Structure, dynamics, and antimicrobial and immune modulatory activities of human LL-23 and its single-residue variants mutated on the basis of homologous primate cathelicidins

Abstract

LL-23 is a natural peptide corresponding to the 23 N-terminal amino acid residues of human host defense cathelicidin LL-37. LL-23 demonstrated, compared to LL-37, a conserved ability to induce the chemokine MCP-1 in human peripheral blood mononuclear cells, a lack of ability to suppress induction of the pro-inflammatory cytokine TNF-α in response to bacterial lipopolysaccharides (LPS), and reduced antimicrobial activity. Heteronuclear multidimensional nuclear magnetic resonance (NMR) characterization of LL-23 revealed similar secondary structures and backbone dynamics in three membrane-mimetic micelles: SDS, dodecylphosphocholine (DPC), and dioctanoylphosphatidylglycerol. The NMR structure of LL-23 determined in perdeuterated DPC contained a unique serine that segregated the hydrophobic surface of the amphipathic helix into two domains. To improve our understanding, Ser9 of LL-23was changed to either Ala or Val on the basis of homologous primate cathelicidins. These changes made the hydrophobic surface of LL-23 continuous and enhanced antibacterial activity. While identical helical structures did not explain the altered activities, a reduced rate of hydrogen-deuterium exchange from LL-23 to LL-23A9 to LL-23V9 suggested a deeper penetration of LL-23V9 into the interior of the micelles, which correlated with enhanced activities. Moreover, these LL-23 variants had discrete immunomodulatory activities. Both restored the TNF-α dampening activity to the level of LL-37. Furthermore, LL-23A9, like LL-23, maintained superior protective MCP-1 production, while LL-23V9 was strongly immunosuppressive, preventing baseline MCP-1 induction and substantially reducing LPS-stimulated MCP-1 production. Thus, these LL-23 variants, designed on the basis of a structural hot spot, are promising immune modulators that are easier to synthesize and less toxic to mammalian cells than the parent peptide LL-37.

FIGURE 1. Production of the pro-inflammatory cytokine TNF-α (A) or the chemokine MCP-1 (CCL2) (B) in response to peptides in the presence or absence of bacterial

LPS. Human PBMC were treated with 20 μg/ml of peptide in the presence or absence of 20 ng/ml of LPS and after 24 h the production of TNF-α or MCP-1 was assessed by ELISA. The data in panel (A) demonstrated that none of the peptides was intrinsically pro-inflammatory and only LL-23 failed to suppress LPS-mediated TNF-α production. The data in panel (B) demonstrated that all of the peptides except LL-23V9 were able to induce substantial levels of baseline MCP-1 but differed in their ability to suppress LPS mediated MCP-1 production. Results represent the means ± SD of 5 experiments.

FIGURE 2. Depolarizing activity of the LL-37 derived peptides

E. coli DC2 susceptible strain was used in these experiments (23). The percent dequenching of fluorescence of the membrane-potential sensitive fluorophore diSC35 was assessed as a function of peptide concentrations of 8 (white bars), 16 (light gray bars) and 32 (dark gray bars) μg/ml. (These concentrations correspond to 3, 6, and 12 μM for the LL-23 series and 2, 4, and 8 μM in the case of the LL-37). As a positive control gramicidin S (GMS) was used at 16 μg/ml.

FIGURE 3

HSQC spectra of ¹⁵N-labeled LL-23 (A) in water, in complex with (B) SDS, (C) D8PG, or (D) DPC at pH 6 and 35°C. The peptide concentration was ~0.5 mM (1.5 mg/ml). For peptide/lipid ratios and other data, see Table 3. For clarity peaks are partially labeled. There are two sets of peaks for R23. Side-chain signals are boxed or connected by a line.

FIGURE 4. Structure and dynamics of ¹⁵N-labeled LL-23 in complex with SDS, D8PG, or DPC micelles

(A) The H^α chemical shift plot of LL-23 in SDS (open squares), D8PG (open circles), or DPC (filled triangles). (B) The ¹⁵N NOE values of LL-23 measured in complex with SDS, D8PG, or DPC micelles under the conditions described in Fig. 3.

FIGURE 5. Solution structures of human LL-23 and its single residue variants in complex with deuterated DPC at pH 5.4 and 25°C

Shown are ensembles of 20 backbone structures of LL-23 (A), LL-23A9 (C), and LL-23V9 (E) with amino acid residues 2–20 superimposed, and ribbon diagrams of LL-23 (B), LL-23A9 (D), and LL-23V9 (F) with side chains labeled. In panels B, D, and F, the residue at position 9 is bolded.

FIGURE 6

Space-filling models of LL-23 (A), LL-23A9 (B) and LL-23V9 (C) in complex with membrane-mimetic DPC micelles. Color code: oxygen, red; nitrogen, blue; carbon and hydrogen, white; and hydrophobic side chains, green. The hydrophobic defect at residue Ser9 of LL-23 (pointed by an arrow) is a hot spot, which formed the basis for structure-based peptide design in panels (B) and (C).

FIGURE 7

Structural comparison of human LL-37 with LL-23 in complex with micelles. The structure of LL-37 (PDB entry: 2K6O) was reported previously (18). The space-filling and ribbon diagram models of LL-37 are shown in panels (A) and (B), while a ribbon diagram of LL-23 (PDB entry: 2LMF) is given in panel (C). Both LL-37 and LL-23 consist of an amphipathic helix followed by a short disordered tail at the C-terminus. Note that only the amphipathic helix portion is required to associate with bacterial membranes. Also in both structures, a hydrophilic Ser9 (in gold) is located on the hydrophobic surface, leading to two hydrophobic domains in each. The two-domain structure (18) explains the cooperative LPS binding of LL-37 (56) and weak LPS binding of LL-23 (see the text).