Research Resource for Integrated Glycotechnology:

Robert J. Woods

Computational Studies of Carbohydrate-Protein Interactions

The aims of this project are; (1) Extend the GLYCAM force field to charged glycosyl residues (sialic and uronic acids, sulfates); (2) Apply ensemble averaging in the calculation of molecular properties; (3) Improve predicted geometries and interaction energies for carbohydrate-protein complexes; (4) Apply these theoretical methods to galectin-oligosaccharide complexes.

Parameterization and Simulations of Anionic Glycosides. Our simulations with homogalacturonan are being used to investigate the mechanism of enzyme activity for a homogalacturonan-degrading enzyme (EPGII). These simulations have suggested the presence of a previously unreported secondary binding site, which is being investigated through mutagenesis experiments in collaboration with Dr. Carl Bergmann (CCRC). The focus of the anionic bacterial polysaccharides has been shifted to the Neisseria meningitidis system, for which we have access to polysaccharide samples through collaboration with Dr. H.J. Jennings (NRC, Canada) [1]. This biologically diverse group shares many structural features and will serve as an excellent set of molecules to evaluate the abilities of MD simulations to assist in interpreting the antigenic and immunogenic properties of these molecules.

Ensemble Averaged Molecular Properties. We are applying this method for the first time to homo- and heteronuclear J-couplings computed by ab initio quantum mechanical methods. At present we have adopted a rotational isomeric state (RIS) approach to identifying the relevant conformational state, followed by quantum calculations at the UHF B3LYP level, with our own basis set. This work has extremely broad implications in the conformational analysis of flexible molecules. We recently published an application of ensemble averaging in the calculation of partial atomic charges [2], and a study of carbohydrate flexibility employing these charges [3].

Carbohydrate-Protein Simulations. Pursuant to our collaboration with Dr. Boons (CCRC) on a study of the Con-A - trimannoside complex [4], we have completed an X-ray diffraction analysis of the complex [5]. The experimental structure is in excellent agreement with the predicted interactions in this system. The Con-A - trimannoside work clearly showed the usefulness of MD simulation in predicting ligand-protein interactions. However, some discrepancies were observed, as concerned positioning of conserved water molecules. This feature will require further study.

Structure-Affinity Relations in Galectin-Oligosaccharide Complexes. The MD simulations have been completed for Galectin-1 in complex with a number of biologically significant ligands, including LacNAc, 3'-OSO3-LacNAc, Neu5NAc-a-(2-3)-LacNAc and (LacNAc)₂ [6]. Each of these ligands can stably occupy the known LacNAc binding site. Seperate Biacore data suggests that each of these ligands show similar affinities. The mechanism of binding is being confirmed experimentally using X-ray crystallography and a combination of NMR techniques, such as ¹³C relaxation and residual dipolar coupling measurements. These measurements are facilitated by using isotopically-enriched Galectin-1, which we have expressed and purified. Our data suggests that Galectin-1 can mediate heterotypic binding, which may go some way toward explaining its diverse biological functions [6].

Publications:

[1] Basma, M., B. Reuhs, and R.J. Woods. 2002. Manuscript in preparation.

[2] Basma, M., S. Sundara, D. Calgan, T. Varnali, and R.J. Woods. 2001. J. Comput. Chem. 22:1125-1137.

[3] Kirschner, K. and R.J. Woods. 2001. Proc. Natl. Acad. Sci. USA 98:10541-10545.

[4] Clarke, C., R.J. Woods, A. Cooper, M.A. Nutley, and G.-J. Boons. 2001. J. Am. Chem. Soc. 123:12238-12247.

[5] Kadirvelraj, R., C. Clarke, G.-J. Boons, and R.J. Woods. 2002. Manuscript in preparation.

[6] Ford, M.F., T. Weimar, T. Kohli, J.H. Prestegard, and R.J. Woods. 2002. Manuscript in preparation.

[7] Kirschner, K. and R.J. Woods. 2002. J. Am. Chem. Soc. Manuscript submitted.