MOVEMENT OF A LOOP IN DOMAIN-3 OF AEROLYSIN IS REQUIRED FOR CHANNEL FORMATION

Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure o f the proaerolysin dimer and electron microscopic images of the oligom er indicated that a loop in domain 3 must move away from the P-sheet t hat forms the main body of the protein before oligomerization can proc eed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cy steines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disul fide bridge. The double mutant was unable to oligomerize, and it was c ompletely inactive, showing not only that the bridge had formed but al so that movement of the loop was essential for formation of the oligom er. The existence of the bridge was confirmed by X-ray crystallography . The reduced form of the protein and the single mutants T253C and A30 0C were as active as wild type, indicating that the amino acid replace ments themselves had no functional consequences. Labeling studies usin g an environment-sensitive fluorescent sulfhydryl-reactive probe confi rmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which a lso must precede oligomerization.