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J. Biol. Chem., Vol. 283, Issue 27, 18636-18645, July 4, 2008

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Origin of Low Mammalian Cell Toxicity in a Class of Highly Active Antimicrobial Amphipathic Helical Peptides^*

Ayman Hawrani^¶, Robin A. Howe, Timothy R. Walsh^¶, and Christopher E. Dempsey^||1

From the ^||Biochemistry Department and Department of Cellular and Molecular Medicine, Bristol University, Bristol BS8 1TD, National Public Health Service Cardiff, University Hospital of Wales, Cardiff CF14 4XW, and the ^¶Department of Medical Microbiology, Cardiff University, Cardiff CF14 4XN, United Kingdom

We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316–12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.

Received for publication, November 7, 2007 , and in revised form, March 31, 2008.

^* This work was supported by a Wellcome Trust university translation award (to T. R. W., R. A. H., and C. E. D.). 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 To whom correspondence should be addressed: Biochemistry Dept., School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. Fax: 117 3312168; E-mail: c.dempsey{at}bris.ac.uk.

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