Conformational Dynamics of the Lipopolysaccharide from Escherichia coli O91 Revealed by Nuclear Magnetic Resonance Spectroscopy and Molecular Simulations.

Abstract

The outer leaflet of the outer membrane in Gram-negative bacteria contains lipopolysaccharides (LPS) as a major component, and the outer membrane provides a physical barrier and protection against hostile environments. The enterohemorrhagic Escherichia coli of serogroup O91 has an O-antigen polysaccharide (PS) with five sugar residues in the repeating unit (RU), and the herein studied O-antigen PS contains ∼10 RUs. 1H-13C HSQC-NOESY experiments on a 1-13C-labeled PS were employed to deduce 1H-1H cross-relaxation rates and transglycosidic 3JCH related to the ψ torsional angles were obtained by 1H-1H NOESY experiments. Dynamical parameters were calculated from the molecular dynamics (MD) simulations of the PS in solution and compared to those from 13C nuclear magnetic resonance (NMR) relaxation studies. Importantly, the MD simulations can reproduce the dynamical behavior of internal correlation times along the PS chain. Two-dimensional free energy surfaces of glycosidic torsion angles delineate the conformational space available to the O-antigen. Although similar with respect to populated states in solution, the O-antigen in LPS bilayers has more extended chains as a result of spatial limitations due to close packing. Calcium ions are highly abundant in the phosphate-containing core region mediating LPS-LPS association that is crucial for maintaining bilayer integrity, and the negatively charged O-antigen promotes a high concentration of counterbalancing potassium ions. The ensemble of structures present for the PS in solution is captured by the NMR experiments, and the similarities between the O-antigen on its own and as a constituent of the full LPS in a bilayer environment make it possible to realistically describe the LPS conformation and dynamics from the MD simulations.