Binding of sugar ligands to Ca(2+)-dependent animal lectins. I. Analysis of mannose binding by site-directed mutagenesis and NMR.
The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.
Iobst
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Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Wormald
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MR
Weis
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Dwek
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RA
Drickamer
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K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008358:Mannose / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D051381:Rats
D002352:Carrier Proteins; D037102:Lectins; D037241:Mannose-Binding Lectins; D009838:Oligodeoxyribonucleotides; D008358:Mannose; D002118:Calcium