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Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

View Article: PubMed Central - PubMed

ABSTRACT

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures—formed by a single sequence—may be a key feature in preventing bacterial resistance and could explain why sequence–function relationships in AMPs remain elusive.

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Related in: MedlinePlus

Thermally stabilized maculatin.(a) CD spectra show that wild-type (WT) maculatin starts to denature at elevated temperatures in the presence of 100 nm POPC LUVs (peptide-to-lipid ratio=1/100). (b) WT maculatin-induced LUV leakage is reduced for bilayers containing phosphatidylcholine (PC) lipids with longer hydrophobic tails (14:1 Δ9-cis; 16:1 Δ9-cis; 18:1 Δ9-cis; 20:1 Δ11-cis; 22:1 Δ13-cis). (c) The single-mutation P15A stabilizes maculatin against thermal denaturation, with no detectable loss of helicity even at 74 °C. The temperatures shown were the temperature measured for the cuvette, with 74 °C corresponding to a cell-holder temperature of 95 °C, the highest setting possible. (d) P15A induces similar but less liposomal leakage as WT maculatin, with similar lipid tail length dependence. (e) The pore-sizing assay measures the leakage of dyes of increasing size (400–10,000 Da) from 0.5 mM POPC LUVs (diameter=100 nm) after addition of 0.5 μM peptide (that is, P/L=1/1,000) using fluorescence spectroscopy. Hundred per cent leakage was determined using 10 vol.% Triton X-100. Pore size and leakage efficiency of the P15A single and P15A-E19Q double mutants are similar to that of WT. (f) Octameric pores formed by P15A-E19Q during assembly simulations were found to spontaneously conduct both ANTS and DPX dyes using unbiased conductance simulations. Error bars are s.e.m.
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f1: Thermally stabilized maculatin.(a) CD spectra show that wild-type (WT) maculatin starts to denature at elevated temperatures in the presence of 100 nm POPC LUVs (peptide-to-lipid ratio=1/100). (b) WT maculatin-induced LUV leakage is reduced for bilayers containing phosphatidylcholine (PC) lipids with longer hydrophobic tails (14:1 Δ9-cis; 16:1 Δ9-cis; 18:1 Δ9-cis; 20:1 Δ11-cis; 22:1 Δ13-cis). (c) The single-mutation P15A stabilizes maculatin against thermal denaturation, with no detectable loss of helicity even at 74 °C. The temperatures shown were the temperature measured for the cuvette, with 74 °C corresponding to a cell-holder temperature of 95 °C, the highest setting possible. (d) P15A induces similar but less liposomal leakage as WT maculatin, with similar lipid tail length dependence. (e) The pore-sizing assay measures the leakage of dyes of increasing size (400–10,000 Da) from 0.5 mM POPC LUVs (diameter=100 nm) after addition of 0.5 μM peptide (that is, P/L=1/1,000) using fluorescence spectroscopy. Hundred per cent leakage was determined using 10 vol.% Triton X-100. Pore size and leakage efficiency of the P15A single and P15A-E19Q double mutants are similar to that of WT. (f) Octameric pores formed by P15A-E19Q during assembly simulations were found to spontaneously conduct both ANTS and DPX dyes using unbiased conductance simulations. Error bars are s.e.m.

Mentions: On contact with phosphatidylcholine liposomes, maculatin peptides, which are unstructured in solution, fold into continuous α-helices that form pores in the liposome membrane. First, we used circular dichrosim (CD) spectroscopy to establish experimentally that the P15A mutation, which is active against Gram-positive bacteria489, stabilizes membrane-bound maculatin against thermal denaturation without affecting pore size or pore-forming capactiy for a range of different lipids and peptide-to-lipid ratios (Fig. 1). This thermally stabilized mutant allowed us to elevate the temperatures of unbiased long-timescale folding-partitioning molecular dynamics simulations to 90–150 °C, increasing sampling by 2–3 orders of magnitude, without significantly affecting the thermodynamics of the system1011121314. Next, we experimentally determined the leakage of fluorescent dyes of increasing molecular weight from liposomes treated with maculatin (Fig. 1). This revealed that only the smallest dyes encapsulated (8-aminonaphthalene-1,3,6-trisulphonic acid/p-Xylene-bis-N-pyridinium bromide; ANTS/DPX), with molecular weights of ∼400 Da, are able to leak out of the liposome through pores formed by maculatin peptides, allowing us to estimate the approximate number of peptides forming a pore, by assuming simple channel geometries. Using this information, we built atomic detail systems of up to 16 maculatin peptides placed in phosphatidylcholine lipid bilayers of different acyl chain lengths (Supplementary Table 1).


Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide
Thermally stabilized maculatin.(a) CD spectra show that wild-type (WT) maculatin starts to denature at elevated temperatures in the presence of 100 nm POPC LUVs (peptide-to-lipid ratio=1/100). (b) WT maculatin-induced LUV leakage is reduced for bilayers containing phosphatidylcholine (PC) lipids with longer hydrophobic tails (14:1 Δ9-cis; 16:1 Δ9-cis; 18:1 Δ9-cis; 20:1 Δ11-cis; 22:1 Δ13-cis). (c) The single-mutation P15A stabilizes maculatin against thermal denaturation, with no detectable loss of helicity even at 74 °C. The temperatures shown were the temperature measured for the cuvette, with 74 °C corresponding to a cell-holder temperature of 95 °C, the highest setting possible. (d) P15A induces similar but less liposomal leakage as WT maculatin, with similar lipid tail length dependence. (e) The pore-sizing assay measures the leakage of dyes of increasing size (400–10,000 Da) from 0.5 mM POPC LUVs (diameter=100 nm) after addition of 0.5 μM peptide (that is, P/L=1/1,000) using fluorescence spectroscopy. Hundred per cent leakage was determined using 10 vol.% Triton X-100. Pore size and leakage efficiency of the P15A single and P15A-E19Q double mutants are similar to that of WT. (f) Octameric pores formed by P15A-E19Q during assembly simulations were found to spontaneously conduct both ANTS and DPX dyes using unbiased conductance simulations. Error bars are s.e.m.
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f1: Thermally stabilized maculatin.(a) CD spectra show that wild-type (WT) maculatin starts to denature at elevated temperatures in the presence of 100 nm POPC LUVs (peptide-to-lipid ratio=1/100). (b) WT maculatin-induced LUV leakage is reduced for bilayers containing phosphatidylcholine (PC) lipids with longer hydrophobic tails (14:1 Δ9-cis; 16:1 Δ9-cis; 18:1 Δ9-cis; 20:1 Δ11-cis; 22:1 Δ13-cis). (c) The single-mutation P15A stabilizes maculatin against thermal denaturation, with no detectable loss of helicity even at 74 °C. The temperatures shown were the temperature measured for the cuvette, with 74 °C corresponding to a cell-holder temperature of 95 °C, the highest setting possible. (d) P15A induces similar but less liposomal leakage as WT maculatin, with similar lipid tail length dependence. (e) The pore-sizing assay measures the leakage of dyes of increasing size (400–10,000 Da) from 0.5 mM POPC LUVs (diameter=100 nm) after addition of 0.5 μM peptide (that is, P/L=1/1,000) using fluorescence spectroscopy. Hundred per cent leakage was determined using 10 vol.% Triton X-100. Pore size and leakage efficiency of the P15A single and P15A-E19Q double mutants are similar to that of WT. (f) Octameric pores formed by P15A-E19Q during assembly simulations were found to spontaneously conduct both ANTS and DPX dyes using unbiased conductance simulations. Error bars are s.e.m.
Mentions: On contact with phosphatidylcholine liposomes, maculatin peptides, which are unstructured in solution, fold into continuous α-helices that form pores in the liposome membrane. First, we used circular dichrosim (CD) spectroscopy to establish experimentally that the P15A mutation, which is active against Gram-positive bacteria489, stabilizes membrane-bound maculatin against thermal denaturation without affecting pore size or pore-forming capactiy for a range of different lipids and peptide-to-lipid ratios (Fig. 1). This thermally stabilized mutant allowed us to elevate the temperatures of unbiased long-timescale folding-partitioning molecular dynamics simulations to 90–150 °C, increasing sampling by 2–3 orders of magnitude, without significantly affecting the thermodynamics of the system1011121314. Next, we experimentally determined the leakage of fluorescent dyes of increasing molecular weight from liposomes treated with maculatin (Fig. 1). This revealed that only the smallest dyes encapsulated (8-aminonaphthalene-1,3,6-trisulphonic acid/p-Xylene-bis-N-pyridinium bromide; ANTS/DPX), with molecular weights of ∼400 Da, are able to leak out of the liposome through pores formed by maculatin peptides, allowing us to estimate the approximate number of peptides forming a pore, by assuming simple channel geometries. Using this information, we built atomic detail systems of up to 16 maculatin peptides placed in phosphatidylcholine lipid bilayers of different acyl chain lengths (Supplementary Table 1).

View Article: PubMed Central - PubMed

ABSTRACT

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures—formed by a single sequence—may be a key feature in preventing bacterial resistance and could explain why sequence–function relationships in AMPs remain elusive.

No MeSH data available.


Related in: MedlinePlus