THE SOLUTION STRUCTURE OF A CYTOTOXIC RIBONUCLEASE FROM THE OOCYTES OF RANA-CATESBEIANA (BULLFROG)

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a lectin possessing potent cell cytotoxicity. It was isolated from the o ocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of H-1 homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carr ied out with the program X-PLOR using a total of 951 restraints includ ing 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected f rom a total of 150 calculated structures, the root-mean-square deviati on from the average structure for the backbone heavy-atoms involved in well-defined secondary structure is 0.48 Angstrom, while that for all backbone heavy-atoms is 0.91 Angstrom. The structure of RC-RNase cons ists of three alpha-helices and two triple-stranded anti-parallel beta -sheets and folds in a kidney-shape, very similar to the X-ray crystal structure of a homologous protein, onconase isolated from Rana pipien s. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(2' --> 5')guanosine (2',5'-CpG) and 2'-deoxycyti dylyl(3' --> 5')-2'-deoxyguanosine (3',5'-dCpdG). Based on the ligand- induced chemical shift changes in RC-RNase and the NOE cross-peaks bet ween RC-RNase and the inhibitors, the key residues involved in protein -inhibitor interaction have been identified. The inhibitors were found to bind in a ''retro-binding'' mode, with the guanine base bonded to the B-1 subsite. The His103 residue was found to occupy the B state wi th the imidazole ring pointing away from the active site. The structur e coordinates and the NMR restraints have been deposited in the Brookh aven Protein Data Bank (1bc4 and 1bc4mr, respectively). (C) 1998 Acade mic Press.