On the mechanism of pore formation by melittin

Abstract

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.

FIGURE 1.

Models of pore formation by melittin in membranes composed of zwitterionic PC lipids. A, existing model. Step 1: At low concentrations, melittin (∼) binds to the membrane and forms an amphipatic α-helix oriented parallel to the membrane. Step 2: If the melittin concentration reaches above a certain threshold, melittin inserts in the membrane and the orientation shifts to largely perpendicular, causing pore formation. B, new model. Melittin (∼) binds and forms an amphipatic α-helix and can be oriented either parallel (step 1) or perpendicular (step 2) to the plane of the membrane. The perpendicular orientation leads to membrane insertion and pore formation, whereas the parallel orientation is inactive and prevents other melittin molecules from forming pores, hence protecting the membrane (dotted line).

FIGURE 2.

Calcein dequenching as a function of lipid (liposome) concentration. A, calcein dequenching from liposomes composed of pure DOPC was measured for various melittin concentrations. The final lipid concentrations were 0.25 μm (1, ▪), 0.5 μm (2, ○), 1 μm (3, ▴), 2 μm (4, ▾), 4 μm (5, ♦), 8 μm (6, ◂), and 16 μm (7, ▸). Leakage took place within 5 min and was stable for over 24 h. The typical error from at least two independent experiments is indicated. The solid lines present fits with a cumulative log normal distribution, and these allowed us to estimate the concentration of melittin where 50% of the calcein leaked out (C_1/2, dotted lines) (18). B, the C_1/2 values for the various lipid concentrations obtained from A are shown. The dashed line shows the result of the linear regression analysis, with a slope of 11 nm melittin/μm lipids and an offset of 21 nm (dotted line). C, CD spectra of melittin are shown. The mean residue ellipticity [θ] is plotted as a function of the wavelength for 44 μm melittin in 10 mm potassium phosphate, pH 7, plus various concentrations of NaCl. Melittin is in the unfolded conformation in the absence of NaCl (solid line); 2.5 m NaCl stabilizes the α-helical conformation of melittin (dotted line) (25). At the NaCl concentration used in this study (150 mm, dashed line), melittin is predominantly in the unfolded conformation.

FIGURE 3.

Two-step leakage experiments with melittin and DOPC liposomes. A, calcein dequenching experiments. At time t₁, 1 μm total lipid concentration in the form of liposomes were added to the cuvette. The liposomes were loaded with either 100 mm calcein (solid, dashed curves) or did not contain calcein (dotted curve). At time t₂, 35 nm melittin was added to the cuvette, and leakage was determined from the changes in fluorescence. For the solid and dotted curves, a second batch (1 μm) of liposomes loaded with calcein was added at time t₃. At time t₄ (solid, dotted) or time t₃ (dashed), 0.03% (w/v) of Triton X-100 was added to determine the 100% level of leakage. B, two-step calcein dequenching experiments similar to A, with various concentrations of melittin. The leakage of the first (solid line, ▪) and second batch of liposomes (dotted line, •) is indicated. C, similar to B but using dual-color fluorescence-burst analysis (18, 20) instead of the calcein dequenching assay, and using 250 μm lipids. The experiments from A–C show that melittin is capable of pore formation in freshly added liposomes but not in liposomes already present in solution. D, reversible binding of melittin to the membranes. DOPC liposomes were equilibrated with 44 μm of melittin, and the fractions of liposome-bound and free melittin were determined (x-axis, lipid concentration). The liposomes were harvested and washed by centrifugation and then dissolved with 1% (w/v) n-dodecyl-β-d-maltoside. The melittin concentrations of the supernatant (solid line), washing solution (dotted line), and pellet (dashed line) were determined by tryptophan fluorescence. Error bars are from at least 2 independent experiments.

FIGURE 4.

Two-step leakage experiments with melittin and DOPG liposomes. A, calcein dequenching experiments similar to Fig. 3A, but using 1 μm DOPG lipids in the form of liposomes and 250 nm melittin. The decrease in fluorescence at time t₂ is not caused by photobleaching but probably by fusion of the liposomes (18). B, two-step calcein dequenching experiments similar to A, with various concentrations of melittin. The leakage of the first (solid line, ▪) and second batch of liposomes (dotted line, •) is indicated. C, same as Fig. 3D, but for liposomes composed of pure DOPG instead of DOPC. The experiments from A–C indicate that melittin did not cause leakage of the second batch and that all melittin irreversibly bound to the first batch of DOPG liposomes. Error bars are from at least two independent experiments.