The Nature of Daptomycin Aggregates

Abstract

Daptomycin is the first FDA-approved member of a new structural class of antibiotics_the cyclic lipopeptides. It is an important drug against multidrug-resistant gram-positive pathogens. The peptide interacts with the lipid matrix of cell membranes, inducing membrane permeability to ions, but its molecular mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-defense antimicrobial peptides, daptomycin does not induce molecular-leaking pores in the cell membranes--no calcein leakage was detected from lipid vesicles. Thus how it affects the membrane permeability to ions is not clear. The antibacterial activity and induced ion leakage by daptomycin correlate with the target membrane's content of phosphatidylglycerol (PG) and occur only in the presence of Ca2+ ions. Fluorescence resonance energy transfer (FRET) experiments and the recently discovered lipid extracting effect have shown daptomycin aggregation in membranes, but the chemical structure of daptomycin gives no clue to the nature of daptomycin aggregates. Jung et al discovered an inversion of the CD spectrum of daptomycin that occurs only in the simultaneous presence of PG and Ca++. We have correlated the inversion of the CD with the lipid extracting effect of daptomycin. Here we make use of the CD spectral change with Ca2+ ion concentration and with PG concentration to analyze the nature of the daptomycin-Ca++-PG aggregates. The data shows a sharpe threshold (or micellization) effect on the Ca2+ ion concentration dependence, indicating that each aggregate contains 20 or more calcium ions. This result shows that the aggregates are large, inconsistent with the idea of daptomycin aggregates forming oligomeric pores, since such a pore must be made of a relatively small number of daptomycin. Once we realize the micellization effect, the stoichiometry of the daptomycin-Ca++-PG aggregates can be analyzed from the calcium and PG concentration dependence.