Streptolysin O: Inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation

Streptolysin O is a four-domain protein toxin that permeabilizes animal cel l membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is med iated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by introduci ng single mutant cysteine residues that were then thiol-specifically deriva tized with the environmentally sensitive fluorophor acrylodan, Membrane bin ding of the labeled proteins was accompanied by spectral shifts of the prob e fluorescence, suggesting that the toxin molecule had undergone a conforma tional change. Here we provide evidence that this change corresponds to an allosteric transition of the toxin monomer that is required for the subsequ ent oligomerization and pore formation. The conformational change is revers ible with reversal of binding, and it is related to temperature in a fashio n that closely parallels the temperature-dependency of oligomerization. Fur thermore, we describe a point mutation (N402E) that, while compatible with membrane binding, abrogates the accompanying conformational change. At the same time, the N402E mutation also abolishes oligomerization. These finding s corroborate the contention that the target membrane acts as an allosteric effector to activate the oligomerizing and pore-forming capacity of strept olysin O.