Abstract
In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.
Highlights
3100A0-11173). □S The on-line version of this article contains supplemental Tables S1, S2, and S10 –S13, Figs
Manno-oct-2-ulosonate; PMX, polymyxin; Dab, diaminobutyric acid; NOE, nuclear Overhauser effect; Re-LPS, LPS from the deep rough mutant D31m4 of E. coli; HPLC, high-performance liquid chromatography; DPC, LPS are high molecular weight, strongly negatively charged molecules, which for smooth LPS can be divided in three regions: the lipid A portion of LPS inserts into the phospholipidic membrane and in many Gram-negative bacteria consists of a di-glucosamine diphosphate with 5 to 7 fatty acid chains extending to one side of the disaccharide
Because interactions with the charged phosphate groups were proposed to be important for binding antimicrobial peptides, we developed an HPLC-based method that allows purification of LPS in its natural form (Fig. 1). 13C,15N-Labeled LPS from the deep rough mutant strain was isolated and purified to chemical homogeneity
Summary
Purification from the membrane fraction of the corresponding cells was described in literature. To facilitate purification by reversed-phase HPLC, the phosphate groups were methylated (17). The modified LPS was investigated in detail by NMR (18). Because interactions with the charged phosphate groups were proposed to be important for binding antimicrobial peptides, we developed an HPLC-based method that allows purification of LPS in its natural (non-methylated) form (Fig. 1). 13C,15N-Labeled LPS from the deep rough mutant strain was isolated and purified to chemical homogeneity. Extensive use of heteronuclear solution NMR techniques allowed characterization of LPS embedded in DPC micelles and facilitated the study of its interactions with polymyxins from different organisms. The interaction studies relied on chemical shift mapping techniques and isotope-filtered NOEs and allowed direct probing for the interaction sites
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