Structure and characterization of RNase H3 from Aquifex aeolicus.
The crystal structure of ribonuclease H3 from Aquifex aeolicus (Aae-RNase H3) was determined at 2.0 Å resolution. Aae-RNase H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus stearothermophilus RNase H3 (Bst-RNase H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase H3 is relatively long and flexible, while that of Aae-RNase H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase H3 is important for substrate binding, and uses the flat surface of the β-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Nujarin
N
Jongruja
Dong-Ju
DJ
You
Clement
C
Angkawidjaja
Eiko
E
Kanaya
Yuichi
Y
Koga
Shigenori
S
Kanaya
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012260:Ribonucleases; C117079:ribonuclease HIII