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 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 alpha-helices and two sets of antiparallel beta-sheets. Superimposition of each structure with the mean structure yielded an average root mean square deviation (RMSD) of 0.72(+/-0.14)A for the backbone atoms, and 1.42(+/-0.19)A 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.
Hsu
Chun-Hua
CH
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC.
Liao
You-Di
YD
Pan
Yun-Ru
YR
Chen
Lih-Woan
LW
Wu
Shih-Hsiung
SH
Leu
Ying-Jen
YJ
Chen
Chinpan
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004527:Egg Proteins / Q000737:chemistry* / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011892:Rana catesbeiana / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D004527:Egg Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases