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Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS*
  • Wilaiwan Sriwimol
    Affiliations
    Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakornpathom 73170, Thailand

    Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand
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  • Aratee Aroonkesorn
    Affiliations
    Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand
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  • Somsri Sakdee
    Affiliations
    Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand
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  • Chalermpol Kanchanawarin
    Affiliations
    Laboratory of Theoretical and Computational Biophysics, Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
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  • Takayuki Uchihashi
    Affiliations
    Department of Physics and Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
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  • Toshio Ando
    Affiliations
    Department of Physics and Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan
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  • Chanan Angsuthanasombat
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand

    Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development, Bangkok 10160, Thailand
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  • Author Footnotes
    * This research work was supported in part by Thailand Research Fund Grants IRG 57-8-0009 and BRG-58-8-0002) in cooperation with the Commission of Higher Education, Ministry of Education (Thailand). This work was also supported by a scholarship from the Commission of Higher Education, Ministry of Education (Thailand) (to W. S.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:June 25, 2015DOI:https://doi.org/10.1074/jbc.M114.627554
      The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-β-d-glucoside micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than a toxin monomer in membrane perturbation of calcein-loaded liposomes. A three-dimensional model of toxin trimer obtained by negative-stain EM in combination with single-particle reconstruction at ∼5 nm resolution showed a propeller-shaped structure with 3-fold symmetry. Fitting the three-dimensional reconstructed EM map with a 100-ns molecular dynamics-simulated Cry4Ba structure interacting with an octyl-β-d-glucoside micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy imaging at nanometer resolution and a subsecond frame rate demonstrated conformational transitions from a propeller-like to a globularly shaped trimer upon lipid membrane interactions, implying prepore-to-pore conversion. Real-time trimeric arrangement of monomers associated with l-α-dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid bicelle membranes was also envisaged by successive high-speed atomic force microscopy imaging, depicting interactions among three individual subunits toward trimer formation. Together, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for the molecular assembly of a prepore trimer capable of inserting into target membranes to generate a lytic pore.

      Introduction

      Pore-forming Cry δ-endotoxins produced by Bacillus thuringiensis (Bt)
      The abbreviations used are: Bt
      Bacillus thuringiensis
      Bti
      Bacillus thuringiensis subsp. israelensis
      Aedes-mALP
      Aedes aegypti membrane-bound alkaline phosphatase
      DMPC
      l-α -dimyristoylphosphatidylcholine
      CHAPSO
      3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid
      HS-AFM
      high-speed atomic force microscopy
      OG
      octyl-β-d-glucoside
      CMC
      critical micelle concentration
      Cry
      crystal
      CAT
      chloramphenicol acetyltransferase
      LUV
      large unilamellar vesicle
      MD
      molecular dynamics
      Sf9
      Spodoptera frugiperda 9.
      are highly toxic toward a wide range of insect larvae (
      • Schnepf E.
      • Crickmore N.
      • Van Rie J.
      • Lereclus D.
      • Baum J.
      • Feitelson J.
      • Zeigler D.R.
      • Dean D.H.
      Bacillus thuringiensis and its pesticidal crystal proteins.
      ). The 130-kDa Cry4Ba toxin from Bt subsp. israelensis (Bti), for instance, is specifically active against the larvae of Aedes and Anopheles mosquitoes, the most important vectors for the life-threatening diseases of dengue viral hemorrhagic fever and malaria, respectively (
      • Angsuthanasombat C.
      Structural basis of pore formation by mosquito-larvicidal proteins from Bacillus thuringiensis.
      ,
      • Becker N.
      • Margalit J.
      ). Biochemically, these insecticidal proteins are produced as insoluble protoxins within crystalline inclusions that require dissolution in the target larval midgut lumen (usually at an alkaline pH for dipteran and lepidopteran insect larvae) prior to being processed by gut proteases to yield ∼65-kDa active portions that are resistant to further proteolysis (
      • Schnepf E.
      • Crickmore N.
      • Van Rie J.
      • Lereclus D.
      • Baum J.
      • Feitelson J.
      • Zeigler D.R.
      • Dean D.H.
      Bacillus thuringiensis and its pesticidal crystal proteins.
      ,
      • Angsuthanasombat C.
      Structural basis of pore formation by mosquito-larvicidal proteins from Bacillus thuringiensis.
      ,
      • Pardo-López L.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.
      ). For numerous Bt-Cry toxins, it has been shown that the activated toxins bind specifically to various types of receptors lining the apical brush-border membrane of target midgut cells (
      • Pigott C.R.
      • Ellar D.J.
      Role of receptors in Bacillus thuringiensis crystal toxin activity.
      ). For instance, in our previous works, two different glycosylphosphatidylinositol-anchored proteins, i.e. glycosylphosphatidylinositol-anchored alkaline phosphatase (ALP) and glycosylphosphatidylinositol-anchored aminopeptidase N, were identified as potential receptors mediating Cry4Ba toxicity in Aedes mosquito larvae (
      • Dechklar M.
      • Tiewsiri K.
      • Angsuthanasombat C.
      • Pootanakit K.
      Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti midgut larvae: a Bacillus thuringiensis Cry4Ba toxin-receptor.
      ,
      • Thammasittirong A.
      • Dechklar M.
      • Leetachewa S.
      • Pootanakit K.
      • Angsuthanasombat C.
      Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin.
      • Aroonkesorn A.
      • Pootanakit K.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.
      ). The toxin-receptor interactions are believed to facilitate toxin insertion into the target cell membrane to form ion leakage pores, resulting in osmotic cell lysis and, eventually, death of the insect larvae (
      • Schnepf E.
      • Crickmore N.
      • Van Rie J.
      • Lereclus D.
      • Baum J.
      • Feitelson J.
      • Zeigler D.R.
      • Dean D.H.
      Bacillus thuringiensis and its pesticidal crystal proteins.
      ,
      • Pardo-López L.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.
      ,
      • Pigott C.R.
      • Ellar D.J.
      Role of receptors in Bacillus thuringiensis crystal toxin activity.
      ). However, the exact mechanisms underlying the toxicity exerted by these insecticidal Bt-Cry toxins still remain to be explored.
      Crystal structures of almost all major specificity classes of the Bt-Cry toxins, including the Cry4Ba mosquito-specific toxin, reveal a strong overall structural resemblance suggestive of the same general mechanism of toxicity (
      • Angsuthanasombat C.
      Structural basis of pore formation by mosquito-larvicidal proteins from Bacillus thuringiensis.
      ,
      • Li J.D.
      • Carroll J.
      • Ellar D.J.
      Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution.
      ,
      • Boonserm P.
      • Davis P.
      • Ellar D.J.
      • Li J.
      Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications.
      ). All individual structures display a wedge-shaped form (estimated dimensions, 55 × 65 × 75 Å) with three structurally distinctive domains (DI-III): from the N to the C terminus, an α-helical bundle (DI), a β sheet prism (DII), and a β sheet sandwich (DIII). Unlike DIII, whose function is still ambiguous, both DI and DII have been clearly defined regarding membrane pore formation and receptor recognition, respectively (
      • Angsuthanasombat C.
      Structural basis of pore formation by mosquito-larvicidal proteins from Bacillus thuringiensis.
      ,
      • Pardo-López L.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.
      ,
      • Li J.D.
      • Carroll J.
      • Ellar D.J.
      Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution.
      ). In our earlier studies, the role of Cry4Ba-DI in membrane-inserted pore formation has been studied intensively. Of particular interest, we have strengthened the proposed “umbrella-like” mechanistic model (
      • Angsuthanasombat C.
      Structural basis of pore formation by mosquito-larvicidal proteins from Bacillus thuringiensis.
      ,
      • Gazit E.
      • La Rocca P.
      • Sansom M.S.
      • Shai Y.
      The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “umbrella-like” structure of the pore.
      ) by providing direct evidence for liposomal membrane-perturbing activity of the purified Cry4Ba pore-forming fragment, i.e. α4-loop-α5 hairpin (
      • Leetachewa S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Novel preparation and characterization of the α4-loop-α5 membrane perturbing peptide from the Bacillus thuringiensis Cry4Ba δ-endotoxin.
      ). Additionally, one highly conserved residue, Asn183, located in the middle of the transmembrane α5, has been found to play an important role in Cry4Ba toxicity and is essentially involved in toxin-pore oligomerization (
      • Likitvivatanavong S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.
      ). Moreover, two highly conserved aromatic residues (Tyr249 and Phe264) of Cry4Ba-α7 have been found to play a vital role in toxin-membrane interactions, conceivably needed for lipid-induced conformational transitions prior to an efficient insertion of the transmembrane α4-loop-α5 hairpin into the lipid bilayers (
      • Tiewsiri K.
      • Angsuthanasombat C.
      Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin.
      ,
      • Tiewsiri K.
      • Fischer W.B.
      • Angsuthanasombat C.
      Lipid-induced conformation of helix 7 from the pore forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for toxicity mechanism.
      ). More recently, we have demonstrated that the polarity of the Cry4Ba α4-α5 loop residue Asn166 is an important element for ion permeation through the toxin-induced pore (
      • Juntadech T.
      • Kanintronkul Y.
      • Kanchanawarin C.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Importance of polarity of the α4-α5 loop residue-Asn166 in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for ion permeation and pore opening.
      ). We have also verified the functional significance of intrinsic stability toward the Pro-rich cluster (Pro193-Pro194_Pro196) which is present exclusively in the long loop connecting α4 and α5 of Cry4Aa, another Bti toxin closely related to Cry4Ba (
      • Imtong C.
      • Kanchanawarin C.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Bacillus thuringiensis Cry4Aa insecticidal protein: functional importance of intrinsic stability of the unique α4-α5 loop comprising the Pro-rich sequence.
      ).
      For mechanisms of oligomerization and pore formation, one of the proposed models is that active toxin subunits would rearrange upon receptor binding, therefore inducing conformational changes to form an oligomeric prepore capable of penetrating the target membrane to generate a lytic pore (
      • Gómez I.
      • Sánchez J.
      • Miranda R.
      • Bravo A.
      • Soberón M.
      Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin.
      ,
      • Bravo A.
      • Gómez I.
      • Conde J.
      • Muñoz-Garay C.
      • Sánchez J.
      • Miranda R.
      • Zhuang M.
      • Gill S.S.
      • Soberón M.
      Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains.
      ). Alternatively, the activated toxins could individually insert into the target membrane, where they subsequently assemble into an oligomeric pore complex, causing cell lysis (
      • Likitvivatanavong S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.
      ,
      • Groulx N.
      • McGuire H.
      • Laprade R.
      • Schwartz J.L.
      • Blunck R.
      Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
      ). However, the precise molecular description of either model still remains to be critically verified. We have previously employed detergent dialysis-driven two-dimensional crystallization to directly demonstrate, for the first time, a symmetrical trimeric structure of the l-α -dimyristoylphosphatidylcholine (DMPC) lipid-associated Cry4Ba toxin complex (
      • Ounjai P.
      • Unger V.M.
      • Sigworth F.J.
      • Angsuthanasombat C.
      Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba δ-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation.
      ). Although the trimeric form has been supported by other studies (
      • Muñóz-Garay C.
      • Portugal L.
      • Pardo-López L.
      • Jiménez-Juárez N.
      • Arenas I.
      • Gómez I.
      • Sánchez-López R.
      • Arroyo R.
      • Holzenburg A.
      • Savva C.G.
      • Soberón M.
      • Bravo A.
      Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.
      ,
      • Gómez I.
      • Sánchez J.
      • Muñoz-Garay C.
      • Matus V.
      • Gill S.S.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity.
      ), the tetrameric complex of Cry toxin-induced pores has also been reported recently through other approaches (
      • Groulx N.
      • McGuire H.
      • Laprade R.
      • Schwartz J.L.
      • Blunck R.
      Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
      ,
      • Lin X.
      • Parthasarathy K.
      • Surya W.
      • Zhang T.
      • Mu Y.
      • Torres J.
      A conserved tetrameric interaction of Cry toxin helix α3 suggests a functional role for toxin oligomerization.
      ). In this study, further attempts were made to provide more critical insights into our trimeric pore model by employing two direct rendering techniques, i.e. single-particle EM and HS-AFM, for visualizing the three-dimensional structure and trimeric assembly of the membrane-associated Cry4Ba toxin. We clearly showed that a membrane-induced state of toxin monomers is a critical prerequisite for the formation of a potential prepore trimer.

      Discussion

      So far, the step at which the Bt-Cry oligomerization pathway might occur, either in solution (
      • Gómez I.
      • Sánchez J.
      • Miranda R.
      • Bravo A.
      • Soberón M.
      Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin.
      ,
      • Gómez I.
      • Sánchez J.
      • Muñoz-Garay C.
      • Matus V.
      • Gill S.S.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity.
      ,
      • Lin X.
      • Parthasarathy K.
      • Surya W.
      • Zhang T.
      • Mu Y.
      • Torres J.
      A conserved tetrameric interaction of Cry toxin helix α3 suggests a functional role for toxin oligomerization.
      ,
      • Guo S.
      • Zhang Y.
      • Song F.
      • Zhang J.
      • Huang D.
      Protease-resistant core form of Bacillus thuringiensis Cry1Ie: monomeric and oligomeric forms in solution.
      ) or in a membrane-bound state (
      • Likitvivatanavong S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.
      ,
      • Groulx N.
      • McGuire H.
      • Laprade R.
      • Schwartz J.L.
      • Blunck R.
      Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
      ), remains to be clearly elucidated. In this study, we demonstrated that no defined oligomeric complex was observed for the purified trypsin-activated Cry4Ba toxin in carbonate-based solution (toxin solubilization buffer (pH 9.0)), as analyzed via a seminative PAGE system and size exclusion chromatography. Therefore, these results indicate that the 65-kDa Cry4Ba monomer is unable to self-assemble to form an oligomeric complex in solution, implying a need of a particular driving force in promoting a molecular recognition between toxin monomers required for the oligomeric assembly. In other words, a proper conformational transition of the toxin monomer could be a critical prerequisite for formation of the oligomeric pore complex.
      We clearly demonstrated that the 65-kDa monomers were able to assemble into a ∼200-kDa stable trimer upon incubation with non-ionic OG micelles. In addition, the amount of toxin trimer obtained appeared to be CMC-dependent, implying that a higher OG-CMC could perhaps provide a better membrane-like environment. It has been shown recently that the chymotrypsin-treated Cry4Ba toxin, upon interacting with lipid vesicles, was able to assemble into a ∼250-kDa oligomer without the need of the Aedes-cadherin receptor fragment, rather dissimilar to Cry11Aa (another mosquito-active toxin from Bti) (
      • Rodríguez-Almazán C.
      • Reyes E.Z.
      • Zúñiga-Navarrete F.
      • Muñoz-Garay C.
      • Gómez I.
      • Evans A.M.
      • Likitvivatanavong S.
      • Bravo A.
      • Gill S.S.
      • Soberón M.
      Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae.
      ) and other lepidopteran active Cry1A toxins that require the cadherin-binding for oligomerization (
      • Gómez I.
      • Sánchez J.
      • Miranda R.
      • Bravo A.
      • Soberón M.
      Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin.
      ). Therefore, this could corroborate our findings that toxin-membrane interactions are most likely to be involved in the Cry4Ba-trimer assembly.
      We also revealed that the Cry4Ba trimer assembly is not covalently mediated by intermolecular disulfide bonds and that the only two Cys residues (Cys27 in DI and Cys525 in DIII, Fig. 3D) are unlikely to be involved in Cry4Ba trimer formation. Moreover, this trimeric complex seems to be relatively stable at a moderately high temperature as well as resistant to SDS-induced dissociation. Although, at this stage, a detailed description of an SDS-resistant intermonomeric interface is still unclear, 200-kDa OG-induced trimer formation could likely be mediated by intermolecular non-covalent interactions. It has been suggested that multiple polar interactions, especially a network of hydrogen bonds and ionic interactions, appeared to display a key role in the stability of oligomer assemblies to the dissociation by SDS (
      • Hotze E.M.
      • Heuck A.P.
      • Czajkowsky D.M.
      • Shao Z.
      • Johnson A.E.
      • Tweten R.K.
      Monomer-monomer interactions drive the prepore to pore conversion of a β-barrel-forming cholesterol-dependent cytolysin.
      ). We have demonstrated previously, via combined MD and continuum solvent studies, that the Cry4Aa toxin, which is very much related to Cry4Ba, could form a stable trimer in aqueous solution, as primarily attributed to the intersubunit interactions through certain polar uncharged and charged residues in the pore-forming domain DI (
      • Taveecharoenkool T.
      • Angsuthanasombat C.
      • Kanchanawarin C.
      Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore.
      ). We have also shown, via mutagenesis studies, that one highly conserved polar residue, Asn183, situated in DI-α5, exerts a vital role in Cry4Ba trimer prepore formation and, therefore, larvicidal activity (
      • Likitvivatanavong S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.
      ). Therefore, a major driving force in facilitating the molecular recognitions between micelle-associated monomers required for trimer assembly could possibly be a network of H-bonds and ionic interactions.
      Results from cytotoxicity assays indicated that the SDS-resistant Cry4Ba trimer obtained via OG micelle induction appeared to be a potential preformed trimer because it was able to interact with Aedes-mALP (a potential Cry4Ba receptor expressed on Sf9 cells) to exert cytolytic activity to an extent similar to that seen with the monomer. Although it is still unclear whether the similar cytotoxicity observed between trimers and monomers is due to the role of such a Cry4Ba receptor expressed on the target cells, the preformed trimer clearly induced membrane permeability of receptor-free LUVs and much more efficient than the toxin monomer. Moreover, hyperbolic kinetics of calcein release can be observed with this potential prepore trimer even at a 10-fold lower concentration than the monomer, which, instead, displayed an apparent sigmoidal pattern containing an initial lag phase. Therefore, these findings corroborate that the OG micelle-induced trimer is indeed a potential prepore state that can readily incorporate into the lipid membrane, whereas the toxin monomers have to insert into the lipid membrane prior to assembly into a prepore/active pore trimer for inducing membrane permeability. Another possibility is that the membrane-interacting capability of the prepore trimer with the receptor-free LUVs is greater than that of the toxin monomer, which may, perhaps, require a specific docking molecule for promoting interactions with the membrane surface. In this context, the results from toxin-lipid interaction assays via toxin-LUV cosedimentation as well as HS-AFM imaging substantiated that the Cry4Ba preformed trimer indeed exhibits a much higher binding capability than the monomer toward artificial membranes, thereby acting faster to induce LUV permeability.
      Previously, although two symmetrical trimeric conformations of the membrane-associated Cry4Ba complex, i.e. propeller-like and pinwheel-like, could be revealed by electron crystallography, these two-dimensional data were inadequate to provide more insights into the molecular organization of the pore architecture (
      • Ounjai P.
      • Unger V.M.
      • Sigworth F.J.
      • Angsuthanasombat C.
      Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba δ-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation.
      ). In this study, although the three-dimensional reconstructed model revealed only the propeller-like shape, its reconstructed EM map fitted with a 100-ns MD simulated Cry4Ba structure interacting with an OG micelle could give an idea about the relative positioning of individual domains in the context of the trimeric prepore complex. In line with several previous studies as well as the established mechanistic models for the membrane-bound state of Bt-Cry toxins, a defined hairpin structure of α4-loop-α5 within DI is conceivably required for membrane insertion and pore formation (
      • Becker N.
      • Margalit J.
      ,
      • Pardo-López L.
      • Soberón M.
      • Bravo A.
      Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.
      ,
      • Li J.D.
      • Carroll J.
      • Ellar D.J.
      Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution.
      ,
      • Leetachewa S.
      • Katzenmeier G.
      • Angsuthanasombat C.
      Novel preparation and characterization of the α4-loop-α5 membrane perturbing peptide from the Bacillus thuringiensis Cry4Ba δ-endotoxin.
      ). However, the fact that the hydrophobic faces of the outer amphipathic helices of DI face inwards, these three-domain Cry toxins must go through conformational changes, particularly within DI, to convert this pore-forming domain into a transmembrane pore in which the hydrophobic surfaces would be in close contact with the membrane lipids (
      • Becker N.
      • Margalit J.
      ,
      • Li J.D.
      • Carroll J.
      • Ellar D.J.
      Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution.
      ). Accordingly, the individual protomeric subunits within the propeller-shaped trimer that shows an apparent protrusion from the pore-forming domain could be expected to have an overall conformation different from the water-soluble monomer.
      One caveat in this study with the use of HS-AFM is that its images, particularly measured in the x-y plane, might contain some structural features that are not present on the sample in actuality because their measurements are often influenced by tip sample convolution effects. Even with such common artifacts arising in the particle image diameter, a significant increase observed in height (z axis) of the Cry4Ba trimer could still possibly be a sign of conformational differences between the monomer and each subunit in the trimeric propeller-shaped complex. Therefore, this suggests a change in toxin conformation upon trimeric organization. Regardless of the molecular details of this complex that could not be revealed at this stage, such a conformational change may perhaps occur through a protrusion of a transmembrane part, possibly either the α4-loop-α5 hairpin (
      • Gazit E.
      • La Rocca P.
      • Sansom M.S.
      • Shai Y.
      The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “umbrella-like” structure of the pore.
      ) or lipid-sensing α7 (
      • Tiewsiri K.
      • Fischer W.B.
      • Angsuthanasombat C.
      Lipid-induced conformation of helix 7 from the pore forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for toxicity mechanism.
      ), from the pore-forming domain. However, our findings via HS-AFM were rather different from those of the previous AFM studies of Cry1Aa (
      • Vie V.
      • van Mau N.
      • Pomarede P.
      • Schwartz J.L.
      • Laprade R.
      • Frutos R.
      • Rang C.
      • Masson L.
      • Heitz F.
      • Le Grimellec C.
      Lipid-induced pore formation of the Bacillus thuringiensis Cry1Aa insecticidal toxin.
      ) and Cry4Ba (
      • Puntheeranurak T.
      • Stroh C.
      • Zhu R.
      • Angsuthanasombat C.
      • Hinterdorfer P.
      Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes.
      ) via a conventional tapping mode because a tetramer rather than a trimer has been proposed as the preferred oligomerization state of the membrane-associated pore complex, although there is no direct evidence for its functional relevance.
      Moreover, our HS-AFM imaging at nanometer resolution and a subsecond frame rate clearly demonstrated conformational transitions from a propeller-like to globularly shaped trimer upon lipid membrane interactions, conceivably implying prepore-to-pore conversion. Albeit probe artifacts, a significant increase in size (diameter) together with a height decrease (∼2 nm) observed for the globularly shaped complex may perhaps reflect an opening (increased diameter) and/or membrane-inserting (decreased height) feature of the trimeric pore. Nevertheless, the detailed perception of the two different trimeric conformations (prepore and membrane-associated pore) remains to be further verified by performing a more in-depth structural characterization.
      The real-time trimeric arrangement of monomers associated with bicelle membranes can also be visualized by our successive HS-AFM imaging, depicting interactions among three individual subunits toward trimer assembly. This important evidence therefore further strengthens the notion proposed here for the structural requirement of a membrane-associated state of the Cry4Ba active toxin for molecular assembly of a potential prepore trimer that would subsequently transform into a membrane-lytic pore. Likewise, other recent studies also attempted to give details for an oligomerization pathway of the Cry1Aa toxin using single-molecule analysis via photobleaching (
      • Groulx N.
      • McGuire H.
      • Laprade R.
      • Schwartz J.L.
      • Blunck R.
      Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
      ). Despite the fact that the toxin molecule itself could not be visualized directly, the authors suggested that oligomerization of the Cry1Aa toxin is a highly dynamic process occurring after insertion of monomers into the lipid membrane to form an oligomeric complex (
      • Groulx N.
      • McGuire H.
      • Laprade R.
      • Schwartz J.L.
      • Blunck R.
      Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
      ). Taken together, our findings provide pivotal insights, for the first time, into the structural requirement of membrane-induced conformational changes for the formation of a potential prepore trimer of such insecticidal pore-forming proteins from Bt biopesticides.

      Author Contributions

      W. S., T. U., T. A., and C. A. designed research. W. S. performed most of the experiments for her Ph.D. study and performed research. A. A., S. S., C. K., and T. U. performed research. W. S., C. K., T. U., T. A., and C. A. analyzed data. W. S. and C. A. wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Prof. Prapon Wilairat and Drs. Lerson Tanasugarn, Chamras Promtmas, Kusol Pootanakit, Gerd Katzenmeier, Somphob Leetachewa, and Taweetham Limpanuparb for their critical comments. We also thank Dr. Puey Ounjai (Department of Biology, Faculty of Science, Mahidol University) for making available facilities for EM studies.

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