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Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses

¹ Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan
² Department of Structural Biology, Biomolecular Engineering Research Institute, Osaka 565-0874, Japan

Reprint requests to: Dr. Kosuke Morikawa, Department of Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan; e-mail: morikawa{at}beri.co.jp; fax: 81-6-6872-8219.

(RECEIVED November 13, 2000; FINAL REVISION January 2, 2001; ACCEPTED January 2, 2001)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.48001.

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.

Keywords: Ribonuclease H; DNA/RNA hybrid; polynucleotidyl transferase family; crystal structure; mutational analysis

Abbreviations: RNase, ribonuclease • DNA, deoxyribonucleic acid • RNA, ribonucleic acid • MIR, multiple isomorphous replacement • PEG, polyethylene glycol • MES, 2-(N-morpholino)ethanesulfonic acid • MALDI, matrix-assisted laser desorption ionization • TOF, time-of-flight

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Ribonuclease H (RNase H) has a unique function to degrade specifically the RNA moiety of DNA/RNA hybrid duplexes (Hausen and Stein 1970). Biochemistry of this divalent metal-dependent endonuclease has been studied extensively because of its involvement in important biological processes such as DNA replication and repair (Kogoma and Foster 1998). Genes encoding RNase H have been found ubiquitously in viruses, bacteria, archaea, and eucarya, including human (Ohtani et al. 1999a). Because of the unique biochemical function, RNase H could be a potential target for medical applications. For example, RNase H is one of the therapeutic targets for AIDS (acquired immunodeficiency syndrome). The enzyme is also involved in antisense gene therapy.

RNase H is classified into two major types, according to their sequence similarity (Ohtani et al. 1999a, b). The biochemical and structural properties of the Type 1 RNase H, including the mechanisms of specific recognition of DNA/RNA hybrids and the catalytic reaction, have been studied extensively (Kanaya 1998; Morikawa and Katayanagi 1998). On the other hand, less information is available for the Type 2 enzyme, although a recent crystallographic study revealed the three-dimensional structure of an archaeal RNase HII (Lai et al. 2000). It would be interesting to understand how these two types of enzymes, which do not seem to share a common ancestor, catalyze the same hydrolytic reaction.

Haruki and coworkers (1998) previously cloned the rnhB gene encoding RNase HII (Tk-RNase HII) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, and overexpressed the protein in Escherichia coli. The recombinant Tk-RNase HII, which will be simply designated as Tk-RNase HII hereafter, shows a relatively broad divalent cation specificity. This enzyme does not cleave the RNA-DNA junction itself, but cleaves to leave a single ribonucleotide at the 5'-end of the RNA-DNA junction.

We have determined the X-ray crystal structure of Tk-RNase HII. In combination with the characterization of mutant proteins, the crystal structure allows identification of the substrate recognition interface and the catalytic center.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Truncation of the protein for crystallization
Tk-RNase HII was crystallized initially by the vapor diffusion method using 5% PEG6000 as a precipitant. The crystal provided a diffraction pattern, which represents the primitive hexagonal lattice with unit cell dimensions of a = b= 62 (Å) and c = 115 (Å). Unfortunately, Bragg reflections were observed up to 3.5 Å resolution, which is insufficient for a structure determination at an atomic level.

In parallel with the optimization of the crystallization condition, limited proteolysis was examined to define the core fragments, which may yield a higher resolution crystal. Digestions of Tk-RNase HII with chymotrypsin and trypsin produced distinct fragments whose N-terminal sequences are the same as that of the full-length Tk-RNase HII, indicating preservation of the N-terminus in both fragments. The molecular mass of the chymotrypsin fragment was determined by MALDI-TOF mass spectroscopy to be 22,250 Daltons. Taking the preference of the protease into account, the fragment is presumed to range from residues 1–198. Similarly, the trypsin fragment, with a molecular mass of 24,488 Daltons, appeared to range from residues 1–217. Interestingly, the trypsin fragment retained nearly full activity, whereas the chymotrypsin fragment was almost completely inactive. These results suggest that the segment between 199 and 217 is crucial for the RNase H activity.

We have prepared five different truncated proteins for the crystallization trial: residues 1–198 (Tk-RNase HII-198), 1–203 (Tk-RNase HII-203), 1–207 (Tk-RNase HII-207), 1–213 (Tk-RNase HII-213), and 1–217 (Tk-RNase HII-217). Among them, Tk-RNase HII-213 produced rod-shaped crystals in a solution containing 15% (w/v) PEG6000 buffered by 200 mM MES at pH 6.5. The crystal, which belongs to the space group P2₁2₁2₁ with unit cell dimensions of a = 43 (Å), b = 73 (Å), c = 78 (Å), diffracted to 2Å resolution with synchrotron radiation at the Photon Factory (Sakabe 1991). We attempted to determine the structure of this crystal form by the MIR method.

Overall structure
The folding of Tk-RNase HII-213 (the full-length molecule is composed of 228 amino acids) is shown in Figure 1. The structure is composed of two distinct domains. The N-terminal domain (residues 1–162) has a central core of a five-stranded ß-sheet, in which only ß2 is antiparallel to the other four. This sheet is surrounded by seven -helices: 1–7. As a whole, this domain with the /ß structure forms the well-known RNase H fold, which was found in the crystal structures of bacterial RNase HI (Katayanagi et al. 1990; Yang et al. 1990; Ishikawa et al. 1993), reverse transcriptase (Davies et al. 1991), integrase (Dyda et al. 1994; Bujacz et al. 1995), transposase (Rice and Mizuuchi 1995), and RuvC Holliday-junction resolvase (Ariyoshi et al. 1994). The C-terminal domain (residues 163–213) forms a compact structure consisting of two -helices: 8 and 9. Unlike the N-terminal domain, bacterial RNases HI do not possess this domain. In the full-length enzyme, the 9 helix is followed by 15 amino acid residues (Fig. 2A), of which conformation is likely to be flexible, as revealed from the susceptibility to trypsin digestion.

View larger version (99K): [in this window] [in a new window]   Fig. 1. Stereodiagram showing the backbone structure of Tk-RNase HII-213. Helices, strands, and loops are shown in red, cyan, and orange, respectively. Green triangles with labeled amino acids indicate the sites in which the C-terminal truncations (Tk-RNase HII-198, Tk-RNase HII-203, Tk-RNase HII-207, and Tk-RNase HII-213) were constructed.

View larger version (73K): [in this window] [in a new window]   Fig. 2. (A) Amino acid sequence alignment of Tk-RNase HII (Tk) and Mj-RNase HII (Mj) based on the comparison of their three-dimensional structures. The range of the secondary structures of Tk-RNase HII are shown above the sequence (white box for -helix and black arrow for ß-strand). Bold letters indicate amino acid residues conserved between the two proteins. The amino acid residues shown in red represent the mutated residues in the present study. The amino acid residues shown in blue are the truncated residues 214–228 in Tk-RNase HII-213. The truncation sites are indicated by green triangles. (B) Stereodiagram of the structures of Tk-RNase HII-213 (blue), Mj-RNase HII (red), and Ec-RNase HI (yellow). Root mean square displacements of the superimposition on the Tk-RNase HII structure are 1.2 Å for the 190 C atoms of Mj-RNase HII, and 2.0 Å for the 47 C atoms consisting of the core ß-sheet of Ec-RNase HI. Active-site residues are shown by white stick models. The "basic protrusion" of Ec-RNase HI is indicated by a yellow arrow. (C) A close-up view of the active-site of the three RNases H. The view direction is the same as in the panel B and in Figure 1.

Lai and coworkers (2000) reported that Mj-RNase HII forms a dimer in the crystal. In contrast, the Tk-RNase HII structure is obviously monomeric, even in the crystal. In agreement with this, both analyses by size exclusion chromatography and dynamic light scattering indicated that the Tk-RNase HII is present as a monomer in solution (data not shown). Thus, we believe that dimer formation is not essential, at least, for the RNase H activity.

Active-site
Lai and coworkers (2000) proposed possible active-site residues of Mj-RNase HII by comparing its structure with those of E. coli RNase HI (Ec-RNase HI) (Katayanagi et al. 1992) and retroviral integrase (Bujacz et al. 1995). The sequence alignment between Mj-RNase HII and Tk-RNase HII indicates perfect conservation of the proposed residues (Fig. 2A), suggesting that the catalytic mechanisms of these two enzymes are the same. Actually, their putative active-site residues are well superimposed onto each other with a root-mean-square displacement of 0.67 Å, including the side chain atoms (Fig. 2C). To obtain more convincing experimental evidence for the catalytic residues, we constructed five Tk-RNase HII mutants, in which the putative catalytic residues, Asp7, Glu8, Asp105, His132, and Asp135, were individually replaced by Ala.

The far- and near-UV CD (circular dichroism) spectra of the five mutants were nearly identical to those of the wild-type protein (data not shown), suggesting that the conformation of the protein was not changed markedly by these mutations. Table 1 summarizes the kinetic parameters of the five mutants for the hydrolysis of M13 DNA/RNA hybrid. The D7A and D105A mutants showed no detectable activity. The D135A mutant showed only a trace of the activity. These three residues possess highly conserved carboxyl groups required for polynucleotide transfer reaction (Yang and Steitz 1995; Mizuuchi 1997; Morikawa 1998).

Kashiwagi and coworkers (1996) proposed a catalytic mechanism of Ec-RNase HI, in which His124 acts as a general base with the aid of Asp134. Assuming the participation of the histidine residue in the catalytic reaction of the Type 2 RNase H, as found in the catalytic reaction of the Type 1 RNase H, His132 is the only possible candidate for the same role. However, the H132A mutant retained substantial activity, although it was lower than that of the wild-type protein (Table 1), indicating that His132 is not essential for the activity. Consistent with this, His132 is conserved only in limited organisms (Ohtani et al. 1999a). As shown in Figure 3, the side chain of His132 forms hydrogen bonds with the main chain atoms of the loop between the ß4 strand and the 5 helix, which contains the catalytically important Asp105. Therefore, it is likely that the decrease in the activity shown by the His132 Ala protein is attributable to a change of the active-site conformation.

View larger version (92K): [in this window] [in a new window]   Fig. 3. A representative 2Fo-Fc electron density map after refinement. The model of a protein portion around His132 is fitted into density contoured at the 1.0 level. The imidazole group can assume two conformations, both of which form hydrogen bond(s) with the loop adjacent to the catalytic residue Asp105.

It has been proposed that Ser159 of Mj-RNase HII (Ser145 in Tk-RNase HII) is involved in the catalytic reaction, because it is located close to the active-site residues and forms a hydrogen bond with Asp149 (Asp135 in Tk-RNase HII) (Lai et al. 2000). However, this serine residue is not fully conserved in the Type 2 RNase H sequences. For example, Bacillus subtilis RNase HII, which has Ala at the corresponding position, has been shown to exhibit the RNase H activity (Ohtani et al. 1999b). These results strongly suggest that the serine residue is not involved in the catalytic reaction of Type 2 RNase H.

The endonulease activity of Tk-RNase HII requires a divalent metal cation, such as Mg^{2 +}, Mn^{2 +}, or Co^{2 +} (Haruki et al. 1998). To identify metal binding sites, these metal ions were soaked into the crystal. However, no significant electron density was observed around the active-site in difference Fourier maps. Thus, the affinity of the metals with Tk-RNase HII appears to be low in the absence of DNA/RNA hybrids.

Substrate binding
Limited proteolytic analyses indicated that the segment 199–217 is important for the RNase H activity. To investigate the functional role of this segment, the enzymatic properties of the five truncated proteins were analyzed. The far-UV CD spectra of these truncated proteins were nearly identical to that of the wild-type protein (data not shown), suggesting that the protein conformation was not markedly changed by the C-terminal truncations. The kinetic parameters of the truncated proteins are compared with those of the wild-type protein in Table 1. The kinetic parameters of Tk-RNase HII-217 were almost identical to those of the wild-type protein. The additional truncation of the four residues did not seriously affect the K_m and k_cat values. However, further truncations resulted in a dramatic increase in the K_m value. They affected the k_cat value as well, but much less seriously. As a result, the K_m values of Tk-RNase HII-207 and Tk-RNase HII-203 were increased by 17- and 90-fold, respectively, as compared to that of the wild-type protein. Thus, the deletion of residues 199–213, which form the 9 helix (Fig. 1), seriously affected the K_m value, rather than the k_cat value. The interactions between the truncated proteins and the substrate were analyzed using surface plasmon resonance (Fig. 4). Tk-RNase HII-213 and Tk-RNase HII-217 with the intact 9 helix retained almost full substrate binding affinity. In contrast, this affinity decreased significantly as the length of the 9 helix was reduced by the truncation. Consequently Tk-RNase HII-198, which completely lost the 9 helix, did not bind to the substrate at all. Tk-RNase HII-203 apparently did not bind to the substrate, although it retained RNase H activity. The interaction between this protein and the substrate may be too weak to be detected by the BIAcore analysis. Taken together, these results suggest strongly that the 9 helix plays an important role in substrate binding. Because this helix contains many basic residues (Fig. 2A), it is likely to contact the negatively charged DNA/RNA hybrid by electrostatic force.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Surface plasmon resonance analysis of the proteins (100 nM) as reported by Haruki and coworkers (1997). Black, blue, green, orange, red, and cyan lines are sensorgrams for the wild-type protein, Tk-RNase HII-217, Tk-RNase HII-213, Tk-RNase HII-207, Tk-RNase HII-203, and Tk-RNase HII-198, respectively.

View larger version (77K): [in this window] [in a new window]   Fig. 5. (A) A hypothetical docking model of the DNA/RNA hybrid on Tk-RNase HII-213. This model was generated by superposition of the obtained crystal structure onto the Ec-RNase HI-DNA/RNA model proposed by Nakamura and coworkers (1991). Yellow and green ribbons indicate RNA and DNA strands, respectively. Blue and red surfaces represent the segment 199–213 and the active-site residues, respectively. (B) A putative substrate recognition by Tk-RNase HII. The C-terminal domain is rotated by 70° so that the basic 9 helical segment can make contact with phosphate backbones of the DNA/RNA hybrid duplex.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

Briefly, the E. coli cells that overproduced the protein were harvested and suspended in 10mM Tris-HCl at pH7.5 containing 1mM EDTA. After sonication and then incubation at 90°C, nucleic acid was removed by polyethyleneimine treatment, followed by ammonium sulfate precipitation (70% sat.). Finally, the protein was purified to homogeneity by chromatography on HiTrap heparin and MonoQ columns (Amersham Pharmacia Biotech). The purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentrations of the wild-type, mutant, and truncated proteins were estimated by assuming A_280nm of 0.63 for 1 mg/ml protein.

The five C-terminal truncated proteins were constructed by introducing a stop codon (TAA) at the corresponding position. Overproduction and purification of these truncated proteins were performed as reported for the wild-type protein (Haruki et al. 1998).

X-ray crystallography
Tk-RNase HII-213 was crystallized by the hanging drop vapor diffusion method with 15%(w/v) PEG6000 and 200 mM MES at pH 6.5. Two sets of X-ray diffraction data were collected for the native crystal (Table 2). The initial phases were estimated by the MIR method using the first data set and three kinds of heavy atom derivatives (Table 2). The density modification (Cowtan 1994) extended the resolution to 3.5 Å and effectively improved the electron density map. After 80 % of the amino acid residues were assigned, the structure factor data were swapped for the second data set, because of the higher resolution limit. The CNS refinement (Brünger et al. 1998) with manual model correction reduced the R-factor and the free R to 0.233 and 0.278, respectively, with the 15–2.0 Å resolution data. The refinement and the final model statistics are summarized in Table 3. The atomic coordinates have been deposited to the Protein Data Bank (accession code: 1IO2).

	Acknowledgments

 
We thank Mr. Kochi for technical assistance with biochemical experiments.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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