Structure and orientation of bovine lactoferrampin in the mimetic bacterial membrane as revealed by solid-state NMR and molecular dynamics simulation

Abstract

Bovine lactoferrampin (LFampinB) is a newly discovered antimicrobial peptide found in the N1-domain of bovine lactoferrin (268-284), and consists of 17 amino-acid residues. It is important to determine the orientation and structure of LFampinB in bacterial membranes to reveal the antimicrobial mechanism. We therefore performed (13)C and (31)P NMR, (13)C-(31)P rotational echo double resonance (REDOR), potassium ion-selective electrode, and quartz-crystal microbalance measurements for LFampinB with mimetic bacterial membrane and molecular-dynamics simulation in acidic membrane. (31)P NMR results indicated that LFampinB caused a defect in mimetic bacterial membranes. Ion-selective electrode measurements showed that ion leakage occurred for the mimetic bacterial membrane containing cardiolipin. Quartz-crystal microbalance measurements revealed that LFampinB had greater affinity to acidic phospholipids than that to neutral phospholipids. (13)C DD-MAS and static NMR spectra showed that LFampinB formed an α-helix in the N-terminus region and tilted 45° to the bilayer normal. REDOR dephasing patterns between carbonyl carbon nucleus in LFampinB and phosphorus nuclei in lipid phosphate groups were measured by (13)C-(31)P REDOR and the results revealed that LFampinB is located in the interfacial region of the membrane. Molecular-dynamics simulation showed the tilt angle to be 42° and the rotation angle to be 92.5° for Leu(3), which are in excellent agreement with the experimental values.

Figure 1

Plot of association constants (K_a) of LFampinB against DMPG weight percentage in the DMPG and DMPC mixed membranes.

Figure 2

Temperature variation of ³¹P NMR spectra of LFampinB-bacterial membrane systems. (a) Acidic phospholipids bilayer with a weight percentage of 65% DMPG, 10% CL, and 25% DMPC. (b) Addition of LFampinB.

Figure 3

¹³C NMR spectra of [1-¹³C] Ala⁷-LFampinB incorporated in the bacterial membrane. (a) DD-MAS (4 kHz) NMR spectrum of hydrated bacterial membrane. (b) DD-static NMR spectrum of hydrated bacterial membrane. (c) Side-band pattern of the CP-MAS (2 kHz) NMR spectrum of lyophilized powder sample (signals with asterisks indicate those of lipid molecules).

Figure 4

(Color figure online.) Contour plots of RMSD against ζ and γ_L3 for N-terminal α-helix of LFampinB (a and b). (a) Chemical shift anisotropies of four amino-acid residues (Leu³, Leu⁴, Ala⁷, Gln⁸) are considered for the RMSD calculation. (b) Chemical shift anisotropies of six amino-acid residues (Leu³, Leu⁴, Ala⁷, Gln⁸, Phe¹¹, Gly¹²) considered for the RMSD calculation. (c) Chemical shift oscillation curve for the N-terminal α-helix of LFampinB calculated by taking into account four amino-acid residues (Leu³, Leu⁴, Ala⁷, Gln⁸). Experimentally obtained values are also shown (solid circles).

Figure 5

Schematic representation of membrane-bound structure for LFampinB by solid-state NMR. (a) Most probable alignment and structure bound to bacterial membrane. (b) Helix wheel and plane angle for carbonyl carbons. The X axis indicates the direction from bilayer normal to the helix axis. Carbonyl plane normal shows 93° from the Y axis.

Figure 6

(Color figure online.) Snapshots of LFampinB in the DMPG membrane (a) before and (b) after 10-ns simulation. The hydrophilic side chains of Lys, Glu, Arg, and α-helix conformation of the peptide backbone are shown (blue, red, cyan, and orange, respectively). Lipid headgroups of phosphatidylglycerol parts, myristic acid chain parts, and water molecules are shown (green, gray, and red, respectively). For simplicity, the hydrophobic side chains of LFampinB, sodium, and chloride ions are not shown.

Figure 7

Tilt-angle ζ of LFampinB (a) and distance between the center of mass of LFampinB helix and membrane center (b) as a function of simulation time. The tilt angle ζ was calculated between the principal axis of LFampinB helix and the Z′ axis, which is normal to the membrane. The principal axis of LFampinB helix was calculated using the backbone main-chain atoms that keep the α-helical structure from residues 2–10. Time dependence of the plane angle γ of carbonyl carbon atom of Leu³ from Y axis in helix wheel (c). The angle was calculated as a clockwise angle from the Y helix axis.