STRUCTURE OF A GALACTOSE-SPECIFIC C-TYPE ANIMAL LECTIN. Anand R. Kolatkar and William I. Weis, Stanford University, Dept. Structural Biology, Stanford, CA

Galactose-binding C-type lectins function in serum glycoprotein clearance, tumor cell recognition, and organization of the extracellular matrix. The crystal structure of a galactose-binding mutant of a C-type animal lectin has been solved unliganded and in complex with galactose and N-acetylgalactosamine (GalNAc). Three amino acid substitutions and insertion of a glycine-rich loop in wild-type mannose-binding protein A (MBP-A) gives a mutant (QPDWG) that exhibits specificity and affinity for galactose similar to naturally-occurring galactose-binding C-type lectins. The 3- and 4- OH groups of galactose coordinate the Ca²⁺ at site 2 and form hydrogen bonds with amino acid residues that also coordinate the Ca²⁺. Galactose specificity is conferred by a glycine-rich loop which holds Trp¹⁸⁹ in a position optimal for packing against the apolar face of the galactose ring, and which prevents mannose binding by steric exclusion. The structure of the N-acetylgalactosamine/QPDWG complex shows that the 2-acetamido group of GalNAc is oriented such that it could interact with the amino acid positions identified by site-directed mutagenesis (Iobst, S.T. & Drickamer, K., J. Biol. Chem., 271, 1996, in press) as being important in GalNAc-specific C-type lectin binding sites. An additional mutation of Thr²⁰² --> His in QPDWG (to produce QPDWGH) exhibits an 8-fold increase in GalNAc specificity over galactose. The GalNAc/QPDWGH structure is currently being refined, and the preliminary results indicate that His²⁰² is too distant from the acetamido group of GalNAc to make direct contact. It is possible that His²⁰² affects GalNAc binding through either a bridging water or by interaction with an amino acid residue that does make direct contact with GalNAc.