Characterization of daptomycin oligomerization with perylene excimer fluorescence: Stoichiometric binding of phosphatidylglycerol triggers oligomer formation

Abstract

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.

The action mechanism of daptomycin

“…85 Proximity also exists between the fatty acyl tails of adjacent oligomer subunits. This is evident from the excimer fluorescence of perylene or pyrene incorporated into the acyl residues of daptomycin 51 and A54145, 84 respectively (see Figure 7). In spite of their direct attachment to Trp (1) , excimer fluorescence-and thus, aggregation of fatty acyl residues-arises already in the partially Ca 2+ -saturated state, 84 while Trp (1) responds only at full Ca 2+ saturation.…”

“…26 Replacement of the decanoyl residue with pyrene-or perylene-butanoic acid-performed for the sake of fluorescence studies, see section 10.1-caused only minor reduction in antimicrobial activity. 51 Overall, it appears that the specific structure of the fatty acyl residue is less important than its overall hydrophobicity.…”

“…Fluorescence studies using perylene-labelled daptomycin and a lipid bicelle system consisting of DMPC, DMPG, and dihexanoyl-PC produced a maximal perylene excimer signal at an equimolar ratio of peptide and PG, suggesting that the interaction is stoichiometric. 51 In contrast, isothermal titration calorimetry (ITC) using daptomycin and DOPC/DOPG liposomes suggests that each daptomycin molecule binds two molecules of PG. 63 One possible explanation for the discrepancy is that the lipopeptide molecules interact with both leaflets of the bicelle but with only the outer leaflet of the liposome.…”

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

“…In NBD-daptomycin, the free amino group of ornithine is modified with NBD. In perylene-daptomycin, the N-terminally attached decanoyl residue is replaced by perylene-butanoic acid (13). Kynurenine has intrinsic fluorescence that can be used for FRET experiments in conjunction with NBD (15).…”

“…Measurements of Perylene Excimer Fluorescence-Perylenedaptomycin was prepared as described before (13). The compound was added to LUV at 2 M. Calcium was added to 5 mM, the samples were incubated for 5 min, and the fluorescence emission were recorded with excitation at 430 nm.…”