Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis.
Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.
Promdonkoy
Boonhiang
B
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Pathumthani, Thailand. boonhiang@biotec.or.th
Ellar
David J
DJ
D000330:Aedes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D002462:Cell Membrane / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology* / Q000633:toxicity; D006460:Hemolysin Proteins; D007814:Larva / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis