Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.
The success of knowledge-based homology modelling is critically dependent on the predictive potency of the program structure-based calculations, which attempt to translate homologous sequences into three-dimensional structures, and on the actual relevance of the crystal structure for the protein topology. As quality control, experimental data for selected parameters of the protein's conformation are required. Using the crystal structure of the sialidase of Salmonella typhimurium as framework for model building of the homologous enzyme from Clostridium perfringens, a set of energy-minimised conformers is derived. These proteins present e.g. Tyr, Trp and His residues with an assessable area on the surface, since the side chains of these amino acid residues are responsive to chemically induced dynamic nuclear polarization (CIDNP), monitored by NMR. Hence, as first lesson, a comparative analysis for model-derived and experimentally determined values can be performed. The second lesson of this study concerns the notable impact of single amino acid substitutions (Tyr/Phe, Cys/Ser) on the surface accessibility of the CIDNP-reactive amino acid side chains in mutant forms of the sialidase. Corroborating the predictions from the theoretical calculations, the spectra of the engineered mutants reveal marked and non-uniform alterations. Thus, the effect of apparently rather conservative amino acid substitutions on a distinct conformational aspect of this protein, even at distant sites, should not be underestimated.
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