title: Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS.
abstract: 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.
authors.affiliation: 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.
authors.forename: Wilaiwan
authors.initials: W
authors.lastname: Sriwimol
authors.affiliation: Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand.
authors.forename: Aratee
authors.initials: A
authors.lastname: Aroonkesorn
authors.affiliation: Department of Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Nakornpathom 73170, Thailand.
authors.forename: Somsri
authors.initials: S
authors.lastname: Sakdee
authors.affiliation: Laboratory of Theoretical and Computational Biophysics, Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Chalermpol
authors.initials: C
authors.lastname: Kanchanawarin
authors.affiliation: Department of Physics and Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uchihashi
authors.affiliation: Department of Physics and Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Ando
authors.affiliation: 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. Electronic address: chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000166:cytology / Q000187:drug effects; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005960:Glucosides / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007306:Insecticides / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D008081:Liposomes / Q000737:chemistry; D008823:Micelles; D056004:Molecular Dynamics Simulation; D010572:Pest Control, Biological; D011487:Protein Conformation; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D061987:Sf9 Cells / Q000166:cytology / Q000187:drug effects*; D018411:Spodoptera / Q000166:cytology / Q000187:drug effects; D013451:Sulfonic Acids / Q000737:chemistry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D005960:Glucosides; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007306:Insecticides; D008081:Liposomes; D008823:Micelles; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C018619:octyl-beta-D-glucoside; D004134:Dimyristoylphosphatidylcholine
keywords: EM; bacillus; bacterial toxin; conformational transition; gel electrophoresis; high-speed atomic force microscopy; micelle-induced trimerization; propeller-like shape; protein assembly; three-dimensional reconstructed prepore