NMR structure of active and inactive forms of the sterol‐dependent antifungal antibiotic bacillomycin L

Abstract

The antifungal antibiotic lipopeptide bacillomycin L [cyclo-(l-Asp1-d-Tyr2-d-Asn3-l-Ser4-l-Gln5-d-Ser6-lThr7-b-amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (l-Asp1, d-Tyr2) di-O-methylated form. The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral f and c angles defining a type-II H b-turn including amino acids 5±8, in certain cases stabilized by a 8H N -5CO hydrogen bond, whereas a minority of structures adopt an inverse g-turn including amino acids 6±8, stabilized in all cases by an 8H N -6CO hydrogen bond. The di-O-methylation of l-Asp1 and d-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved d-Tyr2 position.Keywords: antibiotics; antifungals; bacillomycin L; NMR.Among the antifungal antibiotic substances, there is a specific family of molecules that acts on the biomembrane of infectious agents with an established sterol dependency. This family comprises very different molecules including the polyene macrolides as the widely used nystatins or amphotericins in pharmacotherapy (for reviews see [1,2]). The antifungal activity is believed to rely on a better interaction with ergosterol, which is more abundant in the lipid composition of fungal membranes, than cholesterol present in other organisms. However, the fine nature of the molecular interaction and the origin of this selectivity at atomic resolution remain unknown [3±6]. Lipidic cyclopeptides (lipopeptides) isolated from cultures of bacterial strains of Bacillus subtilis also exhibit such a general property. Their peptidic nature potentially makes them more adapted for a drug-design approach or a combinatorial-chemistry search for more active or more suitable molecules. These peptides include iturins [7±9], bacillomycins [10±14] and mycosubtilin [15]. All antibiotics of the iturinic group (Fig. 1) are characterized by an invariant lddlldl amino acid sequence alternation with a conserved tyrosine residue at position 2, and variable hydrophobic tails [16] with the R absolute configuration at C 3 [17] linked into the cyclic peptide through a b-peptide bond. The molecular structure of two members, mycosubtilin [18] and iturin A [19,20], have already been studied mainly using solution NMR spectroscopy.All these lipopeptides are also haemolytic [21±23], very potent against fungi [24] and different yeasts [25±27], but...

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