Solid-State NMR Investigations of Peptide-Lipid Interaction and Orientation of a -Sheet Antimicrobial Peptide, Protegrin † Satoru Yamaguchi, ‡ Teresa Hong, § Alan Waring, § Robert I. Lehrer, § and Mei Hong* ,‡ Department of Chemistry, Iowa State UniVersity, Ames, Iowa 50011, and Department of Medicine, UniVersity of California at Los Angeles School of Medicine, Los Angeles, California 90095 ReceiVed March 12, 2002; ReVised Manuscript ReceiVed May 11, 2002 ABSTRACT: Protegrin-1 (PG-1) is a broad-spectrum -sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using 2 H, 31 P, 13 C, and 15 N solid-state NMR spectroscopy. 2 H spectra of mechanically aligned and chain- perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the 31 P spectra of POPC and the 2 H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The 13 CO and 15 N chemical shifts of Val-16 labeled PG-1 indicate that the -strand axis is tilted by 55° ( 5° from the bilayer normal while the normal of the -sheet plane is 48° ( 5° from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized -sheet membrane peptide has been determined by solid- state NMR. Antimicrobial peptides are evolutionarily conserved ele- ments of the innate immune system produced by mammals, amphibians, and insects to kill a broad range of pathogens such as bacteria, viruses, and fungi (1). In general, they act with high potency and speed by disrupting the cell mem- branes of the invading organisms. Since microorganisms cannot easily develop resistance to these compounds, anti- microbial peptides provide a promising alternative to con- ventional antibiotics. However, for many antimicrobial peptides, the structural basis of their antimicrobial action is still incompletely understood. Antimicrobial peptides share some common structural characteristics. They tend to be short, consisting of 15-45 residues. They are rich in cationic residues Arg and Lys. They are often amphipathic, as manifested by the helical wheel diagrams of helical peptides such as magainin (2). Most of these peptides can be sorted into three subclasses according to their predominant conformation: R-helical peptides such as cecropins and melittin; -sheet peptides such as defensins, protegrins, and tachyplesins; and peptides with mixed conformations or rich in -turn structures, such as nisin and tritrpticin (3). While structurally diverse, antimicrobial peptides generally kill microbes by disrupting the cell membranes. This conclusion is based on the observation that many all-D-amino acid analogues of these peptides possess the same antibiotic activities as the parent L-enantiomers (4, 5); thus chiral receptors cannot be involved in antimicrobial activities. Specific models of antimicrobial peptide-lipid interaction have been proposed (6). The barrel-stave model postulates that a bundle of helical peptides forms a membrane-spanning aqueous pore, thus destroying the membrane potential. The main evidence for this model comes from conductance measurements on alamethicin (7-9), a mostly hydrophobic peptide. The toroidal or wormhole model postulates that the peptides form a dynamic supramolecular complex with lipids, with the polar faces of the peptides associated with the polar headgroups of the lipids (2). In contrast to the barrel-stave model, the lipids in these toroidal openings tilt from the lamellar normal and connect the two leaflets of the mem- brane. This model is supported by measurements of the lipid flip-flop rate, peptide translocation rate (2), and neutron diffraction experiments (10). In the carpet model, aggregated helices lie in the plane of the bilayer, due to the favorable electrostatic interaction between the cationic peptide and the † M.H. acknowledges the Arnold and Mabel Beckman Foundation for a Young Investigator Award and the Sloan Foundation for a Research Fellowship. Support for this study was also provided by Grants AI 22839 and AI-37945 from the National Institutes of Health. * Corresponding author. E-mail: mhong@iastate.edu. Tel: (515) 294-3521. Fax: (515) 294-0105. ‡ Iowa State University. § University of California at Los Angeles School of Medicine. 9852 Biochemistry 2002, 41, 9852-9862 10.1021/bi0257991 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/16/2002