Interactions of lipopolysaccharide and polymyxin studied by NMR spectroscopy

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 E. coli. A method for efficient purification of biosynthetically produced LPS using RP-HPLC in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine (DPC) 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 NOESY experiments, reveal the mode of interaction of LPS with polymyxins. Using MD calculations the complex of LPS with PMX-B in the presence of DPC micelles was modelled using restraints derived from chemical shift mapping data and intermolecular NOEs. In the modelled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar sidechains are formed to GlcN-A and Kdo-C, while hydrophobic sidechains penetrate the acyl chain region.