A CONSERVED TRYPTOPHAN IN PNEUMOLYSIN IS A DETERMINANT OF THE CHARACTERISTICS OF CHANNELS FORMED BY PNEUMOLYSIN IN CELLS AND PLANAR LIPID BILAYERS

Pneumolysin is one of the family of thiol-activatable, cytolytic toxin s. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is cons erved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligo mers in the target cell membrane. However, the mutations did affect ha emolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers . Of eight modified pneumolysins examined, Trp-433 --> Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibit ion by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433 --> Phe was least s ensitive to cation inhibition. The ion-conducting channels formed acro ss planar lipid bilayers exhibit small (less than 30 pS), medium (30 p S-1 nS) and large (more than 1 nS) conductance steps. Small-and medium -sized channels were preferentially closed by bivalent cations. In con trast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433 --> Phe formed large channels that were in sensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysac charide of up to 40 kDa did not prevent haemolysis by Trp-433 --> Phe. Electron microscopy revealed that Trp-433 --> Phe formed oligomeric a re and ring structures with dimensions identical with those of wildtyp e toxin, and that the ratio of arcs to rings formed was the same for w ild-type toxin and the Trp-433 --> Phe variant. We conclude that the c hange Trp-433 --> Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and th at the size of are and ring structures revealed by electron microscopy does not reflect the functional state of the channels.