Abstract
The asialoglycoprotein receptors and many other C-type (Ca2+-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta-methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild-type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca2+ and form hydrogen bonds with amino acids that also serve as Ca2+ ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca2+ coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site.
Highlights
The atomic coordinates and structure factors (codes 1AFA (MeGal complex), 1AFB (GalNAc complex), and 1AFD) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY
Insertion of a glycine-rich loop found in the major form of the rat asialoglycoprotein receptor, rat hepatic lectin-1 (RHL-1), and other Gal-binding C-type lectins that display strong discrimination against mannose results in a mutant (“QPDWG”) with galactose affinity and selectivity comparable with RHL-1 [13]
We describe the structure of a trimeric fragment of QPDWG containing the neck and COOH-terminal carbohydrate-recognition domains (CRDs) [14], both alone and complexed with -methyl galactoside (MeGal) and N-acetylgalactosamine (GalNAc)
Summary
23.4 28.2 22.1 26.8 21.7 26.1 a Rsym ϭ ⌺h ⌺i Ii(h) ϪI(h)͘ /⌺h ⌺i Ii(h), where Ii(h) is the ith measurement andI(h)͘ is the weighted mean of all measurements of I(h). b R ϭ ⌺h F(h)obs Ϫ F(h)calc /⌺h F(h)obs for reflections in the working set (Rcryst) or in the test set (Rfree) [19]. 23.4 28.2 22.1 26.8 21.7 26.1 a Rsym ϭ ⌺h ⌺i Ii(h) ϪI(h)͘ /⌺h ⌺i Ii(h), where Ii(h) is the ith measurement andI(h)͘ is the weighted mean of all measurements of I(h). All residues except Lys152 of one protomer of the unliganded cl-QPDWG model, which lies in a poorly ordered turn, fall within the allowed regions of the Ramachandran plot. No significant differences in structure are observed among the different copies, except in regions of lattice contacts. The side chains of residues Ser and His in protomer 1 and Met103 in protomer 2 were modeled in two conformations in QPDWG ϩ MeGal. The side chains of residues His in protomer 1 and Ser102, Met103, and Ser129 in protomer 2 were modeled in two conformations in QPDWG ϩ GalNAc. RMSD, root-mean-square deviation
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