Solution structure of a pentasaccharide representing the repeating unit of the O‐antigen polysaccharide from Escherichia coli O142: NMR spectroscopy and molecular simulation studies

Abstract

Conformational studies have been performed of a pentasaccharide derived from the O-polysaccharide from Escherichia coli O142. The polymer was selectively degraded by anhydrous hydrogen fluoride and reduced to yield an oligosaccharide model of its repeating unit, which in the branching region consists of four aminosugars. A comparison of (1)H and (13)C chemical shifts between the pentasaccharide and the polymer showed only minor differences, except where the cleavage had taken place, indicating that the oligomer is a good model of the repeating unit. Langevin dynamics and molecular dynamics simulations with explicit water molecules were carried out to sample the conformational space of the pentasaccharide. For the glycosidic linkages between the hexopyranoside residues, small but significant changes were observed between the simulation techniques. One-dimensional (1D) (1)H,(1)H double pulsed field gradient spin echo (DPFGSE) transverse rotating-frame Overhauser effect spectroscopy (T-ROESY) experiments were performed, and homonuclear cross-relaxation rates were obtained. Subsequently, a comparison of interproton distances from NMR experiment and the two simulation approaches showed that in all cases the use of explicit water in the simulations resulted in better agreement. Hydrogen-bond analysis of the trajectories from the molecular dynamics simulation revealed interresidue interactions to be important as a cluster of different hydrogen bonds and as a distinct highly populated hydrogen bond. NMR data are consistent with the presence of hydrogen bonding within the model of the repeating unit.

Transient hydrogen bonding in uniformly 13C,15N‐Labeled Carbohydrates in Water

“…The ψ E fluctuates as would be anticipated near a saddle point on the potential energy surface, whereas the ψ B populates a major conformational state (ψ B < 0°) with excursions to a minor conformational state with ψ B > 0°. Effective trans‐glycosidic proton–proton distances were calculated for comparison to the previously reported data6 and were found to be in good agreement (Table I) thereby lending credence to the novel force field used.…”

“…Here, we use HNCA,22 HNCO,23 and HSQC24 NMR experiments at 280 K to assign NH peaks in a uniformly 13 C‐ and 15 N‐labeled O142 PS. With these assignments, we demonstrate that amide NH and 1 J NH temperature coefficients obtained by measurements from 270 to 300 K support the presence of hydrogen bonds that were predicted from the 2‐ns MD simulation6 and in a new 105‐ns simulation.…”

“…The pentasaccharide derived from the E. coli O142 O‐antigen PS was first studied by an MD simulation that was extended by two orders of magnitude compared with the previous molecular simulation 6. Importantly, a novel force field developed for carbohydrates30 was used in the present MD simulation.…”

“…The bacteria were grown to late exponential phase, checked for purity at the end of the growth cycle, and killed with 1% (v/v) formaldehyde. Details of the pentasaccharide and polysaccharide preparation were previously described by Landersjö et al5, 6 The samples were lyophilized and stored in powder form.…”

“…The O‐antigenic PS from enteropathogenic E. coli O142 consists of pentasaccharide repeating units depicted in Figure 1, in which four residues are amino sugars. Previously, Landersjö et al characterized this PS by NMR spectroscopy in natural abundance,5 generated the pentasaccharide repeat unit, measured NOEs (nuclear Overhauser effects), and analyzed its structure using molecular dynamics (MD) simulations 6. The 2‐ns trajectory yielded glycosidic torsion angle values and predicted that NH groups in the pentasaccharide GalNAc residues act as interresidue hydrogen bond donors, which may play an important role in its three‐dimensional structure.…”