Membrane Binding and Oligomerization of the Lipopeptide A54145 Studied by Pyrene Fluorescence

Abstract

A54145 is a lipopeptide antibiotic related to daptomycin that permeabilizes bacterial cell membranes. Its action requires both calcium and phosphatidylglycerol in the target membrane, and it is accompanied by the formation of membrane-associated oligomers. We here probed the interaction of A54145 with model membranes composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, using the steady-state and time-resolved fluorescence of a pyrene-labeled derivative (Py-A54145). In solution, the labeled peptide was found to exist as a monomer. Its membrane interaction occurred in two stages that could be clearly distinguished by varying the calcium concentration. In the first stage, which was observed between 0.15 and 1 mM calcium, Py-A54145 bound to the membrane, as indicated by a strong increase in pyrene monomer emission. At the same calcium concentration, excimer emission increased also, suggesting that Py-A54145 had oligomerized. A global analysis of the time-resolved pyrene monomer and excimer fluorescence confirmed that Py-A54145 forms oligomers quantitatively and concomitantly with membrane binding. When calcium was raised beyond 1 mM, a distinct second transition was observed that may correspond to a doubling of the number of oligomer subunits. The collective findings confirm and extend our understanding of the action mode of A54145 and daptomycin.

Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145

“…The figure also includes the key findings of another recent study, in which pyrene moieties were semisynthetically incorporated into the fatty acyl tail of A54145 [16]. As in the current study, two successive calcium-dependent transitions were observed, which occurred at calcium concentrations very similar to those reported here.…”

The action mechanism of daptomycin

“…Intriguingly, global analysis of time-resolved excimer fluorescence indicates that, even at partial Ca 2+ saturation, membrane-bound A54145 already exists as an oligomer, which however has a lower number of subunits that the one observed at full saturation. 84 This observation may be related to the proposed occurrence of daptomycin tetramers as intermediates of pore formation (see Figure 6). In this context, it is also worth mentioning that daptomycin may interact with membranes even in the absence of calcium, as shown by fluorescence polarization anisotropy in an early study, 38 although this observation does not indicate oligomer formation.…”

“…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.…”

“…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. Intriguingly, global analysis of time-resolved excimer fluorescence indicates that, even at partial Ca 2+ saturation, membrane-bound A54145 already exists as an oligomer, which however has a lower number of subunits that the one observed at full saturation.…”

“…C: A tetrameric form that is confined to the outer leaflet has been observed on membranes containing cardiolipin 66 and may also occur at sub-saturating calcium concentrations. 84 across the two membrane leaflets, the positions of individual residues relative to the bilayer, and the mutual proximity of residues within the oligomer. While daptomycin will initially interact with the outer membrane leaflet, the fact that it permeabilizes the target membrane suggests that it will ultimately penetrate the inner leaflet also.…”