Simulation studies of the interaction of antimicrobial peptides and lipid bilayers

Experimental studies of a number of antimicrobial peptides are sufficiently detailed to allow computer simulations to make a significant contribution to understanding their mechanisms of action at an atomic level. In this review we focus on simulation studies of alamethicin, melittin, dermaseptin and related antimicrobial, membrane-active peptides. All of these peptides form amphipathic alpha-helices. Simulations allow us to explore the interactions of such peptides with lipid bilayers, and to understand the effects of such interactions on the conformational dynamics of the peptides. Mean field methods employ an empirical energy function, such as a simple hydrophobicity potential, to provide an approximation to the membrane. Mean field approaches allow us to predict the optimal orientation of a peptide helix relative to a bilayer. Molecular dynamics simulations that include an atomistic model of the bilayer and surrounding solvent provide a more detailed insight into peptide-bilayer interactions. In the case of alamethicin, all-atom simulations have allowed us to explore several steps along the route from binding to the membrane surface to formation of transbilayer ion channels. For those antimicrobial peptides such as dermaseptin which prefer to remain at the surface of a bilayer, molecular dynamics simulations allow us to explore the favourable interactions between the peptide helix sidechains and the phospholipid headgroups.     

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A.

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the self-assembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCl, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.     

Pore formation by LamB of Escherichia coli in lipid bilayer membranes.

Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.     

Determining the mechanism of membrane permeabilizing peptides: Identification of potent, equilibrium pore-formers

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.     

Poisson-Nernst-Planck Models of Nonequilibrium Ion Electrodiffusion through a Protegrin Transmembrane Pore

Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP) calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of −100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD) simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290–680 pS. Better agreement with the experimental range of 40–360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (I_Cl−/I_K+) on the order of 10³. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their conductance characteristics.     

Ion channels of alamethicin dimer N-terminally linked by disulfide bond

A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore.     

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of beta-barrel pore-forming toxins (beta-PFTs). By contrast, other beta-PFTs form homo-oligomeric pores; for example, the staphylococcal alpha-hemolysin (alpha HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the alpha HL pore. It is also pertinent to the mechanism of assembly of beta-PFT pores and suggests new possibilities for engineering these proteins.     

Toroidal pores formed by antimicrobial peptides show significant disorder

A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism.     

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.     

pH-triggered pore-forming peptides with strong composition-dependent membrane selectivity

Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ～6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.     

Structure-function relationship of model Aib-containing peptides as ion transfer intermembrane templates

Peptaibols comprise a family of peptide antibiotics with high contents of 2-aminoisobutyric acid (Aib) residues and C-terminal amino alcohols. These peptides form alpha-helical structures leading to voltage-gated ion channels in lipid membranes. In the present study, amphiphilic helical Aib-containing peptides of various chain-lengths, Ac-(Aib-Lys-Aib-Ala)n-NH2 (n = 1-5), were designed to investigate the mechanisms of the aggregation and transmembrane orientation of helical motifs in lipid bilayer membranes. Peptide synthesis was performed by the conventional stepwise Fmoc solid-phase method. The crude peptides were obtained in high yields (66-85%) with high purities (69-95%). Conformational analysis of the synthetic peptides was performed by CD spectroscopy. It was found that these peptides take on highly helical structures, and the helicity of the peptides increases with an increase in chain-length. The longest peptide, Ac-(Aib-Lys-Aib-Ala)5-NH2, self-aggregates and adopts a barrel-stave conformation in liposomes. Ac-(Aib-Lys-Aib-Ala)5-NH2 exhibited potent antimicrobial activity against Gram-positive bacteria. Patch-clamp measurements revealed that this peptide can form well-defined ion channels with a long lifetime at relatively low transbilayer potentials and peptide concentrations. For this peptide, the single-channel conductance of the most frequent event is 227 pS, which could be related to a single-state tetrameric pore.     

The nicotinic acetylcholine receptor: from molecular model to single-channel conductance

The nicotinic acetylcholine receptor (nAChR) is the archetypal ligand-gated ion channel. A model of the α7 homopentameric nAChR is described in which the pore-lining M2 helix bundle is treated atomistically and the remainder of the molecule is treated as a “low resolution” cylinder. The surface charge on the cylinder is derived from the distribution of charged amino acids in the amino acid sequence (excluding the M2 segments). This model is explored in terms of its predicted single-channel properties. Based on electrostatic potential profiles derived from the model, the one-dimensional Poisson-Nernst-Planck equation is used to calculate single-channel current/voltage curves. The predicted single-channel conductance is three times higher (ca. 150 pS) than that measured experimentally, and the predicted ion selectivity agrees with the observed cation selectivity of nAChR. Molecular dynamics (MD) simulations are used to estimate the self-diffusion coefficients (D) of water molecules within the channel. In the narrowest region of the pore, D is reduced ca. threefold relative to that of bulk water. Assuming that the diffusion of ions scales with that of water, this yields a revised prediction of the single-channel conductance (ca. 50 pS) in good agreement with the experimental value. We conclude that combining atomistic (MD) and continuum electrostatics calculations is a promising approach to bridging the gap between structure and physiology of ion channels. Received: 2 August 1999 / Revised version: 5 November 1999 / Accepted: 9 November 1999     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Identification of a new pore in the mitochondrial outer membrane of a porin-deficient yeast mutant

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant and of its parent strain. The addition of the detergent-solubilized material resulted in a strong increase in the membrane conductance which was not observed if only the detergent was added to the aqueous phase. Surprisingly, the membrane conductance induced by the detergent extracts of the mutant membrane was only a factor of 20 less than that caused by the outer membrane of the parent strain under otherwise identical conditions. Single-channel recordings of lipid bilayer membranes in the presence of mitochondrial outer membranes of the yeast mutant suggested the presence of a transient pore. The reconstituted pores had a single-channel conductance of 0.21 nS in 0.1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. The pores present in the mitochondrial outer membranes of the yeast mutant shared some similarities with the pores formed by mitochondrial and bacterial porins although their effective diameter is much smaller than those of the 'normal' mitochondrial porins which have a single-channel conductance of about 0.4 nS in 0.1 M KCl, corresponding to an effective diameter of 1.7 nm. Zero-current membrane-potential measurements suggested that the second mitochondrial porin is slightly cation-selective. Its possible role in the metabolism of mitochondria is discussed.     

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.     

Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

1/f-Noise of Open Bacterial Porin Channels

General diffusion pores and specific porin channels from outer membranes of gram-negative bacteria were reconstituted into lipid bilayer membranes. The current noise of the channels was investigated for the different porins in the open state and in the ligand-induced closed state using fast Fourier transformation. The open channel noise exhibited 1/f-noise for frequencies up to 200 Hz. The 1/f-noise was investigated using the Hooge formula (Hooge, Phys. Lett. 29A: 139–140 (1969)), and the Hooge parameter α was calculated for all bacterial porins used in this study. The 1/f-noise was in part caused by slow inactivation and activation of porin channels. However, when care was taken that during the noise measurement no opening or closing of porin channels occurred, the Hooge Parameter α was a meaningful number for a given channel. A linear relationship was observed between α and the single-channel conductance, g, of the different porins. This linear relation between single-channel conductance and the Hooge parameter α could be qualitatively explained by assuming that the passing of an ion through a bacterial porin channel is—to a certain extent—influenced by nonlinear effects between channel wall and passing ion. Received: 8 May 1996/Revised: 27 January 1997