Streptolysin O: the C-terminal, tryptophan-rich domain carries functional sites for both membrane binding and self-interaction but not for stable oligomerization

Streptolysin O belongs to the class of thiol-activated toxins, which are si ngle chain, four-domain proteins that bind to membranes containing choleste rol and then assemble to form large oligomeric pores. Membrane binding invo lves a conserved tryptophan-rich sequence motif located within the C-termin ally located domain 4. In contrast, sites involved in oligomerization and p ore formation have been assigned to domains 1 and 3, respectively. We here examined the functional properties of domain 4, which was recombinantly exp ressed with an N-terminal histidine tag for purification and an additional cysteine residue for covalent labeling. The fluorescently labeled fragment readily bound to membranes, but it did not form oligomers nor lyse cell mem branes. Moreover, the labeled fragment did not detectably become incorporat ed into hybrid oligomers when combined with lytically active full-length to xin. However, when present in large excess over the active toxin, the domai n 4 fragment effected reduction of hemolytic activity and of functional por e size, which indicates interference with oligomerization of the lytically active species. Our findings support the notion that domain 4 of the strept olysin O molecule may fold autonomously, is essential for membrane binding and is capable not of irreversible but of reversible association with the e ntire toxin molecule. (C) 2001 Elsevier Science B.V. All rights reserved.