The Identification and Structure of the Membrane-spanning Domain of the Clostridium septicum Alpha Toxin

Jody A Melton,Michael W Parker,Jamie Rossjohn,J Thomas Buckley,Rodney K Tweten

doi:10.1074/jbc.m313758200

Abstract

Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins. The location and structure of the transmembrane domains of these toxins have remained elusive. Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic beta-hairpin of AT has been identified. Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane beta-hairpin. AT derivatives that contained deletions that removed up to 90% of the beta-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism. Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the beta-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced. Together these studies revealed the location and structure of the membrane-spanning domain of AT.

Highlights

Clostridium septicum is a Gram-positive anaerobe that typically causes fulminant, often fatal infections such as nontraumatic gas gangrene or necrotizing enterocolitis in compromised patients [1]
The small lobe of aerolysin is missing from the amino terminus in Alpha toxin (AT), and this implies that AT is a single-lobed structure consisting of three domains that are homologous to D2–D4 of aerolysin
The fluorescence intensity (FI) of NBD is quenched when it is in an aqueous environment, such as the channel of the pore, but increases when it is in a nonpolar environment, such as the core of the bilayer

Summary

Introduction

Clostridium septicum is a Gram-positive anaerobe that typically causes fulminant, often fatal infections such as nontraumatic gas gangrene or necrotizing enterocolitis in compromised patients [1]. Two structural motifs used by pore-forming toxins to span membranes are amphipathic ␤-strands and ␣-helices. Using multiple fluorescent and biochemical approaches, we have confirmed that this amphipathic loop in D2 of AT, comprised of residues Lys-203 to Gln-232, forms an amphipathic ␤-hairpin that spans the membrane and is necessary for pore formation.

Results

Conclusion