Conformational Investigation of a Cyclic Enterobacterial Common Antigen Employing NMR Spectroscopy and Molecular Dynamics Simulations

Abstract

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.

Identification and Biosynthesis of Cyclic Enterobacterial Common Antigen in Escherichia coli

“…(37) and Yersina pestis (39) and also with the 13 C assignments reported for Plesiomonas shigelloides ECA PG (7) ( Table 2). However, a comparison of these chemical shift assignments of ECA CYC revealed small variations that are likely due to heterogeneity occurring from natural modifications, possible degradation during purification, and variations in experimental conditions.…”

“…In addition to ECA PG , the occurrence of ECA LPS has also been reported in certain members of the Enterobacteriaceae, including E. coli K-12 (15,16). However, it is important to emphasize that the occurrence of ECA CYC has been demonstrated in only a few organisms (11,16,19,37,39), and it has not been identified in E. coli. Therefore, we conducted experiments to determine if indeed the water-soluble ECA that was present in HIFd preparations obtained from E. coli B was ECA CYC .…”

“…This is due, in large part, to the general belief that the occurrence of ECA CYC within members of the Enterobacteriaceae is rather restricted, since it has only been found in cell extracts of Shigella sonnei phase I (11,19), Yersinia pestis (39), and Plesiomonas shigelloides (7,37); the last organism has now been included in the Enterobacteriaceae.…”

Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12

“…The lack of a convenient method for the detection of ECA CYC is largely responsible for the fact that ECA CYC has been demonstrated only in the cell extracts of a few gramnegative organisms (6,11,22,42,43). Accordingly, the possibility that it is present in all members of the Enterobacteriaceae has remained open.…”

“…In addition to containing ECA PG on the cell surface, cell extracts prepared from Shigella sonnei phase I (11,22), Yersinia pestis (43), and Plesiomonas shigelloides contain water-soluble cyclic polysaccharides comprised of ECA trisaccharide repeat units (6,42). These polysaccharides have been designated cyclic ECA (ECA CYC ).…”