NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles

Abstract

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca2+ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca2+ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374-85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca2+ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.

Lipopeptides as anti-infectives: a practical perspective

“…The arrangement into micelles is not accompanied by a change in lipopeptide conformation, as originally proposed [36]. Indeed, making a comparison with addition of magnesium instead of calcium to a daptomycin solution, the NOESY spectra closely resembled the NOESY spectra obtained for apo-daptomycin [51]. Also, in the presence of 1 eq of Ca 2+ , two new structures quite similar to the apo-form are present [35], suggesting that the interaction of daptomycin with Ca 2+ is weak and nonperturbing.…”

“…Finally, to determine whether the interaction of daptomycin with lipids is accompanied by a change in structure, NMR TOCSY and NOESY experiments were carried out in the presence of Ca 2+ and DHPC (dihexanoylphosphatidylcholine) micelles. Results suggested that daptomycin undergoes only a minor conformational rearrangement upon binding to DHPC in the presence of Ca 2+ [51]. Daptomycin is marketed under the name Cubicin by Cubist Pharmaceuticals (www.cubist.com).…”

The action mechanism of daptomycin

“…The Straus group has reported the structure of daptomycin in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles in the presence of calcium, 47 which resembled the apo-daptomycin structure. Therefore, it was assumed that daptomycin experiences only a minor conformational rearrange-ment upon binding to DHPC micelles in the presence of Ca 2+ .…”

Mechanism of Action and Limited Cross-Resistance of New Lipopeptide MX-2401

“…In model membranes containing phosphatidylglycerol, daptomycin also causes membrane fusion in the presence of Ca 2ϩ (18). The perturbation of the bacterial cell membrane and its depolarization are proposed to lead to intracellular ion (14,32). It is thought that the highly dynamic nature of daptomycin enables it to adapt to these different environments and to effectively interact with the bacterial cell membrane.…”

“…The perturbation of the bacterial cell membrane and its depolarization are proposed to lead to intracellular ion leakage and rapid cell death (36). Daptomycin adopts similar structures in the apo form, in the presence of one or more equivalents of Ca 2ϩ , and in the presence of phosphatidylcholine/Ca 2ϩ (14,32). It is thought that the highly dynamic nature of daptomycin enables it to adapt to these different environments and to effectively interact with the bacterial cell membrane.…”