Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Abstract

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 αD-β4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on αE. 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 αA, αB, αC, αD and the loops between αA and αB, αD and β5, β6 and β7 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.

• Bacillus thuringiensis
• cytolytic δ-endotoxin
• Cyt2Aa1 toxin
• reversion mutagenesis
• transmembrane pore