Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution

Katayanagi, Katsuo; Miyagawa, M.; Matsushima, Masaaki; Ishikawa, Mariko; Kanaya, Shigenori; Nakamura, Haruki; Ikehara, Morio; Matsuzaki, Toshiake; Morikawa, Kosuke

doi:10.1016/0022-2836(92)90260-q

Cited by 228 publications

(269 citation statements)

References 51 publications

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“…Although Mn 2ϩ and Mg 2ϩ are similar in size, the metals exhibit different coordination properties. For example, the coordination of these metals with Escherichia coli RNase H shows a single Mg 2ϩ ion at the catalytic site of the enzyme, whereas the same site was able to accommodate two Mn 2ϩ ions (35)(36)(37). The Mg 2ϩ ion formed an outer sphere (water-mediated) coordination with the E. coli enzyme, whereas the Mn 2ϩ ions formed an inner sphere (non-watermediated) coordination, resulting in smaller distances between the Mn 2ϩ ion and the coordinating amino acids relative to Mg 2ϩ (37).…”

Section: Discussionmentioning

confidence: 99%

Binding and Cleavage Specificities of Human Argonaute2

Lima

Nichols

et al. 2009

Journal of Biological Chemistry

114

107

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RNA interference is a mechanism by which double-stranded RNA triggers the loss of RNA of homologous sequence (1). Long double strand RNAs are processed by the double strand endonuclease Dicer into short RNA duplexes (siRNA) 2 ranging from 21 to 23 nucleotides in length (2). The double strand RNA-binding proteins Dicer and human immunodeficiency virus, type 1, trans-activating response RNA-binding protein (TRBP) transfer the siRNAs to the RNA-induced silencing complex (RISC) (3). The antisense strand of the siRNA binds to the RISC endonuclease Argonaute 2 (Ago2), which then cleaves the target mRNA at a single phosphodiester bond bridging the ribonucleotides opposing the 10th and 11th nucleotide from the 5Ј terminus of the antisense strand (4 -11).The structure-activity relationships of siRNAs in human cultured cells have been studied extensively, but these types of studies offer few insights into the underlying mechanisms contributing to the observed activities of the siRNA and, in particular, their interaction with the RISC endonuclease human Ago2. Surprisingly, the little that is known about the interaction between human Ago2 and the substrate comes from a single report describing the preliminary characterization of recombinant human Ago2 (11). Specifically, human Ago2 cleavage activity was magnesium-dependent, and the antisense RNA containing a phosphate at the 5Ј terminus exhibited greater cleavage activity compared with the antisense RNA with a 5Ј-hydroxyl. The enzyme was unable to cleave a DNA target or use a DNA antisense strand to trigger the cleavage of a complementary RNA (11). In addition, UV cross-linking experiments showed that single strand but not double strand RNA was able to cross-link with the recombinant enzyme. Finally, unlike RISC activity from cellular extracts, which has been shown to catalyze multiple rounds of cleavage, recombinant Ago2 exhibited single-turnover kinetics (11,12).The architecture of the human Ago2 protein consists of a PIWI domain at the amino terminus, a centrally located Mid domain and a PAZ domain at the carboxyl terminus (13-17). The PIWI domain constitutes the catalytic domain of the enzyme and exhibits a three-dimensional structure similar to RNase H, sharing the same aspartic acid-aspartic acid-glutamic acid (DDE) catalytic triad and metal cofactor requirements (10,16,17). Recently, the structures of argonaute from Thermus thermophilus and Archaeoglobus fulgidus bound to the antisense strand have been solved (15,18). The structures show that the PAZ, Mid, and PIWI domains form an extended nucleic acid binding surface for the antisense strand. In addition, a basic binding pocket positioned within the Mid domain and a basic cleft in the PIWI domain were shown to bind, respectively, the 5Ј-terminal phosphate and the backbone at the 5Ј-pole of the antisense strand (15,18). Aside from the two 3Ј-terminal nucleotides of the antisense strand, which were shown to bind a hydrophobic pocket within the PAZ domain, no interactions were observed between the enzyme and the 3Ј-pol...

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Section: Discussionmentioning

confidence: 99%

Binding and Cleavage Specificities of Human Argonaute2

Lima

Nichols

et al. 2009

Journal of Biological Chemistry

114

107

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“…All of these residues are almost fully buried inside the protein molecule. The hydroxyl group of Thr 9 does not face the cavity but forms the hydrogen bond with the hydroxyl group of Thr 69 (46). For a comprehensive analysis of the effect of the mutation at the cavity on the protein stability, we have constructed a series of single mutant proteins, in which Ala 52 is replaced by the 19 other amino acid residues.…”

Section: Methodsmentioning

confidence: 99%

“…We have used this protein for studies of protein stability (14,(35)(36)(37)(38)(39)(40)(41)(42)(43) and folding (44,45). This protein is suitable for these studies for the following reasons: (i) highly refined coordinates of this protein are available (46), (ii) an overproduction system for this protein is available (36), and (iii) this protein reversibly unfolds in a single cooperative fashion with thermal and chemical denaturations (38).…”

mentioning

confidence: 99%

Conformational Stabilities of Escherichia coli RNase HI Variants with a Series of Amino Acid Substitutions at a Cavity within the Hydrophobic Core

Akasako¹,

Haruki²,

Oobatake³

et al. 1997

Journal of Biological Chemistry

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Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala 52 and Val 74 , resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala 52 , and double mutant proteins, in which Ala 52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val 74 was replaced by Ala or Leu. All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified. Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains. A correlation was observed between the protein stability and the hydrophobicity of the substituted residue. As a result, a number of the mutant proteins were more stable than the wild-type protein. The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins. However, examination of the farand near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein. These results suggest that the cavity in the hydrophobic core of E. coli RNase HI is conformationally fairly stable. This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein.

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“…These three residues provide a binding site for Mg*+, which is required for RNase H activity. The carboxyl oxygens of Glu48 and Asp70 are located 0.523 nm and 0.390nm away from the carboxyl oxygen of AsplO, respectively [12]. The accurate configuration of these residues, which might be influenced by the negative charge repulsions among them, seems to be important for the effective binding of Mg2+.…”

Section: The Functional Role Of Asp134mentioning

confidence: 98%

“…The corresponding residues in HIV-1 reverse-transcnptase RNase H are Asp443, Glu478, Asp498, and Asp549, respectively. The crystal structure of E. coli RNase HI [12] and HIV-1 reverse-transcriptase RNase H [14] are deposited in the Protein Data Bank with accession numbers 2RN2 and IHRH, respectively.…”

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confidence: 99%

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

Haruki

Noguchi

Nakai

et al. 1994

European Journal of Biochemistry

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The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the nV helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. cali strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins rD134HIRNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp1 34 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the nV helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its 61 position impacts catalysis.Ribonuclease H (RNase H), which degrades only the RNA strand of an RNA . DNA hybrid in the presence of divalent cations, such as Mg" or Mn", is widely found in various organisms [ 1 -31. Eschericlziu coli RNase HI, which had been designated as RNase H until a second RNase H (RNase HII) was isolated from E. coli 141, is composed of a single polypeptide chain with 155 amino acid residues [S] and has a PI value of 9.0 [6]. The similarities in the primary structures of this enzyme, reverse transcriptase RNase H 17, 81, Thermus thermophilus RNase H [91, and yeast RNase H [lo] strongly suggest that these enzymes are evolutionally related. In fact, the three-dimensional structure of E. coli RNase HI [ll-131 is similar to that of the reverse-transcriptase RNase H from human immunodeficiency virus-1 (HIV-1) [14-161. Therefore, it has been anticipated that detailed analyses of the structure/function relationships of E. Correspondence to S . Kanaya, Protein Engineering ResearchFax: +81 6 872 8210. Abbreviutions. GdnHCI, guanidine hydrochloride; HIV-1, human immunodeficiency virus-1 ; PCR, polymerase chain reaction; t,,*, temperature at which an enzyme loses 50% of its activity; P,, conformational parameter for u-helical residues in proteins ; RNase, ribonuclease; [DI 34XIRNase H, mutant ribonuclease H in which aspartic acid at position 134 is replaced by a specified amino acid.Institute,

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Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution

Cited by 228 publications

References 51 publications

Binding and Cleavage Specificities of Human Argonaute2

Binding and Cleavage Specificities of Human Argonaute2

Conformational Stabilities of Escherichia coli RNase HI Variants with a Series of Amino Acid Substitutions at a Cavity within the Hydrophobic Core

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis

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