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J. Biol. Chem., Vol. 283, Issue 8, 4632-4642, February 22, 2008

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How Interaction of Perfringolysin O with Membranes Is Controlled by Sterol Structure, Lipid Structure, and Physiological Low pH

INSIGHTS INTO THE ORIGIN OF PERFRINGOLYSIN O-LIPID RAFT INTERACTION^*

Lindsay D. Nelson¹, Arthur E. Johnson, and Erwin London²

From the Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215 and the Dept. of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114

Perfringolysin O (PFO) is a sterol-dependent, pore-forming cytolysin. To understand the molecular basis of PFO membrane interaction, we studied its dependence upon sterol and lipid structure and aqueous environment. PFO interacted with diverse sterols, although binding was affected by double bond location in the sterol rings, sterol side chain structure, and sterol polar group structure. Importantly, a sterol structure promoting formation of ordered membrane domains (lipid rafts) was not critical for interaction. PFO membrane interaction was also affected by phospholipid acyl chain structure, being inversely related to tight acyl chain packing with cholesterol. Experiments using the pre-pore Y181A mutant demonstrated that sterol binding strength and specificity was not affected by whether PFO forms a transmembrane β-barrel. Combined, these observations are consistent with a model in which the strength and specificity of sterol interaction arises from both sterol interactions with domain 4 and sterol chemical activity within membranes. The lipid raft-binding portions of sterol bound to PFO may remain largely exposed to the lipid bilayer. These results place important constraints upon the origin of PFO raft affinity. Additional experiments demonstrated that the structure of membrane-inserted PFO at low and neutral pH was similar as judged by the effect of phospholipid and sterol structure upon PFO properties and membrane interaction. However, low pH enhanced PFO membrane binding, oligomerization, and pore formation. In lipid vesicles mimicking the exofacial (outer) membrane leaflet, PFO-membrane binding was maximal at pH 5.5–6. This is consistent with the hypothesis that PFO function involves acidic vacuoles.

Received for publication, November 19, 2007 , and in revised form, December 11, 2007.

^* This work was supported by National Institutes of Health Grants GM 48596 (to E. L.) and AI 37657 (to R. K. Tweten and A. E. J.), and by the Robert A. Welch Foundation (Chair Grant BE-0017). 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.

¹ Supported by National Research Service Award T32 AI007539 [GenBank] .

² To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215. Tel.: 631-632-8564; Fax: 631-632-8575; E-mail: erwin.london{at}stonybrook.edu.

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