Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR

Abstract

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.

FIGURE 1

Amino acid sequences of retrocyclin-2 and RTD-1. The ¹⁵N-labeled residues in retrocyclin-2 are shown in bold.

FIGURE 2

(a) Definition of the tilt angle τ and rotation angle ρ in a β-strand peptide. (b) Calculated 2D ¹⁵N-¹H/¹⁵N correlation spectra as a function of τ and ρ for an 18-residue β-hairpin molecule, using structure 15 of RTD-1. The resonances of all 18 residues are shown. Filled and open circles represent the resonances of β-turn and β-strand residues, respectively.

FIGURE 3

Calculated 2D ¹⁵N-¹H/¹⁵N correlation spectra for the seven ¹⁵N-labeled residues in retrocyclin-2. The spectra are subsets of those in Fig. 2. Note the clear difference between the transmembrane (τ = 10°, ρ = 10°) and in-plane (τ = 90°, ρ = 90°) orientations.

FIGURE 4

(a) Experimental 2D ¹⁵N-¹H/¹⁵N correlation spectrum of retrocyclin-2 in DLPC bilayers (P/L = 1:25). The relative volumes of the resolved peaks are indicated. (b) Best-fit spectrum (open circles) using the measured ¹⁵N chemical shift principal values of the peptide, which are (42, 86, 202) ppm for Gly and (75, 76, 221) ppm for Ile. Best-fit angles: τ = 20°, ρ = 236°. Resonance assignment is indicated. For comparison, the idealized experimental spectrum (solid circles) is superimposed. (c) Best-fit spectrum using standard ¹⁵N chemical shift tensor values of (64, 77, 217) ppm (23) for all sites. The same best-fit angles as (b) are obtained, but the agreement with the experimental spectrum is better than (c), especially for the Gly-8 position. (d) Simulated 2D spectrum with τ = 30°, ρ = 236°. (e) Simulated 2D spectrum for τ = 20°, ρ = 226°. (f) RMSDs between the experiment and simulations as a function of (τ, ρ) angles. The minimum RMSD occurs at τ = 20°, ρ = 236° (star).

FIGURE 5

Retrocyclin-2 orientation in DLPC bilayers. (a) Viewed from the side of the DLPC bilayer. The end-to-end backbone length of the β-hairpin is 27 Å, comparable to the P-P distance of 31 Å for liquid-crystalline DLPC bilayers. (b) Viewed from the top of the lipid bilayer. The C=O bond of residue 2 used for defining the ρ-angle is highlighted. The β-sheet plane is relatively straight.

FIGURE 6

(a) RMSD between the experiment and simulations using a bent hairpin structure of RTD-1. The minimum RMSD of 0.47, which is significantly higher than the experimental RMS noise of 0.20, occurs at β = 88°, α = 347° (star). (b) Best-fit simulation (open circles) superimposed with the experimental spectrum (solid circles). The two differ significantly. (c) RTD-1 structure 13 used for the simulations, showing significant curvature in the β-hairpin. The peptide is shown in its best-fit orientation, which happens to be transmembrane.

FIGURE 7

(a) Simulated 2D ¹⁵N-¹H/¹⁵N correlation spectra (open circles) of an ideal β-hairpin as a function of (τ, ρ) angles. Only the frequencies of the seven labeled residues are shown. Gray lines illustrate the orientation-dependent elliptical patterns on which the strand resonances fall. The best-fit spectrum, near (τ = 70°, ρ = 220°), does not fit the experimental spectrum (solid circles) well. (b) The ideal hairpin conformation.

FIGURE 8

(a) Experimental 2D ¹⁵N-¹H/¹⁵N correlation spectrum of retrocyclin-2 in POPC bilayers (P/L = 1:25). The peak shift to lower chemical shifts compared to the DLPC spectrum (Fig. 4 a) and the strong overlap both indicate a more in-plane orientation of the peptide. (b) Best-fit spectrum with τ = 65° and ρ = 278°. (c) RMSD between the experiment and simulations as a function of (τ, ρ). The minimum RMSD position is indicated by a star. (d) Orientation of retrocyclin-2 in POPC bilayers.

FIGURE 9

³¹P spectra of retrocyclin-2 bound to various oriented lipid bilayers at a peptide concentration of 4%. (a) POPC. (b) POPC/POPG. (c) POPE/POPG.