Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

Abstract

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions.Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the P0PG:POPC ratio. Vesicles (300 pM POPG) readily aggregated at HNP-2 concentrations above 1 pM, but no mixing of vesicle contents could be detected for concentrations as high as 2 pM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 pM POPG vesicles, 50% of entrapped solute is released by 0.7 p M HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (-400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

Characterization of the Interactions of Alzheimer β‐Amyloid Peptides with Phospholipid Membranes

“…Many peptide-lipid interactions have been investigated for proteins such as mellitin, alamethacin and defensins, by means of synthetic and model membranes. The mechanisms of these interactions fall into the following categories : mernbrane perturbation characterized by disruption of the bilayer lipid organization due to the presence of peptide in the head-group region of the bilayer (Dempsey, 1990;Roux et al, 1994;Wimley et al, 1994), channel formation, which generally occurs at lower peptide concentrations and requires transmembrane potential (Dempsey, 1990;Kagan et al, 1990), and formation and fusion of bilayer micelles characterized by the breakdown of membranes into micelles in a manner similar to that induced by detergent solubilization (Dufourcq et al, 1986;Dempsey, 1990;F u j i et al, 1993). Defensins and mellitin have been shown to be able to fall into all three membrane-disrupting categories : perturbation of lipid order; channel formation ; formation and fusion of bilayer micelles (Demspey, 1990; White et al, 1995).…”

“…for mellitin, defensins and myelin basic protein, binding of lipid membranes may rcsult not only in disruption but in surface binding and aggregation of these membranes (Dempsey 1990;ter Beeat and Hoekstra, 1993;Wimley et al, 1994). To explore these surface effects, we investigated the ability of AAPP peptides to bind and aggregate phospholipids by means of a light- 1.6 scattering assay (Fig.…”

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…Based on this structure, several membrane-binding models were proposed, including wedge, dimer pore, general pore, 86 and multimeric pore. 88 Recently, methods for recombinant expression of correctly folded defensins became available, 89 facilitating high-resolution structural studies of these proteins in membrane-mimetic environments.…”

Functional Determinants of Human Enteric α-Defensin HD5

“…These small, 2-5-kDa peptides are classified into ␣, ␤, and families based on sequence homology and the connectivity of disulfide bonds linking the six conserved cysteine residues (6,7). Defensins bind carbohydrates (8,9), lipids (10,11), and DNA (12) and are active against a wide range of microorganisms, including bacteria (13,14) and viruses (15)(16)(17). Defensins are also capable of interacting with an diverse array of cellular receptors and host proteins, playing important immunomodulatory functions (5,18).…”