The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure

Abstract

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17-29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-37_17-29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-37_17-29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.

Fig. 1. The effect of LL-37_17-29 and its mutants on the growth of M. luteus.

hLL-37_17-29 and single-point mutants were incubated with M. luteus for 24h at a range of concentrations up to 100 µM, and bacterial growth rate was measured by optical density. From the resulting growth curves, the area under the curve (AUC) was calculated. MIC values were defined as the minimal concentration of the peptide which yielded less than 20% of the AUC of the control (bacteria with no added peptides). The mean MIC value of hLL-37_17-29 is 22 µM. Mean MIC values of the F17A and K18A mutants, substituting residues lining the central pore, are 60 µM. Similarly, the mean MIC value gLL-37_17-29 (equivalent to an F17S mutation) is 53 µM. The K18R, K18H and K18Q mutants showed mean MIC values of 25, 40, and >100 µM, respectively. The Q22A mutation, substituting a residue showing minimal contacts with other residues within the fibrillar assembly, showed a mean MIC value of 33 µM. The I24A, I24S, I24K, I24D and I24Q mutations, substituting a residue fully buried in the four-helix bundle, showed a mean MIC value of >100 µM. The F27A mutation, substituting a buried residue contacting other residues within the four-helix bundle and on surrounding helices in the fibrillar assembly, showed a mean MIC value of >100 µM. The experiments were performed at least three times, each on different days. Dots display measured values of individual repeats. Error bars indicate the standard error calculated between all measures. A t-test for two-sample assuming equal variances was performed, asterisk indicates p < 5 × 10⁻⁰³ and double asterisk indicates p < 5 × 10⁻⁰⁵, compared to hLL-37_17-29. Source data are provided as a Source data file.

Fig. 2. Human and gorilla LL-37_17-29 fibrillar assemblies and interactions with bacteria.

a An electron micrograph of 1 mM hLL-37_17-29 incubated for three days. b An electron micrograph of 1 mM gLL-37_17-29 incubated for 3 days. c An electron micrograph of 30 µM hLL-37_17-29 (close to the MIC concentration of 22 µM) incubated with M. luteus for 4 h. d An electron micrograph of 60 µM gLL-37_17-29 (close to the MIC concentration of 53 µM), incubated with M. luteus for 4 h. All scale bars represent 500 nm.

Fig. 3. The crystal structure of hLL-37_17-29.

The crystal structure of hLL-37_17-29 was determined at 1.35 Å resolution. The crystal packing shows self-assembly of amphipathic helices into a densely packed, elongated hexameric fibril with a central pore. The fibril is composed of four-helix bundles with a hydrophobic core that associated via a network of polar interaction (Fig. 5). a The assembly is shown as grey ribbons, with two representative four-helix bundles colored green and purple to emphasize orientation in the fibril. b The view is rotated by 90˚ in relation to panel a, showing the structure along the fibril axis. c The view is rotated by 30˚ along the fibril axis compared to b. d The fibril, in the same orientation as in panel c, is shown in a surface representation, colored by hydrophobicity, according to the scale bar. e An isolated four-helix bundle shown as green ribbons. f The four-helix bundle, in the same orientation as in e, shown in a surface representation colored by hydrophobicity, according to the scale bar.

Fig. 4. Positively charged electrostatic surface of the four-helix bundle of hLL-37_17-29.

a A projection of the electrostatic potential (φ) onto the molecular surface of the four-helix bundle of hLL-37_17-29; the scale bar indicates φ ranges between −10 kT/e (dark red) and 10 kT/e (dark blue). b The bundle is displayed as ribbons, in the same orientation as in panel a, with side chains shown as sticks. The ribbons and carbons of each of the four helices are colored differently (gray, light blue, tan and pink) and non-carbon atoms are colored by atom type (oxygen in red and nitrogen in blue). Residues are labeled. Arg19 and Arg23 (labeled blue) from two helices lie across each side of the bundle.

Fig. 5. Interfaces of the four-helix bundle with surrounding helices in the fibril assembly.

a One representative four-helix bundle is shown by pink ribbons, and surrounding helices are colored grey. Side chains of the “pink” bundle and residues of surrounding helices which contact the bundle are shown as sticks, colored by atom type (oxygen in red and nitrogen in blue). The asymmetric unit of the crystal contains two chains which are almost identical (RMSD of 0.13 Å), thus, the bundle shows almost four identical interfaces. b A zoom-in view. Potential polar interactions (up to 4.3 Å in distance) are indicated by green dotted lines: Asp26 can form inter-helical electrostatic interactions with Arg23 and with Arg29 from different helices, and intra-helical electrostatic interactions with Arg29. In addition, Lys25 can form electrostatic interactions with the negatively charged C-terminus of an adjacent helix. Overall, each helix shows four inter-helical and one intra-helical electrostatic interactions. In addition, Phe27 faces the middle of the interface, contributing to hydrophobic packing.

Fig. 6. Thermostability of hLL-37_17-29 fibrils.

Transmission electron micrographs of 2 mM LL-37_17-29 incubated for 3 days. a The sample was heated to 60 °C for 10 min. b The sample was incubated for additional 24 h at 37 °C after the 60 °C heat shock. c The sample was heated to 80 °C for 10 min All scale bars represent 500 nm.

Fig. 7. Human and gorilla LL-37_17-29 share fibrillary architecture but differ in central pore properties.

Comparison of human (a) and gorilla (b) LL-37_17-29 crystal structures, shown in grey ribbons with side chain shown as sticks colored by atom type (oxygen in red and nitrogen in blue). The view is down the fibril axis, showing the hexametric arrangement and the central pore. The overall structure of the two is highly similar (RMSD of 0.15 Å for the asymmetric unit, comprising two helices and a similar space group and unit cell dimensions (Table 1)). The N-termini of the helices, with Phe17 in hLL-37_17-29 or Ser17 in gLL-37_17-29, and Lys18, line the central pore. The pore of gLL-37_17-29 is more occluded, due to the orientation of the lysine residues extending into the pore. In the hLL-37_17-29 structure, the lysine residues are almost perpendicular to the cross-section of the pore.