CRYSTALLIZATION, STRUCTURAL DETERMINATION AND ANALYSIS OF A NOVEL PARASITE VACCINE CANDIDATE - FASCIOLA-HEPATICA GLUTATHIONE-S-TRANSFERASE

Glutathione S-transferases (GSTs) represent the major class of detoxif ying enzymes from parasitic helminths. As a result, they are candidate s for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cat tle against fasciolosis. This helminth contains at least seven GST iso forms, of which four have been cloned. The cloned isoforms (Fh51, Fh47 , Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. C rystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4(1)22, with one monome r in the asymmetric unit, which corresponds to avery high solvent cont ent of approximately 75%. The physiological dimer is generated via a c rystallographic 2-fold rotation. The three-dimensional structure of Fh 47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: a n N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees i nterdomain rotation in comparison to Sj26, where the equivalent residu e is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh4 7 crystal structure, reveal critical differences in the residues linin g the xenobiotic binding site, particularly at residue positions 9, 10 6 and 204. Ln addition, differences amongst the isoforms in the nonsub strate binding site were noted, which may explain the observed differe ntial binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed reg ions of the molecule. (C) 1997 Academic Press Limited.