title: Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1.
abstract: RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ⩾2M NaCl, ⩾10mM MnCl2, or ⩾300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1 was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn(2+) ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (∼0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Division of Electron Microscopic Research, Korea Basic Science Institute, 169-148 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors: Dong-Ju
authors: DJ
authors: You
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Nujarin
authors: N
authors: Jongruja
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Elias
authors: E
authors: Tannous
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; International College, Osaka University, 1-30 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors: Clement
authors: C
authors: Angkawidjaja
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Yuichi
authors: Y
authors: Koga
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: kanaya@mls.eng.osaka-u.ac.jp.
authors: Shigenori
authors: S
authors: Kanaya
mesh_terms: D020134:Catalytic Domain / Q000187:drug effects; D002712:Chlorides / Q000737:chemistry / Q000494:pharmacology; D006217:Halobacterium / Q000187:drug effects; D015636:Magnesium Chloride / Q000737:chemistry / Q000494:pharmacology; D017895:Manganese Compounds / Q000737:chemistry / Q000494:pharmacology; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects*; D017510:Protein Folding / Q000187:drug effects*; D016914:Ribonuclease H / Q000737:chemistry* / Q000187:drug effects; D012965:Sodium Chloride / Q000737:chemistry / Q000494:pharmacology
chemical_list: D002712:Chlorides; D017895:Manganese Compounds; D015636:Magnesium Chloride; D012965:Sodium Chloride; D016914:Ribonuclease H; C078856:ribonuclease HI; C025340:manganese chloride
keywords: Bi-aspartate site; Crystal structure; Negative charge repulsion; RNase H; Salt-dependent folding