Identification of a Membrane-Spanning Domain of the Thiol-Activated Pore-Forming Toxin Clostridium perfringens Perfringolysin O:  An α-Helical to β-Sheet Transition Identified by Fluorescence Spectroscopy

Abstract

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

Molecular basis of listeriolysin O pH dependence

“…The critical step in the inactivation of the soluble LLO monomer is the premature unfolding of the domain 3 TMHs of LLO, structures that normally insert into the membrane of the cell to form the transmembrane ␤-barrel pore (15,16). The premature unfurling of the TMHs is regulated by the presence of an acidic triad of residues in domain 3.…”

“…The scenario is also consistent with the observation that a high salt concentration suppresses the denaturation of LLO, as would be expected if the carboxylate charge was neutralized. Domain 3 is normally poised to unfold the two ␣-helical bundles that are converted to the two TMHs that span the bilayer during pore formation (15,16). Domain 3 is in a metastable state, the interface between domains 2 and 3 is not complementary, and there is a significant twist to the domain 3 core ␤-sheet.…”

“…The hemolytic activity of each toxin on human RBCs was determined as described in ref. 15, except that the hemolysis buffer (buffer C: 35 mM sodium phosphate͞125 mM NaCl) was adjusted with acetic acid to pH 5.5 or 7.4 as described in ref. 2.…”

“…Studies with PFO have revealed that domain 3 undergoes significant secondary and tertiary structural changes in which six ␣-helices unfurl to form the two TMHs during pore formation (15,16). If these hairpins were to prematurely unravel, it would expose core hydrophobic residues protected at the domain 2-domain 3 interface and would also expose the amphipathic structures of the TMHs to the aqueous milieu.…”

“…The ␤-barrel pore is formed by the contribution of twin transmembrane ␤-hairpins (TMHs) from each CDC monomer in the oligomeric pore complex (15,16). We have now determined that LLO is unstable at neutral pH: A triad of acidic residues within the membrane-spanning domain of LLO triggers the premature unraveling of the TMHs in the LLO monomer, resulting in its inactivation.…”