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J. Biol. Chem., Vol. 279, Issue 14, 14315-14322, April 2, 2004

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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, and Rodney K. Tweten

From the Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, the Biota Structural Biology Laboratory, St Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia, ^||The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, Monash University, School of Biomedical Sciences, Melbourne, Victoria 3168, Australia, and the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada

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 -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 -hairpin. AT derivatives that contained deletions that removed up to 90% of the -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 -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.

Received for publication, December 16, 2003 , and in revised form, January 9, 2004.

^* The work was supported in part by National Institutes of Health Grant AI37657 (to R. K. T.) and a grant from the National Health and Medical Research Council of Australia (to M. W. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ A National Health Medical Research Council Senior Principal Research Fellow.

^** A Wellcome Trust Senior Research Fellow.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 940 Stanton L. Young Blvd, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190. Tel.: 405-271-2133; Fax: 405-271-3117; E-mail: Rod-Tweten{at}ouhsc.edu.

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