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

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. 1 H- 13 C HSQC-NOESY experiments on a 1- 13 C-labeled PS were employed to deduce 1 H- 1 H cross-relaxation rates and transglycosidic 3 J CH related to the ψ torsional angles were obtained by 1 H- 1 H NOESY experiments. Dynamical parameters were calculated from the molecular dynamics (MD) simulations of the PS in solution and compared to those from 13 C 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.