Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides

• Published in: Biophysical journal Vol. 122; no. 6; pp. 950 - 963
• Main Authors: Steigenberger, Jessica, Verleysen, Yentl, Geudens, Niels, Madder, Annemieke, Martins, José C., Heerklotz, Heiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21-03-2023; The Biophysical Society
• Subjects: Cell Membrane Permeability; Liposomes; Peptides, Cyclic - chemistry; Peptides, Cyclic - metabolism; Phosphatidylethanolamines; Phosphatidylglycerols - chemistry; Phosphatidylglycerols - metabolism; Static Electricity
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2022.07.033

Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 μM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 μM) than viscosin (15 μM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the “cation attacks anionic membrane” selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50.