Molecular Basis of LL-A0341β1–Cardiolipin Interactions: Contributions of Δ-Trp7 and d -β-MeTrp8

Abstract LL-A0341β1 (LL) is a cyclic depsipeptide antibiotic that exhibits good activity against Gram-positive bacteria. LL contains several nonproteinogenic amino acids, including Z-dehydrotryptophan (Δ-Trp7) and the exceptionally rare d-erythro-β-methyltryptophan (d-β-MeTrp8). Recent studies have suggested that cardiolipin (CL), a phospholipid abundant in bacterial membranes, is the primary membrane target of LL. Here, we investigate the interaction between LL and tetraoleoyl cardiolipin (TOCL) and use LL analogues containing Trp substitutions at positions 7 (LL-d-Trp7 and LL-l-Trp7) and 8 (LL-d-Trp8) to assess the contributions of Δ-Trp7 and d-β-MeTrp8 to LL activity and TOCL binding. Δ-Trp7 was found to play a critical role in LL activity while d-β-MeTrp8 makes a lesser but still important contribution. We have demonstrated that the interactions of LL and its analogues with TOCL can be investigated using fluorescence spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC) without the need for pre-extruded liposomes or large unilamellar vesicles. However, the results must be interpreted with the recognition that TOCL aggregation and peptide-induced changes in its aggregation state may influence the observed spectroscopic and thermodynamic responses. Fluorescence studies with various phospholipids support our previous findings that CL is the primary membrane phospholipid target of LL. CD spectroscopy revealed that LL lacks a well-defined secondary structure in solution but undergoes a substantial conformational change upon interaction with TOCL. Fluorescence and ITC studies suggest that LL binds strongly to TOCL, with an apparent 1:1 binding stoichiometry that is substantially altered when Δ-Trp7 is replaced with d-Trp. ITC measurements yielded an apparent dissociation constant, KDapp, of 44 nM for the LL–TOCL interaction. Although the KDapp values for LL-d-Trp8 and LL-d-Trp7 were only approximately 3- and 4-fold higher, respectively, and both analogues retained the ability to undergo TOCL-induced conformational changes, their antibacterial activities were markedly reduced relative to LL. These findings suggest that strong binding to CL and the accompanying conformational changes are not sufficient to account for the antibacterial activity of LL.