Solution Structure of the Cytotoxic RNase 4 from Oocytes of Bullfrog Rana catesbeiana

Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to t...

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Bibliographic Details
Published in:Journal of molecular biology 2003-02, Vol.326 (4), p.1189-1201
Main Authors: Hsu, Chun-Hua, Liao, You-Di, Pan, Yun-Ru, Chen, Lih-Woan, Wu, Shih-Hsiung, Leu, Ying-Jen, Chen, Chinpan
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
antitumor
Cell Line
Circular Dichroism
Crystallography, X-Ray
cytotoxicity
Egg Proteins - chemistry
Egg Proteins - metabolism
Endoribonucleases - chemistry
Endoribonucleases - metabolism
Freshwater
Humans
Hydrogen Bonding
Models, Molecular
Molecular Sequence Data
Molecular Structure
NMR
Nuclear Magnetic Resonance, Biomolecular
Oocytes - enzymology
Protein Structure, Secondary
Rana catesbeiana
Rana catesbeiana - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
ribonuclease
ribonuclease 4
Sequence Alignment
solution structure
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Summary:Cytotoxic ribonucleases with antitumor activity are mainly found in the oocytes and early embryos of frogs. Native RC-RNase 4 (RNase 4), consisting of 106 residues linked with four disulfide bridges, is a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana. RNase 4 belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Recombinant RC-RNase 4 (rRNase 4), which contains an additional Met residue and glutamine instead of pyroglutamate at the N terminus, was found to possess less catalytic and cytotoxic activities than RNase 4. Equilibrium thermal and guanidine-HCl denaturation CD measurements revealed that RNase 4 is more thermally and chemically stable than rRNase 4. However, CD and NMR data showed that there is no gross conformational change between native and recombinant RNase 4. The NMR solution structure of rRNase 4 was determined to comprise three α-helices and two sets of antiparallel β-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(±0.14) Å for the backbone atoms, and 1.42(±0.19) Å for the heavy atoms in residues 3–105. A comparison of the 3D structure of rRNase 4 with the structurally and functionally related cytotoxic ribonuclease, onconase (ONC), showed that the two H-bonds in the N-terminal pyroglutamate of ONC were not present at the corresponding glutamine residue of rRNase 4. We suggest that the loss of these two H-bonds is one of the key factors responsible for the reductions of the conformational stability, catalytic and cytotoxic activities in rRNase 4. Furthermore, the differences of side-chain conformations of subsite residues among RNase A, ONC and rRNase 4 are related to their distinct catalytic activities and base preferences.
ISSN:0022-2836
1089-8638
DOI:10.1016/S0022-2836(02)01472-9