An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of a crotalicidin fragment

Abstract

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

Figure 1

Crotalicidin-derived peptides used in this study. (a) Domain structure of crotalicidin pre-pro-peptide (Uniprot U5KJM4) in brackets: length of domains in numbers of amino acids. Flanking the peptide, the pro-peptide features a 35 amino acids long glutamic acid rich domain, whose sequence is also given in red (21 negatively charged residues, 6 positively charged residues), together with that of the full-length crotalicidin peptide in blue (15 positively charged residues). (b) Short model peptides inspired by full-length crotalicidin peptide and pro-peptide, used in this study. The Ctn[15–34] fragment has 7 positively charged residues.

Figure 2

Circular dichroism spectra of the peptides Ctn[15–34] (red), E₁₀-Ctn[15–34] (black) and (GS)₄-Ctn[15–34] (blue). Residue molar ellipticity [θ] in deg.cm²/dmol is plotted versus the wavelength (λ in nm). Measurements were performed at a peptide concentration of 0.2 μg.mL⁻¹ in a 10 mM K₂HPO₄ 50 mM Na₂SO₄ buffer, pH 7.4.

Figure 3

Molecular dynamics simulations in water (100 ns) for the peptides Ctn[15–34] (black curves), E₁₀-Ctn[15–34] (red curves) and (GS)₄-Ctn[15–34] (green curves), yielding the parameters (a) Root mean square deviation (RMSD) of single run, (b) root mean square fluctuations (RMSF) and (c) radius of gyration (R_g). Results are for single runs, three runs were done, for each peptide, with similar results.

Figure 4

Theoretical structures snapshots of (a) Ctn[15–34] (b) E₁₀-Ctn[15–34] and (c) (GS)₄-Ctn[15–34] during 100 ns of dynamics simulations (50 ns intervals). Domain colors: basic domain Ctn[15–34] – dark blue, hydrophobic domain Ctn[15–34] - orange, acidic E₁₀ domain – light blue, control pro-peptide domain (GS)₄ – green.

Figure 5

Toxicity of peptides Ctn[15–34] E₁₀-Ctn[15–34] and GS₄-Ctn[15–34]. (a) In vitro neutral red uptake assay for Caco-2 cells. Viability (fraction of viable cells) as compared to untreated Caco-2 cells as a function of peptide concentration. Diamonds: filled squares: PBS control, open squares: Ctn[15–34] open diamonds: E₁₀-Ctn[15–34] open triangles: (GS)₄-Ctn[15–34]. Error bars are standard deviations of replicate measurements. (b) In vivo toxicity of the peptides for Galleria mellonella larvae. Percentage surviving larvae as a function of time. Filled squares: H₂O control, filled triangles: no treatment control, open triangles: Ctn[15–34] open diamonds: E₁₀-Ctn[15–34] open squares: (GS)₄-Ctn[15–34]. For each peptide a group of n = 15 larvae were used, error bars are standard deviations within the group.

Figure 6

Adsorption of the peptides Ctn, Ctn[15–34] E₁₀-Ctn[15–34] and GS₄-Ctn[15–34] to mixed DOPC:DOPG (7:3 molar ratio) supported lipid bilayers (SLB). Peptide mass Γ (mg/m²) adsorbing to the SLB as a function of time. Curves from top to bottom are for Ctn, Ctn[15–34] GS₄-Ctn[15–34] and E₁₀-Ctn[15–34] Peptide concentrations are 16 μg.mL⁻¹ in 10 mM Tris 100 mM NaCl, pH 7.5.

Figure 7

Liposome leakage assay for the peptides Ctn, Ctn[15–34] E₁₀-Ctn[15–34] and (GS)₄-Ctn[15–34]. Percentage of leakage as a function of time t(s). Mixed DOPC:DOPG (7:3 molar ratio) liposomes with a diameter of 190 nm filled with calcein (70 mM inside the liposomes), at a lipid concentration of 50 μM, were exposed to 128 μg.mL⁻¹ of the peptides. Peptides were injected at t = 2 min (I). At t = 10 min (II) a small amount of 10% (v/v) Triton X-100 was added to release all of the calcein. Two replicates are shown for each peptide with different colors as indicated in the figure.