Tricholongins BI and BII, 19‐residue peptaibols from Trichoderma longibrachiatum

Abstract

Two nonadecapeptides, tricholongins BI and BII, which display antifungal and antibacterial activities, have been isolated from in vitro cultures of the fungus Trichoderma longibrachiatum. The peptides were separated by reversed-phase HPLC; their amino acid compositions were determined by gas chromatography and their sequences by positive-ion fast-atom-bombardment mass spectrometry and high-field NMR. These linear peptides, containing mainly hydrophobic L-amino acids, 8 -9 2-aminoisobutyric acid residues and exhibiting an acetylated N-terminal residue and an amino alcohol C-terminal leucinol belong to the peptaibol class. The methanol solution structure of tricholongins BI and BII has been investigated using both one-and two-dimensional NMR techniques. The total 'H-NMR and I3C-NMR assignments are given. By a combination of the 3 &~,~a~ coupling constant values, temperature coefficients of the NH and CO groups, amide hydrogen/deuterium-exchange rate measurements and NOE data, a secondary structure for tricholongins in solution has been proposed. Both peptides adopt a similar a-helical conformation with a hinge around Pro13 resulting from two 310 bonds. The results suggest that the Nterminus contains mixed a13 lobonds. The membrane permeability modifications induced by tricholongins have been assayed by the use of liposomes composed of egg phosphatidylcholine with 20-30% cholesterol. The peptide-induced leakage of an entrapped fluorescent probe has been followed by fluorescence spectroscopy. In a concentration range of 0.13-0.31 FM, tricholongins induce the leakage of 50% of the entrapped material in 20 min.The peptaibols include the well-known alamethicin from Trichoderma viride [I, 21 and trichorzianines from T. harzianum [3 -51. They are peptides of fungal origin whose mechanism of action is of interest, as they show a broad range of bioactivities. Structurally, these peptides contain an acetylated N-terminal residue, a C-terminal amino alcohol and a high percentage of hydrophobic amino acids including 2-aminoisobutyric acid (Aib).It is likely, therefore, that they are structurally related to melittin [6] or 6-haemolysin, extracted from bee venom and from Staphylococcus aureus, respectively, as they all exhibit a common tendency to form amphiphilic helices. It seems likely, also, that their mechanisms of action are close to those proposed to explain the interaction of melittin with biological membranes [7-101 and also the formation of voltage-gated ion channels [II, 121. We isolated two new groups of peptaibols form T. longibrachiatum (MNHN 3431), one composed of 19-residue neutral peptides and the other consisting of shorter sequences; these were named tricholongins and trichogins, respectively [I 31. These peptides exhibited both antifungal and antibacterial properties against various fungal strains and Grampositive bacteria. In this paper, we describe the isolation, sequence determination and conformational characteristics of the most abundant two peptides of the tricholongin group, tricholongins BI and BII...

Two unprecedented natural Aib-peptides with the (Xaa-Yaa-Aib-Pro) motif and an unusual C-terminus: structures, membrane-modifying and antibacterial properties of pseudokonins KL III and KL VI from the fungus Trichoderma pseudokoningii

“…The 3 J NHChH coupling constants (Table 5) exhibited lower values ( 57.5 Hz), consistent with a helix, for Asn 2 , Ile 3 and Leu 7 , whereas Ile 4 and Leu 8 showed higher values (around 9 Hz). Such high couplings, which are apparently inconsistent with helical structures, are frequently observed for the amino acids flanking Aib-Pro segments in h-helical peptaibols [4,8,37]. The same periodicity of lower and higher values, as regard to the location of the Pro residues has been observed previously for related peptides [7,8,17].…”

Peptaibiomics: Screening for Polypeptide Antibiotics (Peptaibiotics) from Plant-ProtectiveTrichoderma Species

“…Long-chain peptaibols are known to form voltage-gated or non-gated ion channels in bilayer lipid membranes. The carboxyfluorescein leakage in small unilamellar egg phosphatidylcholine vesicles with 20 -30% cholesterol, induced by tricholongins BI and BII, was demonstrated to be higher than that caused by the acidic trichorzianine A IIIc [27]. In single-channel experiments [48], the mode of action of trichorzianine A IIIc was shown to be consistent with the flip-flop gating model introduced in [49].…”

Alamethicin and related peptaibols ? model ion channels

“…The two main forms of alamethicin, Alm-Rf30 and Alm-Rf50 differ only in whether residue 18 is either E or Q respectively. Tricholongin-BI (Rebuffat et al 1991) and trichorzianine-AIIIC (Le Bars et al 1988;Duclohier et al 1989) are 19 residue peptaibols which differ from Alto in the substitution of leucinol or tryptophanol for phenylalaninol at the C-terminus, and of S for G at the position three residues before the central proline. The remaining sequences are for examples of a family of 16 residue peptaibols, the zervamicins (Zrv; Balaram et al 1992).…”

“…Furthermore, T2 measurements provided no evidence for slower motions modulating the alamethicin conformation. A detailed study of the structure and dynamics of a 19 residue peptaibol, tricholongin-B, has been provided by Rebuffat et al (1991), from tH and 13C NMR studies in methanol. The results provided evidence for a helical structure, with a kink in the region of P13.…”