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 oocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of 1H homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carried out with the program X-PLOR using a total of 951 restraints including 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected from a total of 150 calculated structures, the root-mean-square deviation from the average structure for the backbone heavy-atoms involved in well-defined secondary structure is 0.48 A, while that for all backbone heavy-atoms is 0.91 A. The structure of RC-RNase consists 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 homolo gous protein, onconase isolated from Rana pipiens. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(2'-->5')guanosine (2',5'-CpG) and 2'-deoxycytidylyl(3'-->5')-2'-deoxyguanosine (3',5'-dCpdG). Based on the ligand-induced chemical shift changes in RC-RNase and the NOE cross-peaks between 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 B1 subsite. The His103 residue was found to occupy the B state with the imidazole ring pointing away from the active site. The structure coordinates and the NMR restraints have been deposited in the Brookhaven Protein Data Bank (1bc4 and 1bc4mr, respectively).
Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan, 11529, The Republic of China.
C F
CF
Chang
C
C
Chen
Y C
YC
Chen
K
K
Hom
R F
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Huang
T H
TH
Huang
D029845:Amphibian Proteins; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003603:Cytotoxins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry; D004527:Egg Proteins / Q000737:chemistry*; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009865:Oocytes / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011892:Rana catesbeiana
D029845:Amphibian Proteins; D003603:Cytotoxins; D003845:Deoxycytosine Nucleotides; D003849:Deoxyguanosine; D015226:Dinucleoside Phosphates; D004527:Egg Proteins; D004722:Endoribonucleases; D004791:Enzyme Inhibitors; C037781:deoxycytidylyl-(3'-5')-deoxyguanosine; C015772:CpG dinucleotide; C094067:pyrimidine-guanine sequence-specific ribonuclease, Rana catesbeiana