NMR investigations of protein-carbohydrate interactions: Studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N ',N ''-triacetylchitotriose

Model studies on lectins and their interactions with carbohydrate ligands i n solution are essential to gain insights into the driving forces for compl ex formation and to optimize programs for computer simulations. The specifi c interaction of pseudohevein with N,N',N"-triacetylchitotriose has been an alyzed by H-1-NMR spectroscopy. Because of its small size, with a chain len gth of 45 amino acids, this lectin is a prime target tee solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was exte nded 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 n ot affect the pattern of the protein nuclear Overhauser effect signal notic eably, what would otherwise be indicative of a ligand-induced conformationa l change. The average backbone (residues 3-41) RMSD of the 20 refined struc tures was 1.14 Angstrom, whereas the heavy atom RMSD was 2.18 Angstrom. Two different orientations of the trisaccharide within the pseudohevein bindin g 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 co rroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpicall y driven. In relation to hevein, the Trp/Tyrsubstitution in the binding poc ket has only a small effect on the free energy of binding in contrast to en gineered galectin-1 and a mammalian C-type lectin. A comparison of the thre e-dimensional structure of pseudohevein in solution to those reported for w heat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in s olution has been performed, providing a data source about structural variab ility of the hevein domains, The experimentally derived structures and the? values of the solvent accessibilities for several key residues have also b een 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. (C) 2000 Wiley-Liss, Inc.