HIV-protease as a target molecule in the treatment of AIDS

Abstract

The lectin concanavalin A (Con A) sequentially binds a transition metal ion in the metal-binding siteS I and a calcium ion in the metal-binding site S2 in order to form its sacchmide-binding site. Zn2+ or Co2+ soaked into metal-free Con A crystals (apoZnCon 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 towm·ds the holoprotein. In the co-crystals of metal-free Con A with Zn2+ (Zn-Con A), the Zn-ion fully occupies the S 1 site. It orientates Asp I 0 optimally for calcium binding in the S2 site and stabilizes Asp 19 by hydrogen bonding to one of its water ligands. Zn2+ binding inS 1 appm·ently is necessm·y to allow for subsequent Ca2+ binding in the S2 site. Ca2+ binding is c1itical to induce the lm·ge·conformational changes, that comprise the trans to cis isomerization of the Ala207 -Asp208 peptide bond accompanied by the formation of the.sacchmide-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 aisaccharide methyl-3,6-di-0-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside. The mannose on the alpha(l-6) m111 is bound in the p1imm-y or monosacchmide-binding site. The reducing core mannose interacts with the side chains ofTyrl2 and Aspl6 via the hydroxyl groups on C: and C2 respectively. The alpha(l-3) linked mannose shows two distinct conformations, but in each of them the hydroxyl group on the C 3 position makes a hydrogen bond to the main chain cm·bonyl ofPro13. These results are discussed in view of recent thennodynamic data on the ConAtrimannosyl system and in view of differences in oligosaccharide specificity between Con A and the mannose/glucose specific lectins from the Viciae tribe. Salivary ex-amylase (sAmy) is a multifunctional enzyme involved in the initial hydrolysis of starch and in the colonization of bacteria which leads to dental plaque formation. Amylases are monomelic, calcium binding proteins with a single polypeptide chain folded into three domains and contain the (8/ex)s-ban·el topology. Domains A and B appear to be involved in enzymatic activity. The role of the C domain in either of the functions is unknown. To better understllild the functional roles of the vmious domains of this enzyme we have optimized a baculovirus expression system for the production of recombinant amylase (rAmy) with biochemical and biological properties similm· to native sAmy. Crystallization of rAmy was cmied out by vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant in the presence ofCaCh at pH 9.0. The c1-ystals m·e of the space group P212121 with unit cell dimensions of a=52.63, b=75.20 and c=l37.11 A with one molecule per asymmetric unit. A total of 21,505 unique reflections were collected on a R-AXIS II imaging plate system to a resolution of 2.0 A using two crystals ...