1.D.5 The Alamethicin or Peptaibol Antibiotic Channel-forming (Alamethicin) Family
Peptaibols are biologically active peptides containing between seven and twenty amino acyl residues (aas) including α-aminobutyric acid (Aib) along with other unusual aas such as ethylnorvaline, isovaline and hydroxyproline. The N-terminus is acetylated, and the C-terminal amino acid is hydroxylated to an acid alcohol. They are named pebtaibols because they are peptides containing Aib and ending in an alcohol. They are produced by certain fungi, mainly in theTrichoderma genus, as secondary metabolites which function as antibiotics and antifungal agents. Some are referred to as trichorzianines. They are amphipathic which allows them to form voltage-dependent channels in membranes.
Alamethicin (Alm) is a toxic, 20 or 21 amino acid peptide from the fungus, Trichoderma viride, containing nonstandard amino acids α-methyl alanine and Aib. It adopts helical secondary monomeric and dimeric helix-bend-helix structures that self assemble in membranes into ion conducting helix bundles. It inserts via its N-terminus in response to voltage and laterally aggregates to form funnel-shaped pores of varying sizes. The helices within the bundles are probably oriented with the hydrophilic faces of the amphipathic helices facing inwards to form the channel lining and the hydrophobic side chains interacting with the phosphoplipid membrane matrix. The channels formed conduct ions in a non-saturable fashion, suggesting that transport occurs in a diffusional process without ion binding at discrete sites in the channel. The channels are mildly cation-selective, but mildly anion-selective analogues have been synthesized by substituting a glutamine for a lysine residue at position 18. These observations suggest that long-range electrostatic interactions explain the ion selectivity properties of alamethicin channels. The crystal structure of alamethicin has been determined to 1.5 Å resolution. Alamethicin does not alter the majority of the lipids in a bilayer but does reduce the dynamics of a few which serve as peptide anchors (Bertelsen et al. 2012). It shows a strong preference for the inserted over the surface-bound state (Perrin and Pastor 2016).
Alamethicin was the first isolated and is the best characterized member of the peptaibol class of natural linear depsipeptide antibiotics. These compounds are characterized by acetylated N-termini, a high percentage of α-amino-isobutyrate (AIB), and a C-terminal amino alcohol. Most contain 18-20 residues. Because of their high proportion of AIB, they form α-helical structures. Voltage causes them to autoassociate to form ion channels. The alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices (Qian et al., 2008). Many natural members of the peptaibol family are known and others have been synthesized. Natural members include longibrachins I and II, alamethicins F50 and F30, trichocellins TCAII and TCBII, saturnisporin SAIV, and trichosporin TSBVIa. Their sequences are presented in Cosette et al. (1999). Alamethacin has been used to create artificial fusions to ligand binding peptides that render channel activity sensitive to ions such as iron or calcium, thus generating tailored sensor or signal transduction systems (Futaki et al. 2013). Native alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself (Rahaman and Lazaridis 2014).
[Leul'zervamicin (zervamicin Z-L, has the following sequence: Ac-Leu-Ile-Gln-Iva-11e5-Thr-Aib-Leu-Aib-Hypl0-Gln-Aib-Hyp-Aib-Prol5-Phol, where Iva is isovaline, Aib is α-amino isobutyric acid, Hyp is 4-hydroxyproline, and Phol is phenylalaninol. It is a membrane channel-forming polypeptide from Emericellopsis salmosynnemata. It's structure has been determined by x-ray diffraction (Karle et al. 1991), and other pentaibol antibiotics have been determined by NMR. The helical structure is
amphiphilic with all the polar moieties on the convex side of the bent helix. Helices are bent at Hyp'0 from =30° to -450 in the different crystal forms. In all crystal forms, the peptide helices aggregate in a similar fashion to form water channels. Amoung these antibiotics are:
Alamethicin I (11): Ac-Aib-Pro-Aib-Ala-Aib5-Ala(Aib)-Gln-Aib-Val-Aibl O-Gly-Leu-Aib-Pro-Vall 5-Aib-Aib-Glu-Gln-Phol20
Antiamoebin I (II): Ac-Phe-Aib-Aib-Aib-D-Iva5-Gly-Leu-Aib-Aib-Hyp10-Gln-D-Iva-Hyp(Pro)-Aib-Pro 1 5-Phol
Zervamicin ILA (IIB): Ac-Trp-Ile-Gln-Aib(Iva)-Ile5-Thr-Aib-Leu-Aib-Hypl O-Gln-Aib-Hyp-Aib-Prol 5-Phol
Zervamicin Z-L: Ac-Leu-lle-Gln-lva-Ile5-Thr-Aib-Leu-Aib-HypI0-Gln-Aib-Hyp-Aib-Pro 5-Phol
Trichotoxin_A50E is an 18-residue peptaibol antibiotic which forms multimeric transmembrane channels through self-association. The crystal structure of trichotoxin has been determined at a resolution of 0.9 Å (Chugh et al. 2002). The trichotoxin sequence contains nine helix-promoting Aib residues, which contribute to the formation of an entirely helical structure that has a central bend of 8-10º located between residues 10-13. Trichotoxin is the first solved structure of the peptaibol family that is all α-helix as opposed to containing part or all 310-helix. Gln residues in positions 6 and 17 produce a polar face, and are proposed to form the channel lumen. An octameric channel has been constructed from the crystal structure. It has a central pore of ~4-5 Å radius, a size sufficient to enable transport of ions, with a constricted region at one end, formed by a ring of Gln6 residues. Electrostatic calculations are consistent with it being a cationic channel.
In antiamoebin, ions cross the octameric channel by diffusion (Wilson et al., 2011). The tetramer appears to be nonconducting, but the hexamer conducts. These channels are cation selective. The channel can be stabilized in the open configuration by heavy metal (e.g., zinc ion) binding (Noshiro et al. 2010). Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal region. Shenkarev et al. (Shenkarev et al. 2013) studied the Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles) by N-NMR spectroscopy. Interaction with the bicelles stabilized the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-mus time scale.
The trimer and the tetramer of alamethicin (ALM) forms 6Å pores that appear closed while the larger oligomers form open pores at their optimal radii (Kuang et al. 2013). The hexamer in an 8Å pore and the octamer in an 11Å pore give the lowest effective energy per monomer, but all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted 'barrel-stave' model. The N-terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C-terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation (Kuang et al. 2013). In POPC lipid bilayers and at a peptide/lipid ratio of 1:13, molecular-dynamics calculations suggest that the C19F3-labeled derivative assumes dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form (Salnikov et al. 2016).
The conformation and orientation of the 14-residue peptaibol SPF-5506-A4 (SPF) have been studied in bilayers. SPF inserts spontaneously in a transmembrane orientation in both 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers resulting in thinning of the bilayers near the peptides, which drives peptide aggregation. The backbone conformation of the peptide in the bilayer bound state is different from that of the NMR model solved in small bicelles. Mutual adaption between the peptides and the membrane is likely to be important for pore formation (Vestergaard et al. 2017).
The generalized transport reaction is:
Ion (in) → Ion (out)