Molecular Basis for Nanoscopic Membrane Curvature Generation from Quantum Mechanical Models and Synthetic Transporter Sequences

We investigate the physical origin of peptide-induced membrane curvature by contrasting differences between H-bonding interactions of prototypical cationic amino acids, arginine (Arg) and lysine (Lys), with phosphate groups of phospholipid heads using quantum mechanical (QM) calculations of a minimum model and test the results via synthetic oxaorbornene-based transporter sequences without the geometric constraints of polypeptide backbones. QM calculations suggest that although individual Lys can in principle coordinate two phosphates, they are not able to do so at small inter-Lys distances without drastic energetic penalties. In contrast, Arg can coordinate two phosphates down to less than 5 Å, where guanidinium groups can stack “face to face”. In agreement with these observations, poly-Lys cannot generate the nanoscale positive curvature necessary for inducing negative Gaussian membrane curvature, in contrast to poly-Arg. Also consistent with QM calculations, polyguanidine-oxanorbornene homopolymers (PGONs) showed that curvature generation is exquisitely sensitive to the guanidinium group spacing when the phosphate groups are near close packing. Addition of phenyl or butyl hydrophobic groups into guanidine-oxanorbornene polymers increased the amount of induced saddle-splay membrane curvature and broadened the range of lipid compositions where saddle-splay curvature was induced. The enhancement of saddle-splay curvature generation and relaxation of lipid composition requirements via addition of hydrophobicity is consistent with membrane activity profiles. While PGON polymers displayed selective antimicrobial activity against prototypical (Gram positive and negative) bacteria, polymers with phenyl and butyl groups were also active against red blood cells. Our results suggest that it is possible to achieve deterministic molecular design of pore-forming peptides

A Critical Evaluation of Random Copolymer Mimesis of Homogeneous Antimicrobial Peptides

Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a “minimal” mimic, a well-characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small-angle X-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs

Criterion for Amino Acid Composition of Defensins and Antimicrobial Peptides Based on Geometry of Membrane Destabilization

Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small-angle X-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (“negative Gaussian”) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, because it is a necessary condition for processes such as pore formation, blebbing, budding, and vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This “design rule” is consistent with the amino acid compositions of 1080 known cationic AMPs

Apolipoprotein Mimetic Peptide Inhibits Neutrophil-Driven Inflammatory Damage via Membrane Remodeling and Suppression of Cell Lysis

Neutrophils are crucial for host defense but are notorious for causing sterile inflammatory damage. Activated neutrophils in inflamed tissue can liberate histone H4, which was recently shown to perpetuate inflammation by permeating membranes via the generation of negative Gaussian curvature (NGC), leading to lytic cell death. Here, we show that it is possible to build peptides or proteins that cancel NGC in membranes and thereby suppress pore formation, and demonstrate that they can inhibit H4 membrane remodeling and thereby reduce histone H4-driven lytic cell death and resultant inflammation. As a demonstration of principle, we use apolipoprotein A-I (apoA-I) mimetic peptide apoMP1. X-ray structural studies and theoretical calculations show that apoMP1 induces nanoscopic positive Gaussian curvature (PGC), which interacts with the NGC induced by the N-terminus of histone H4 (H4n) to inhibit membrane permeation. Interestingly, we show that induction of PGC can inhibit membrane-permeating activity in general and “turn off” diverse membrane-permeating molecules besides H4n. In vitro experiments show an apoMP1 dose-dependent rescue of H4 cytotoxicity. Using a mouse model, we show that tissue accumulation of neutrophils, release of neutrophil extracellular traps (NETs), and extracellular H4 all strongly correlate independently with local tissue cell death in multiple organs, but administration of apoMP1 inhibits histone H4-mediated cytotoxicity and strongly prevents organ tissue damage