Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.
The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.
Likitvivatanavong
Supaporn
S
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D000330:Aedes / Q000254:growth & development / Q000382:microbiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001704:Biopolymers; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins; D006860:Hydrogen Bonding; D007814:Larva / Q000254:growth & development / Q000382:microbiology; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001704:Biopolymers; D004731:Endotoxins; D006460:Hemolysin Proteins; D008081:Liposomes; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D001216:Asparagine