Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Abstract

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a beta-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the beta-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region

“…Two types of zwitterionic lipid bilayers, containing DPPC and DOPC, and an anionic lipid bilayer containing DOPC/DOPS (mole ratio 4:1) were used in the simulations. Similar anionic lipid bilayers have been extensively simulated (33)(34)(35)(36)(37)(38)(39)(40)(41)(42). For each combination of two HVR peptides with three types of lipid bilayers, six independent simulations labeled HP1 to HP6 were performed.…”

Semen-Derived Enhancer of Viral Infection (SEVI) Binds Bacteria, Enhances Bacterial Phagocytosis by Macrophages, and Can Protect against Vaginal Infection by a Sexually Transmitted Bacterial Pathogen

“…Interestingly, other amyloidogenic peptides such as amyloid-␤ and protegrin-1 (6)(7)(8) have been shown to have bactericidal properties, which are thought to be mediated via a channel-forming mechanism (9). Amyloid-forming peptides have also been shown to mediate antimicrobial effects by binding and sequestering bacteria.…”

Computational Methods for Structural and Functional Studies of Alzheimer’s Amyloid Ion Channels

“…These showed that Aβ channels consisted of β-sheet-rich subunits with morphologies and dimensions in good agreement with the imaged AFM channels [12,29]. The simulations of other amyloids and β-hairpin peptides showed that the subunit-assembly morphology is a common feature for the membrane embedded β-sheet channels [43][44][45].…”

“…PG-1 peptide: A small cationic β-hairpin peptide consisting of 18 amino acids is capable of forming β-sheet channels [44,45]. PG-1 is an antimicrobial peptide (AMP) with a great antibiotic potency [60].…”

“…The designed channels have a perfectly annular shape with the pore-lining inner strands forming a β-sheet through intermolecular backbone hydrogen bonds (H-bonds), but the outer strands do not form a β-sheet due to the larger curvature at the channel periphery. In contrast, β-hairpins generate a single layered annular β-sheet [44,45,59] (Fig. 2c, d).…”