NMR structure of the cathelicidin-derived human antimicrobial peptide LL-37 in dodecylphosphocholine micelles

Abstract

LL-37 is the only cathelicidin-derived polypeptide found in humans. Its eclectic function makes this peptide one of the most intriguing chemical defense agents, with crucial roles in moderating inflammation, promoting wound healing, and boosting the human immune system. LL-37 kills both prokaryotic and eukaryotic cells through physical interaction with cell membranes. In order to study its active conformation in membranes, we have reconstituted LL-37 into dodecylphosphocholine (DPC) micelles and determined its three-dimensional structure. We found that, under our experimental conditions, this peptide adopts a helix-break-helix conformation. Both the N- and C-termini are unstructured and solvent exposed. The N-terminal helical domain is more dynamic, while the C-terminal helix is more solvent protected and structured (high density of NOEs, slow H/D exchange). When it interacts with DPC, LL-37 is adsorbed on the surface of the micelle with the hydrophilic face exposed to the water phase and the hydrophobic face buried in the micelle hydrocarbon region. The break between the helices is positioned at K12 and is probably stabilized by a hydrophobic cluster formed by I13, F17, and I20 in addition to a salt bridge between K12 and E16. These results support the proposed nonpore carpet-like mechanism of action, in agreement with the solid-state NMR studies, and pave the way for understanding the function of the mature LL-37 at the atomic level.

Figure 1

Primary amino acid sequence of LL-37. The regions defined by the arrows indicate the active polypeptides identified by different research groups.

Figure 2

Fingerprint region of LL-37 extracted from a 2D [¹H^–1H] NOESY experiment at 300 ms mixing time. The sample consisted of 1 mM LL-37 reconstituted into phosphate buffer containing 300 mM DPC at pH 6.0.

Figure 3

Summary of the backbone NOESY cross-peaks, Hα CSI, and H/D exchange experiments.

Figure 4

(A) Histogram of NOEs versus residues for LL-37. (B) Histogram of the backbone RMSD versus residues for the final 40 LL-37 conformers. The superpositions of the conformers from residue 4 to residue 33 and from residue 12 to residue 33 are reported.

Figure 5

(A) Representative structure of LL-37 showing the angle between the two helical domains and the break point centered at K12. (B) Ensemble of conformers for LL-37 showing the convergence of the conformers for backbone atoms. (C) Heavy atom representation of the final calculated ensemble of conformers showing the convergence of the side chains. The structures were fit onto the relaxed average conformation.

Figure 6

Kinked region of LL-37. The proximity of K12 and E16 side chains in most of the conformers generated using the simulated annealing procedure suggests the presence of a salt bridge between these residues. Also, the hydrophobic cluster formed by I13, F17, and I20 stabilizes the kink between the two helices.

Figure 7

(A) Intensity retention (I_R) plots for Hα of LL-37 in the presence of 1:2 (blue trace) and 1:4 (red trace) peptide to Gd³⁺ ratios. (B) Selected region of the NOESY spectrum showing the NOEs between the aromatic residues and the methylene detergent resonances.

Figure 8

Molecular model of LL-37 embedded into a DPC micelle. (A) LL-37 orientation obtained from the pseudomicelle restraints. The black sphere represents the target distance (20 Å); the outer and inner spheres represent the lower and upper bounds (±4 Å). (B) LL-37 docked to a DPC micelle. The coordinates of the micelle consisting of 65 DPC lipids and equilibrated with molecular dynamics in the presence of 6305 water molecules were downloaded from the Biocomputing Web Site at the University of Calgary (Department of Biological Sciences at the University of Calgary, http://moose.bio.ucalgary.ca/) and overlaid to the coordinates of the pseudomicelle sphere.