Molecular modeling of the flexible cell wall polysaccharide of Streptococcus mitis J22 on the basis of heteronuclear NMR coupling constants.

A method for constructing conformational models of flexible complex polysaccharides on the basis of NMR data and molecular modeling is described and is applied to a polysaccharide which is a lectin-binding receptor important in coaggregation of oral bacteria. The method involves uniform biosynthetic enrichment of the polysaccharide with 13C which allows accurate measurements of heteronuclear coupling constants from a three-dimensional coupled HMQC-NOESY spectrum. The improved resolution of the 3D spectrum also provides a large number of accurate values of NOE cross peak volumes in a decoupled HMQC-NOESY spectrum. While it was not possible to construct a model for the flexible polysaccharide directly from the NOE data, a model was successfully built from the coupling constant data. Possible values of glycosidic dihedral angles were extracted from the 3JCH data to build models which were evaluated by molecular modeling calculations. A simple average over a linear combination of low-energy conformations was selected which matched the experimental 3JCH data within experimental error. Simulation of the NOE data for this same combination of conformers gave excellent agreement with experimental NOESY data. Molecular dynamics trajectories both with and without coupling constant constraints do not represent the experimental NOE and 3JCH data as well as the linear combination model. While the polysaccharide has some flexibility in the antigenic site, the lectin-binding site, which contains a furanoside with (1-->6)-linkages, provides a more flexible hinge in the polysaccharide.