title: Importance of polarity of the α4-α5 loop residue-Asn(166) in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for ion permeation and pore opening.
abstract: Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150mM KCl, pH8.5), it produced single-channel currents with a maximum conductance of about 425pS comparable to the wild-type toxin. However, maximum conductances for single K(+)-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue-Asn(166) is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface.
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Thanate
authors.initials: T
authors.lastname: Juntadech
authors.forename: Yodsoi
authors.initials: Y
authors.lastname: Kanintronkul
authors.forename: Chalermpol
authors.initials: C
authors.lastname: Kanchanawarin
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000187:drug effects* / Q000254:growth & development; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000378:metabolism / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004553:Electric Conductivity; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D017136:Ion Transport; D007532:Isoleucine / Q000737:chemistry / Q000235:genetics; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010539:Permeability; D010572:Pest Control, Biological; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D005452:Fluoresceins; D006460:Hemolysin Proteins; D008081:Liposomes; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D007532:Isoleucine; D001224:Aspartic Acid; D001216:Asparagine; D000409:Alanine; C007740:fluorexon
keywords: Bacillus thuringiensis; Cry δ-endotoxins; Dye-leakage assay; MD simulations; Planar lipid bilayers; Trimeric pore