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.     

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli.

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.     

Transmembrane pores formed by human antimicrobial peptide LL-37

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23–33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.     

Ion channels formed by amphipathic helical peptides

Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an -helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K⁺ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) -toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K⁺channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. -Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K⁺-channel interactions. Rotation of W15 about its C-C bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.     

Sequencing cyclic peptides by multistage mass spectrometry

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria.     

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.     

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.     

Characterization of pores formed by YaeT (Omp85) from Escherichia coli

Proteins of the Omp85 family play a major role in the biogenesis of the bacterial outer membrane, since they were shown to mediate insertion of outer membrane proteins. The Escherichia coli Omp85 homologue YaeT is essential for viability, but its exact mode of action is not yet elucidated. We could show that YaeT is composed of two distinct domains, an amino-terminal periplasmic and a carboxy-terminal membrane domain. The full length YaeT and the isolated membrane domain induce pores when reconstituted in planar lipid membranes. The pores exhibit a certain variability of conductance indicating a flexible structure, which could be an essential property of a lateral opening channel releasing proteins into the bacterial outer membrane. We could further show that the periplasmic domain proves to be essential for in vivo function of YaeT.     

Thrombin inhibition by cyclic peptides from thrombomodulin.

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM.     

Antimicrobial peptides bind more strongly to membrane pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.     

Ionic mechanism of a voltage-dependent current elicited by cyclic AMP

Intracellular pressure injection of cyclic AMP induces a slow voltage-dependent inward current in some neurons of Aplysia californica.The time course, voltage dependence, and ionic sensitivities of this response are nearly identical to those of the voltage-dependent calcium current induced by serotonin in the same preparation. The response to cyclic AMP is unaffected by changes in the extracellular concentration of chloride or potassium. The current is slowly but minimally reduced by a sodium-free solution. The calcium channel blocker, cadmium, blocks the current elicited by injection of cyclic AMP. The data presented here suggest that cyclic AMP can induce a voltage-dependent calcium current.     

Transbilayer pores formed by beta-barrels: molecular modeling of pore structures and properties.

Transmembrane beta-barrels, first observed in bacterial porins, are possible models for a number of membrane channels. Restrained molecular dynamics simulations based on idealized C alpha beta templates have been used to generate models of such beta-barrels. Model beta-barrels have been analyzed in terms of their conformational, energetic, and pore properties. Model beta-barrels formed by N = 4, 8, 12 and 16 anti-parallel Ala10 strands have been developed. For each N, beta-barrels with shear numbers S = N to 2N have been modeled. In all beta-barrel models the constituent beta-strands adopt a pronounced right-handed twist. Interstrand interactions are of approximately equal stability for all models with N > or = 8, whereas such interactions are weaker for the N = 4 beta-barrels. In N = 4 beta-barrels the pore is too narrow (minimum radius approximately 0.6 A) to allow ion permeation. For N > or = 8, the pore radius depends on both N and S; for a given value of N an increase in S from N to 2N is predicted to result in an approximately threefold increase in pore conductance. Calculated maximal conductances for the beta-barrel models are compared with experimental values for porins and for K+ channels.     

Artificial ion channels formed by a synthetic cyclic peptide.

A new cyclic peptide 1 having an (LLLD)3 configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self-assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1, cyclo[-Trp-Dap-Leu-D-Ala-Trp-Ser-Val-D-Ala-Trp-Ser-Ile-Gly-] (Dap: L-diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the beta-amino group of Dap may play a role in the conductance of the peptide channels. Fourier-transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of beta-structure analogous to that in the gramicidin A dimer and distinct from the H-bonding pattern of the beta-barrels.     

Transport of alpha-helical peptides through alpha-hemolysin and aerolysin pores

A series of negatively charged alpha-helical peptides of the general formula fluorenylmethoxycarbonyl (Fmoc)-D(x)A(y)K(z) were synthesized, where x and z were 1, 2, or 3 and y was 10, 14, 18, or 22. The translocation of the peptides through single pores, which were self-assembled into lipid membranes, was analyzed by measuring the current blockade i(block) and the duration t(block). The pores were either alpha-hemolysin, which has a wide vestibule leading into the pore, or aerolysin, which has no vestibule but has a longer pore of a similar diameter. Many thousands of events were measured for each peptide with each pore, and they could be assigned to two types: bumping events (type I) have a small i(block) and long t(block), and translocation events (type II) have a larger i(block) and shorter t(block). For type-II events, both i(block) and t(block) increase with the length of the peptides on both pores tested. The dipole moment and the net charge of each peptide has a major effect on the transport characteristics. The ratio of type-II/type-I events increases as the dipole moment increases, and uncharged peptides gave mostly type-I events. The structural differences between the two nanopores were reflected in the characteristic values of i(block), and in particular, the vestibule of alpha-hemolysin helps to orient the peptides for translocation. Overall, the results demonstrate that the nanopore technology can provide useful structural information but peptide sequencing will require further improvements in the design of the pores.     

Synthesis of cyclic tryptathionine peptides

The helicity of the tryptathionine moiety of the phallotoxins has been recognized by comparison with cyclic tryptathionine tripeptides. In order to investigate the influence of the configuration of the component amino acids on the conformation of the cyclic peptides, six analogue thioether tripeptides containing L- and D-alanine and L- and D-cysteine, respectively, have been synthesized. The CD spectra of the peptides are very similar to each other, showing mirror images of the CD of phalloidin and, therefore, negative helicity. The spectra of the D-cysteine containing compounds differ from the L-cysteine containing compounds by their weakly positive ellipticity values around 270 nm. The cyclization reaction of Boc-Hpi-D-Ala-D-Cys(STrt)OCH3, along with the cyclic tripeptide, afforded a cyclic hexapeptide by dimerization. The CD spectrum of the dimer is very similar to that of phalloidin, thus pointing to a positive helicity of its two tryptathionine moieties. The dimeric thioether peptide forms a rather strong complex with Cu2+ ions.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.