Binding of Nisin Z to Bilayer Vesicles As Determined with Isothermal Titration Calorimetry

Abstract

Nisin Z, a 34-residue lantibiotic, is secreted by some lactic acid bacteria and exerts its antibacterial activity against various Gram-positive bacteria by permeabilizing the cell membrane. It is a cationic amphiphilic peptide with several unusual dehydro residues and thioether-bridged lanthionines. Isothermal titration calorimetry was used to provide a quantitative thermodynamic description for nisin Z adsorption to and penetration into negatively charged and neutral lipid bilayers. The binding of the cationic peptide (electric charge z approximately 3.8) to anionic membranes was found to be dominated by electrostatic forces which could be described with the Gouy-Chapman theory. For biologically relevant conditions with a membrane surface potential of -40 mV, the peptide concentration near the membrane surface increases by about 2-3 orders of magnitude compared to the bulk concentration. The binding step proper, i.e., the transition from the lipid-water interface into the membrane, is almost exclusively driven by the high surface concentration. Binding can be described by a partition equilibrium of the form X(b) = KC(M) = KC(p,f) exp(-z(p)psi(0)F(0)/RT), where C(M) is the peptide surface concentration, C(p,f) the bulk concentration, and psi(0) the membrane surface potential. The intrinsic partition coefficient (K = 1.8 M(-)(1)) is remarkably small, indicating a correspondingly small hydrophobic energy contribution to the binding process. The electrostatic model was confirmed with nisin Z mutants in which valine-32 was replaced with either lysine (V32K) or glutamate (V32E), increasing or decreasing the electric charge by 1 unit. The extent of peptide binding increased for V32K and decreased for V32E as predicted by the electrostatic theory. In contrast, electrostatic effects were almost negligible for the binding of nisin Z to neutral membranes. However, the binding isotherms were characterized by a distinctly larger intrinsic binding constant K(0) of approximately 540 M(-)(1) and an enhanced hydrophobic free energy of binding. The binding of nisin Z to sonicated lipid vesicles is exothermic with a DeltaH degrees of ca. -9 and -3.4 kcal/mol for charged and neutral membranes, respectively.

Lipid II Is an Intrinsic Component of the Pore Induced by Nisin in Bacterial Membranes

“…For control purposes, nisin was first preincubated with DOPC vesicles lacking Lipid II. In this case, a maximum amount of leakage (about 60%) is obtained as expected, considering the low affinity of nisin for DOPC membranes (22,23). Hardly any activity of nisin was detected if nisin was preincubated with Lipid II variants with prenyl chains of 7 isoprene units or longer, indicating that pores formed by nisin with these variants are stable.…”

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

“…As lipid III and lipid IV are further processed to the linear WTA polymer on the cytoplasmic side, they could only function as targets after internalization of the lantibiotic. Nisin translocation across lipid bilayers, which may occur in the course of pore formation, has been observed in several studies (9,(52)(53)(54). Whether this is true for NAI-107 remains to be determined.…”

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

“…These findings were further supported by isothermal titration calorimetry (ITC) to provide the thermodynamic profile of peptide-membrane interaction [16,17]. ITC has been successfully applied in a number of studies to analyze the membrane interaction of different peptides and proteins [18][19][20][21].…”