title: Origin of low mammalian cell toxicity in a class of highly active antimicrobial amphipathic helical peptides.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Medicine, Bristol University, Bristol BS8 1TD, United Kingdom.
authors.forename: Ayman
authors.initials: A
authors.lastname: Hawrani
authors.forename: Robin A
authors.initials: RA
authors.lastname: Howe
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Walsh
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Dempsey
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D000890:Anti-Infective Agents / Q000138:chemical synthesis / Q000494:pharmacology* / Q000633:toxicity; D001426:Bacterial Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects*; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D020125:Mutation, Missense; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D034361:Streptococcus mitis / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D010455:Peptides