Membrane insertion and bilayer perturbation by antimicrobial peptide CM15

Abstract

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2-9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an alpha-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2-3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

FIGURE 1

Structures of the MTSL and BrMTSL spin labels and the resulting side chains produced by reaction with the peptide cysteine residue.

FIGURE 2

CD spectra of C4BrMTSL in 5 mM phosphate buffer (crosses) in 50% TFE (squares) and in the presence of LUVs at an L/P ratio of 100:1 (triangles). The peptide concentration was 0.1 mM (∼0.2 mg/ml) in each sample.

FIGURE 3

EPR spectra of C4-MTSL in (A) 50 mM MOPS buffer, (B) in 50% TFE, and in the presence of PE/PG/CL (70:25:5) LUVs at L/P ratios of (C) 250:1, (D) 50:1, and (E) 25:1. Spectra for the membrane-bound peptide are presented at a 10-fold higher gain. The scan width is 100 G.

FIGURE 4

EPR spectra of C4-BrMTSL in (A) 50 mM MOPS buffer, (B) in 50% TFE, and in the presence of PE/PG/CL (70:25:5) LUVs at L/P ratios of (C) 250:1, (D) 50:1, and (E) 25:1. Scan width 100 G. Spectra for the membrane-bound peptide are presented at a 10-fold higher gain. Binding to the LUVs results in a pronounced reduction in spin label mobility, and the BrMTSL side chain is significantly more immobilized than the nonbrominated MTSL side chain in the membrane-bound state (compare with Fig. 3).

FIGURE 5

EPR spectra of C4MTSL bound to membranes in the frozen state. C4MTSL was mixed with LUVs at final L/P ratios of (A) 160:1, (B) 80:1, (C) 40:1, and (D) 20:1 and allowed to equilibrate at room temperature for 1 h. The samples were then placed in the EPR cavity and brought to 200 K. Scan widths are 160 G. No changes in line widths or relative amplitudes were observed as a function of the L/P ratio.

FIGURE 6

Accessibilities of C4-MTSL (squares) and C4-BrMTSL (triangles) as a function of the L/P ratio. (A) The change in the EPR saturation parameter, P_1/2, in the presence of 20 mM NiEDDA, (B) the change in P_1/2 upon equilibration with air (20% O₂), and (C) the EPR depth parameter, calculated as described in the text. Error bars indicate standard deviations from at least three separate measurements.

FIGURE 7

EPR spectra of (A) 5PCSL, (B) 7PCSL, and (C) 12PCSL in PE/PG/CL liposomes. In each set of spectra the upper spectrum is in the absence of peptide and the lower spectrum is in the presence of wild-type CM15 at an L/P ratio of 25:1. The increase in 2T_// observed for 5- and 7PCSL indicate a decrease in rotational mobility. For 12PCSL there is no measurable difference in the width of the center line (ΔH₀) or in the relative line amplitudes (A₀ and A₋₁). Scan widths are 100 G for 5- and 7PCSL, and 80 G for 12PCSL.

FIGURE 8

Effect of wild-type CM15 binding on the motion of PCSLs. CM15 was added to PE/PG/CL (70:25:5) LUVs containing 1 mol % PCSL and equilibrated at room temperature overnight before recording the EPR spectrum. The motion parameter 2T_// for 5PCSL (squares) and 7PCSL (triangles) is plotted as a function of the lipid ratio (with ∞ corresponding to control values in the absence of peptide). An increase in 2T_// indicates decreased mobility of the spin label. Error bars indicate the standard deviation from at least three separate experiments.

FIGURE 9

Effect of wild-type CM15 binding on the accessibility of PCSLs to NiEDDA. Large unilamellar liposomes (PE/PG/CL, molar ratio 70:25:5) containing 1 mol % of 5PCSL (squares), 7PCSL (circles), or 12PCSL (triangles) were mixed with CM15 to give the desired L/P ratio, incubated overnight at room temperature and the CW saturation parameter P_1/2 determined under N₂. Final NiEDDA concentration was 20 mM.

FIGURE 10

Effect of wild-type CM15 binding on the EPR depth parameter. The depth parameter for 12PCSL (triangles), 7PCSL (circles), and 5PCSL (squares) is plotted against the L/P molar ratio. The spin labels were at a concentration of 1 mol % in PE/PG/CL LUVs. Error bars represent the standard deviation from at least three independent experiments.