). Cell targeting ofa pore-forming toxin, CytA delta-endotoxin fromBacillus thuringiensis subspecies israelensis, by conju-gating CytA with anti-Thy 1 monoclonal antibodiesand insulin. Bioconjugate Chem. 7, 451–460.
37. Cohen, S., Cahan, R., Ben-Dov, E., Nisnevitch, M.,Zaritsky, A. & Firer, M. A. (2007). Specific targeting tomurine myeloma cells of Cyt1Aa toxin from Bacillusthuringiensis subspecies israelensis. J. Biol. Chem. 282,28301–28308.
38. Gurkan, C. & Ellar, D. J. (2003). Expression in Pichiapastoris and purification of a membrane-acting immu-notoxin based on a synthetic gene coding for theBacillus thuringiensisCyt2Aa1 toxin. Protein Expr. Purif.29, 103–116.
39. Gurkan, C. & Ellar, D. J. (2003). Expression of theBacillus thuringiensis Cyt2Aa1 toxin in Pichia pastorisusing a synthetic gene construct. Biotechnol. Appl.Biochem. 38, 25–33.
40. Shao, Z. & Yu, Z. (2004). Enhanced expression ofinsecticidal crystal proteins in wild Bacillus thuringien-sis strains by a heterogeneous protein P20. Curr.Microbiol. 48, 321–326.
41. Stewart, G. S., Johnstone, K., Hagelberg, E. & Ellar,D. J. (1981). Commitment of bacterial spores to ger-minate. A measure of the trigger reaction. Biochem. J.198, 101–106.
42. Debro, L., Fitz-James, P. C. & Aronson, A. (1986). Twodifferent parasporal inclusions are produced byBacillus thuringiensis subsp. finitimus. J. Bacteriol. 165,258–268.
43. Cantor, C. R. & Schimmel, P. R. (1980). In BiophysicalChemistry, Part II: Techniques for the Study of BiologicalStructure and Function. pp. 380–381, W. H. Freemanand Co., San Francisco, CA.
44. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray Diffraction Data Collected in Oscillation Mode. InMethods in Enzymology (Carter, J. C. W. & Sweet, R. M.,eds.), Vol. 276, pp. 307–326, Academic Press, NewYork.
45. French, G. S. & Wilson, K. S. (1978). On the treatmentof negative intensity observations. Acta Crystallogr.,Sect. A: Cryst. Phys., Diffr., Theor. Gen. Crystallogr. 34,517–525.
46. McCoy, A. J. (2007). Solving structures of proteincomplexes by molecular replacement with Phaser.Acta Crystallogr., Sect. D: Biol. Crystallogr. 63, 32–41.
47. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997).Refinement of macromolecular structures by themaximum-likelihood method. Acta Crystallogr., Sect.D: Biol. Crystallogr. 53, 240–255.
48. Emsley, P. & Cowtan, K. (2004). Coot: model-buildingtools for molecular graphics. Acta Crystallogr., Sect. D:Biol. Crystallogr. 60, 2126–2132.
49. Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein structures.J. Appl. Crystallogr. 26, 283–291.
