CRYSTAL STRUCTURE OF BULLFROG EGG LECTIN. T. Nonaka, E. Hirayama, M. Iwama,* K. Ohgi,* M. Irie,* and Y. Mitsui Department of BioEngineering, Nagaoka University of Technology, Nagaoka, Niigata 940-21, Japan, *Department of Microbiology, Hoshi College of Pharmacy, Shinagawa, Tokyo 142, Japan

Bullfrog (Rana catesbeiana) egg lectin (RCEL) is a member of the pancreatic ribonuclease superfamily. RCEL exhibits RNA-degrading activity as well as multivalent carbohydrate-binding activity (1). Since RCEL preferentially inhibits protein synthesis in tumor cells, it is suggested that RCEL binds cell surface receptors, enters cell cytosol through receptor-mediated endocytosis, then degrades rRNA, and finally kills the cell. RCEL shows 28% and 53% sequence identities respectively to pancreatic ribonuclease A (RNase A) and a cytotoxic ribonuclease (onconase) from frog (Rana pipiens) eggs (2). The amino acid residues, His12, Lys41 and His119 (RNase A numbering), essential for RNA-degrading activity are structurally conserved among RNase A's and onconase. The disposition of the receptor binding sites of RCEL, however, has remained uncertain because the structures of the homologous proteins, human angiogenin and onconase, were revealed as non-liganded forms. Using a hanging-drop vapor diffusion method, crystals of RCEL grew in a few weeks at 20 deg.C under the condition [0.4 mM RCEL, 4 mM sialic acid, 400 mM sodium citrate, 40% (v/v) hexylene glycol, 100 mM HEPES buffer, pH 7.6]. Unit cell dimensions are a = b = 42.24Å, c = 119.4Å (the space group: P3221). X-ray diffraction data was collected up to 1.62 Å resolution [R-merge(I) = 5.23%, completeness = 89.4%, redundancy = 2.67 ] using an in-house Rigaku R-AXIS IIc area detector. The crystal structure of RCEL was solved by molecular replacement techniques using the known structure of onconase as a search model. The current refined model of RCEL revealed, as expected, the catalytic active site similar to those found in the other members of the superfamily and the binding sites for two sialic acid molecules suggesting putative receptor binding sites.

(1) K. Nitta, K. Ozaki et al., Cancer Res. 54, 920-927 (1994). (2) S. C. Mosimann et al., J. Mol. Biol. 236, 1141-1153 (1994).