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NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles
Walter R P Scott1, Seung-Bin Baek, David Jung
1Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
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.
Insights
Daptomycin, a calcium-dependent antibiotic, disrupts bacterial membranes. Its structure in micelles, with and without calcium, was determined using NMR, revealing insights into its membrane-perturbing mechanism.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Daptomycin is a cyclic lipopeptide antibiotic with a unique mechanism of action involving bacterial cell membrane perturbation.
- Calcium ions (Ca2+) are crucial for daptomycin's micellar structure formation and binding to bacterial membranes, suggesting a key role in its activity.
Purpose of the Study:
- To elucidate the three-dimensional structure of daptomycin in the presence of micelles under varying calcium conditions.
- To understand how daptomycin's structural changes influence its membrane-perturbing capabilities.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy was used to determine daptomycin's structure in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles.
- Structures were calculated using molecular dynamics with time-averaged refinement under both calcium-present and calcium-free conditions.
- Lipid flip-flop and calcein release assays were performed on PC liposomes to assess membrane perturbation.
Main Results:
- The three-dimensional structures of daptomycin were determined in DHPC micelles, both in the presence and absence of Ca2+.
- Structural differences were observed depending on the presence or absence of calcium ions.
- Assay data support the role of Ca2+ in daptomycin's membrane interaction and perturbation.
Conclusions:
- The structural studies provide a molecular basis for understanding daptomycin's calcium-dependent mechanism of action.
- These findings offer insights into how daptomycin perturbs bacterial membranes, differentiating it from other antibiotics.
