X-RAY STRUCTURE OF GELONIN AT 1.8 ANGSTROM RESOLUTION

Gelonin is a single chain ribosome inactivating protein (RIP) with pot ential application in the treatment of cancer and AIDS. Diffraction qu ality crystals grown using PEG3350, belong to the space group P2(1), w ith it a = 49.4 Angstrom b = 44.9 Angstrom, c = 137.4 Angstrom and bet a = 98.4 degrees, and contain two molecules in the asymmetric unit. Di ffraction data collected to 1.8 Angstrom resolution has a R(m) value o f 7.3%. Structure of gelonin has been solved by the molecular replacem ent method, using ricin A chain as the search model. Crystallographic refinement using X-PLOR resulted in a model for which the r.m.s deviat ions from ideal bond lengths and bond angles are 0.012 Angstrom and 2. 7 degrees, respectively The final R-factor is 18.4% for 39,806 reflect ions for which I > 1.0 sigma(I). The C-alpha atoms of the two molecule s in the asymmetric unit superpose to within 0.38 Angstrom for 247 ato m pairs. The overall fold of gelonin is similar to that of other RIPs such as ricin A chain and alpha-momorcharin, the r.m.s.d. for C-alpha superpositions being 1.3 and 1.4 Angstrom, respectively The-catalytic residues (Glu166, Arg169 and Tyr113) in the active site form a hydroge n bond scheme similar to that observed in other RIPs. The conformation of Tyr74 in the active site, however, is significantly different from that in alpha-momorcharin. Three well defined water molecules are loc ated in the active site cavity and one of them, X319, superposes to wi thin 0.2 Angstrom of a corresponding water molecule in the structure o f alpha-momorcharin. Any of the three could be the substrate water mol ecule in the hydrolysis reaction catalysed by gelonin. Difference elec tron density for a N-linked sugar moiety has been observed near only o ne of the two potential glycosylation sites in the sequence. The amino acid at position 239 has been established as Lys by calculation of om it electron density maps. The two cysteine residues in the sequence, C ys44 and Cys50, form a disulphide bond, and are therefore not availabl e for disulphide conjugation with antibodies. Based on the structure, the region of the molecule that is involved in intradimer interactions is suggested to be suitable for introducing a Cys residue for purpose s of conjugation with an antibody to produce useful immunotoxins.