BINDING OF SUGAR LIGANDS TO CA2-DEPENDENT ANIMAL LECTINS .1. ANALYSISOF MANNOSE-BINDING BY SITE-DIRECTED MUTAGENESIS AND NMR()

The Ca2+-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesi s to determine the importance of individual residues in ligation of ma nnose and related sugars. The effects of the mutations were assessed b y direct binding assays, competition binding studies, partial proteoly sis, and NMR analysis of sugar-CRD titrations. As suggested by the cry stal structure of the mannose-binding CRD complexed with oligosacchari de ligand, asparagine and glutamic acid residues that interact with hy droxyl groups 3 and 4 of the sugar, as well as with one of the two bou nd Ca2+, are critical for ligand binding. In addition, the beta-carbon of His(189) contributes substantially to the binding affinity, appare ntly through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His(189) and the 2 h ydroxyl group of mannose, and between Ile(207) and C-6 of mannose, obs erved in the crystal structure, contribute less to stability of the li gand complex. The effects of changes at positions 189 and 207 on the a bility of the CRD to distinguish between alpha- and beta-methyl L-fuco sides suggest that fucose may bind in an alternative orientation compa red to the arrangement originally proposed based on the mannose-CRD co mplex.