Crystal structure of type 1 ribonuclease H from hyperthermophilic archaeon Sulfolobus tokodaii: role of arginine 118 and C-terminal anchoring.
The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.
You
Dong-Ju
DJ
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Chon
Hyongi
H
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D019843:Archaeal Proteins / Q000737:chemistry; D001120:Arginine; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry*; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D001120:Arginine; D016914:Ribonuclease H