Abstract
A class of bullfrog ribonucleases has been reported to display cytotoxicity and agglutinating activity toward tumor cells. The co-crystallizing crystal structure of a cytotoxic ribonuclease from bullfrog Rana catesbeiana, RC-RNase 6, complexed with d(ApCpGpA) was determined at 2.0 Å resolution. A new binding site was found near T14, K15, K16, F38, Y64, T83 from one protein, and N54, N56, L58, R60, V62, T83, N84, T89, G102 from two neighboring protein molecules. The binding of d(ApCpGpA) did not belong to the productive or non-productive binding near the active site that was occupied by a sulfate ion in this crystal structure. T14, L58, N84, and G102 among the d(ApCpGpA)-interacting residues were found to be conserved in three different ribonucleases from Rana catesbeina and onconase from Rana pipiens via amino acid sequence alignment. From the 2Fo– Fc electron density map, the last two nucleotides of d(ApCpGpA) were well recognized and refined near T14, K15, K16 and Y64 while the first two residues were wobbling and clamped between three protein molecules. This implies the potent lectin activity of T14, K15, K16 and Y64. Among these four residues, only T14 is conserved in the bullfrog RNases. Therefore, d(ApCpGpA) agglutinating site near T14 and the conservation of T14 suggest that T14 might play a crucial role in antitumor activity of the bullfrog ribonucleases.