MAPPING THE STRUCTURAL FEATURES OF METAL- AND CARBOHYDRATE BINDING TO CONCANAVALIN A. Julie Bouckaert(1), Remy Loris(1), Dominique Maes(1), Freddy Poortmans(2) and Lode Wyns(1); (1)Laboratorium voor Ultrastructuur, VUB, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium and (2)VITO, Boeretang 200, B- 2400 Mol, Belgium

The lectin concanavalin A (Con A) sequentially binds a transition metal ion in the metal-binding site S₁ and a calcium ion in the metal-binding site S₂ in order to form its saccharide-binding site. Zn²⁺ or Co²⁺ soaked into metal-free Con A crystals (apoZn-Con A or apoCo-Con A) bind only partially in the proto-transition metal-binding site, not followed by any conformational changes. These structures can represent the very first step in going from metal-free Con A towards the holoprotein. In the co-crystals of metal-free Con A with Zn²⁺ (Zn-Con A), the Zn-ion fully occupies the S₁ site. It orientates Asp10 optimally for calcium binding in the S₂ site and stabilizes Asp19 by hydrogen bonding to one of its water ligands. Zn²⁺ binding in S₁ apparently is necessary to allow for subsequent Ca²⁺ binding in the S₂ site. Ca²⁺ binding is critical to induce the large conformational changes, that comprise the trans to cis isomerization of the Ala207-Asp208 peptide bond accompanied by the formation of the saccharide-binding site: the co-crystals of metal-free Con A with both Zn²⁺ and Ca²⁺ contain the active holoprotein (Con A ZnCa). Con A interacts with all three saccharide units in the complex with its most specific epitope, the trisaccharide methyl-3,6-di-O-(alpha-D- mannopyranosyl)-alpha-D-mannopyranoside. The mannose on the alpha(1-6) arm is bound in the primary or monosaccharide-binding site. The reducing core mannose interacts with the side chains of Tyr12 and Asp16 via the hydroxyl groups on C₄ and C₂ respectively. The alpha(1-3) linked mannose shows two distinct conformations, but in each of them the hydroxyl group on the C₃ position makes a hydrogen bond to the main chain carbonyl of Pro13. These results are discussed in view of recent thermodynamic data on the Con A-trimannosyl system and in view of differences in oligosaccharide specificity between Con A and the mannose/glucose specific lectins from the Viciae tribe.