Characterization of the structure and membrane interaction of the antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

Abstract

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH(2)), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH(2)), both from Litoria aurea, and a carboxy C-terminal analog of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied to determine which features of this class of peptides are key to activity. Circular dichroism and solution-state NMR data indicate that all three peptides adopt an alpha-helical structure in the presence of trifluoroethanol or lipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide/lipid molar ratios (P/L) = 1:15-1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface adsorbed over a range of low peptide concentrations but insert into the bilayers at high peptide concentrations. This finding is corroborated by (31)P-solid-state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. Oriented circular dichroism data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, show that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15-1:120), whereas the aurein 2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied interact with phosphatidylcholine membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in phosphatidylcholine/phosphatidylglycerol (PC/PG) membranes, whereas aurein 2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations of the three peptides against Staphylococcus aureus (strain C622, ATCC 25923) and Staphylococcus epidermidis (strain C621--clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.

FIGURE 1

Solution CD spectra of the aurein peptides in water/TFE mixtures: (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH (solid black line, 100% H₂O; black dotted line, 75% H₂O/25% TFE; solid gray line, 50% H₂O/50% TFE; gray dotted line, 25% H₂O/75% TFE). The spectra indicate that, in all cases, the peptides are unstructured in water but adopt an α-helical conformation upon addition of TFE.

FIGURE 2

Solution CD spectra of the aurein peptides in DMPC SUVs: (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH (solid black line, P/L = 1:15; dotted line, P/L = 1:50; solid gray line, P/L = 1:100). The spectra indicate that the peptides adopt an α-helical conformation in the presence of DMPC SUVs.

FIGURE 3

Solution CD spectra of the aurein peptides in DMPC/DMPG (1:1) SUVs: (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH (solid black line, P/L = 1:15; dotted line, P/L = 1:50; solid gray line, P/L = 1:100). The spectra indicate that the peptides adopt an α-helical conformation in the presence of DMPC/DMPG SUVs.

FIGURE 4

Fingerprint region of solution NMR NOESY spectra of (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH. The spectra were acquired using a phase-sensitive NOESY experiment, with excitation sculpting with gradients for water suppression (see text). All spectra were acquired at 25°C, using 64 scans and a mixing time of 150 ms. The spectra were referenced to the residual methylene protons present in d3-TFE (3.918 ppm). In b, arrows indicate some of the connectivities use to perform the sequential assignment. In ambiguous cases, the HN-HN region was also used to confirm i to i + 1 connectivities.

FIGURE 5

NMR-derived evidence indicating that the aurein peptides are α-helical: (a) Hα chemical shift differences for Aur2.2-CONH₂ (hashed), Aur2.3-CONH₂ (solid black), and Aur2.3-COOH (open); (b) typical NOE connectivities observed for these peptides—shown here for Aur2.3-CONH₂ only. Solid black bars represent unambiguous NOEs, and gray bars represent connectivities which are present but are ambiguous due to overlap.

FIGURE 6

Oriented CD spectra for (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH in DMPC and (a) Aur2.2-CONH₂, (b) Aur2.3-CONH₂, and (c) Aur2.3-COOH in DMPC/DMPG (1:1). P/L molar ratios = 1:15 (blue), 1:30 (green), 1:40 (red), 1:80 (black), and 1:120 (gray). The spectra were normalized such that the intensities of all spectra at 222 nm are the same. The spectra show that the peptides insert into the DMPC bilayer at threshold P/L* molar ratios between 1:15 and 1:30 for Aur2.2-CONH₂ and Aur2.3-CONH₂, and 1:30 and 1:40 for Aur2.3-COOH. In DMPC/DMPG (1:1), the amidated peptides are inserted over the entire concentration range, whereas the P/L* is between 1:120 and 1:80 for Aur2.3-COOH under these conditions.

FIGURE 7

³¹P-solid-state NMR spectra of all three aurein peptides oriented in DMPC bilayers. The spectra were recorded using 2048 scans at 30°C, oriented such that the membrane normal was parallel to the external magnetic field. The spectra were processed without any line broadening (see text for further experimental details).