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Structure and characterization of RNase H3 from Aquifex aeolicus
Author(s) -
Jongruja Nujarin,
You DongJu,
Angkawidjaja Clement,
Kanaya Eiko,
Koga Yuichi,
Kanaya Shigenori
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08657.x
Subject(s) - aquifex aeolicus , rnase ph , rnase p , rnase mrp , rnase h , active site , ribonuclease , linker , crystallography , chemistry , stereochemistry , biology , biochemistry , enzyme , rna , escherichia coli , computer science , gene , operating system
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.