The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase B

Abstract

The human ABO(H) blood-group antigens are oligosaccharide structures that are expressed on erythrocyte and other cell surfaces. The terminal carbohydrate residue differs between the blood types and determines the immune reactivity of this antigen. Individuals with blood type A have a terminal N-acetylgalactosamine residue and those with blood type B have a terminal galactose residue. The attachment of these terminal carbohydrates are catalyzed by two different glycosyltransferases: an alpha(1-->3)N-acetylgalactosaminyltransferase (GTA) and an alpha(1-->3)galactosyltransferase (GTB) for blood types A and B, respectively. GTA and GTB are homologous enzymes that differ in only four of 354 amino-acid residues (Arg/Gly176, Gly/Ser235, Leu/Met266 and Gly/Ala268 in GTA and GTB, respectively). Diffraction-quality crystals of GTA and GTB have previously been grown from as little as 10 mg ml(-1) stock solutions in the presence of Hg, while diffraction-quality crystals of the native enzymes require much higher concentrations of protein. The structure of a single mutant C209A has been determined in the presence and absence of heavy atoms and reveals that when mercury is complexed with Cys209 it forces a significant level of disorder in a polypeptide loop (amino acids 179-195) that is known to cover the active site of the enzyme. The observation that the more highly disordered structure is more amenable to crystallization is surprising and the derivative provides insight into the mobility of this polypeptide loop compared with homologous enzymes.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

“…For example, AAAA refers to GTA, BBBB refers to GTB, and AABB refers to the chimera with the first two critical residues of GTA and the last two critical residues of GTB. Previous findings implicate the first amino acid in enzyme turnover (12,26), the second and third in acceptor recognition (14,27), and the third and fourth in donor selection (14,17,25,28,29).…”

ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes

“…The first structure consists of residues 175-188 that shows significant flexibility and contains an ␣-helix consisting of residues 180 -187. The second structure consists of residues 189 -195 that adopts an ␣-helical conformation similar to that observed in a mutant GTB structure (20). The nine C-terminal residues remain disordered in the open or semi-closed states but display various levels of order in the closed conformation depending on the presence of substrate and on the identity of Arg 176 .…”

“…The result is a distorted helical structure with mixed ␣-3 10 -␣ character that partially occludes the active site. The mutual repulsion of positively charged residues Lys 179 , Arg 180 , and Arg 188 that held the enzyme in the open state are likely overcome to form the semi-closed conformation through electrostatic interactions with the negatively charged pyrophosphate moiety of bound UDP (20). Interestingly, despite high concentrations of UDP, both the ABBBϩUDP and AABBϩUDP structures show electron density corresponding to ϳ50% occupancy, which correlates to the occupancy in the two observed conformations of the internal loop.…”

“…One region consisted of the last 10 residues of the C terminus, whereas the other was an internal polypeptide loop composed of residues 177-195. Subsequent studies have shown that part of the disorder of the internal loop was because of the presence of a heavy atom, and that crystals of the mutant enzyme GTB/C209A grown in the absence of heavy atoms display a smaller disordered segment of the internal loop consisting of residues 177-187 (20).…”

“…Structural studies have revealed that specific internal sections of polypeptide adjacent to the active site are often observed to be flexible or completely disordered. These internal loops have been suggested to restrict water access to the active site, as well as act in donor recognition and catalysis (3), including the inverting enzymes ␤4Gal-T1 (12), GnT-I (13), GlcAT-I (14), and GlcAT-P (15); the retaining enzymes EXTL2 (16) ␣-(133)-GalT (17,18), GTA, and GTB (19,20); the microbial inverting SpsA (21) and CstII (22); and the retaining microbial enzyme LgtC (23).…”