Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry.
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.
Breukink
E
E
Department of Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
Ganz
P
P
de Kruijff
B
B
Seelig
J
J
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D009561:Nisin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055672:Static Electricity; D013816:Thermodynamics; D017186:Titrimetry
D000900:Anti-Bacterial Agents; D008051:Lipid Bilayers; D009561:Nisin; C071364:nisin Z