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Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site
Biochimica et Biophysica Acta 1547 (2001) 135^142 www.bba-direct.com
Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site Yasuo Tsunaka, Mitsuru Haruki, Masaaki Morikawa, Shigenori Kanaya * Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 29 November 2000; received in revised form 14 February 2001; accepted 28 February 2001
Abstract The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp10 and Glu48 are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011^11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu48 is not responsible for Mg2 binding but is involved in the catalytic function. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: RNase H; Catalytic mechanism; Active site; Site-directed mutagenesis; Substrate binding ; DNA/RNA hybrid
1. Introduction RNase HI from Escherichia coli endonucleolytically hydrolyzes only the RNA strand of DNA/ RNA hybrid at alkaline pH [1]. The enzyme is a basic protein with a pI value of 9.0 and is composed of a single polypeptide chain with 155 amino acid residues. The enzyme has been structurally and functionally well studied [2,3]. The physiological role of the enzyme has also been extensively studied using E. coli rnhA mutants, and the involvement of the enAbbreviations: DTT, dithiothreitol; CD, circular dichroism; ts, temperature-sensitive; ds DNA, double-stranded DNA * Corresponding author. Fax: +81-6-6879-7938; E-mail: [email protected]
zyme in replication and transcription has been proposed [4]. E. coli RNase HI is a member of the polynucleotidyl transferase family [5], which includes RuvC resolvase from E. coli [6], HIV-1 integrase [7], ASV integrase [8], and bacteriophage Mu transposase [9]. A characteristic common to the enzymes of this family is a high negative charge density at the active site, in which divalent cation(s) bind. Two alternative mechanisms have been proposed for the catalytic function of E. coli RNase HI. One is a two-metalion mechanism [10] and the other is a general acid^ base mechanism [2]. However, crystallographic [11], NMR [12], metallobiochemical [13], and enzymatic [14] studies have made the general acid^base mechanism more plausible than the two-metal-ion mecha-
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nism. According to the general acid^base mechanism, one metal ion, instead of two, is required for activity, and the hydroxyl ion, which attacks the phosphate group for the RNA cleavage, is activated by an amino acid residue, instead of the metal ion. According to the latest general acid^base mechanism [15], Asp10 and Asp70 provide ligands for Mg2 binding; His124 accepts a proton from the attacking water molecule which acts as a general base; Asp134 holds this water molecule; Glu48 anchors the water molecule that acts as a general acid. The observation that the carboxyl group of Glu48 directly coordinates with the Mg2 ion [11] is not inconsistent with this mechanism, because the carboxyl group of Glu48 may coincidentally shift the position upon Mg2 binding due to electrostatic attraction. However, the observation that the mutant protein D10R/ E48R, in which Asp10 and Glu48 are both replaced by Arg, exhibited the metal-independent RNase H activity only at acidic pH [16] does not support this mechanism, because it shows that Glu48 is not related to the catalytic function. However, either the digestion products of this unusual RNase H activity or the catalytic residues involved in this unusual RNase H activity remained to be determined. Therefore, it would be informative to analyze the structures and functions of this mutant protein in more detail. Here we report that the mutant protein D10R/ E48R does not show any RNase H activity in our assay system both in vivo and in vitro. Instead, it exhibits high-nucleic-acid-binding a¤nity. These results are consistent with the current model for the catalytic mechanism of E. coli RNase HI, in which Glu48 is related to catalytic function, instead of Mg2 binding. 2. Materials and methods 2.1. Preparation of the mutant protein D10R/E48R The mutant rnhA gene encoding the mutant protein D10R/E48R, in which the codons for Asp10 (GAT) and Glu48 (GAG) are changed to CGT and CCG for Arg, respectively, was constructed by PCR as described previously [17]. This mutant rnhA gene was substituted for the wild-type rnhA gene in plas-
mid pJAL600, which was previously constructed [17], to generate plasmid pJAL10R48R. For overproduction of the mutant protein D10R/E48R, the rnhA mutant strain E. coli MIC3009, which was kindly donated by M. Itaya [18], was transformed with plasmid pJAL10R48R. Overproduction and puri¢cation were performed as described previously [17] with a slight modi¢cation. The cell lysates were subjected to anion-exchange column chromatography, followed by fractionation with ammonium sulfate precipitation, prior to P-11 column chromatography. Anionexchange column chromatography was carried out using a column (3 ml) of DE-52 (Whatman) equilibrated with 10 mM Tris^HCl (pH 7.5) containing 1 mM EDTA (TE bu¡er). The £ow-through fraction, which contains the mutant protein D10R/E48R, was precipitated at 80% saturation level with ammonium sulfate to remove nucleic acid. This latter process was repeated twice. The protein concentration was determined from the UV absorption, assuming that the mutant protein D10R/E48R has the same absorption coe¤cient, A0:1% 280 = 2.0, as that of the wild-type protein [19]. The cellular production level and the purity of the mutant protein were estimated by 15% SDS-PAGE [20], followed by staining with Coomassie brilliant blue. The wild-type protein was previously puri¢ed [17]. 2.2. Biochemical characterizations The molecular weight of the protein was estimated by applying an aliquot of the puri¢ed sample to a column (1.0U60 cm) of Sephacryl S-300 (Pharmacia) equilibrated with 10 mM Tris^HCl (pH 8.0) or 10 mM MES^NaOH (pH 5.5) containing 50 mM KCl. The £ow rate was 0.1 ml min31 , and 1-ml fractions were collected. Bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa) were individually applied to the column as standard proteins. The far-UV (200^260 nm) and near-UV (250^320 nm) circular dichroism (CD) spectra were measured in 20 mM MES^NaOH (pH 5.5) containing 50 mM NaCl at 25³C on a J-725 spectropolarimeter (Japan Spectroscopic Co.), as described previously [21]. The mean residue ellipticity (a, deg cm2 dmol31 ) was calculated by using an average amino acid molecular weight of 110.
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2.3. Complementation assay The rnhA mutant strain E. coli MIC3001, which shows RNase H-dependent temperature-sensitive (ts) growth phenotype and forms colonies at 30³C but not at 42³C, was kindly donated by M. Itaya [18]. Plasmid pBR10R48R was constructed by replacing the wild-type rnhA gene in plasmid pBR860, which was previously constructed [22], with the mutant rnhA gene encoding the mutant protein D10R/ E48R. E. coli MIC3001 was transformed with plasmid pBR10R48R by electroporation with Bio-Rad Gene pulser as described previously [23] and spread on two Luria^Bertani-medium agar plates with 5 g l31 NaCl and 50 mg l31 ampicillin. One plate was incubated at 30³C and the other was incubated at 42³C. 2.4. RNase H activity in vitro The RNase H activity was determined at 30³C for 15 min by using a hybrid between M13 DNA and 3 H-labeled RNA (M13 DNA/RNA hybrid) [24], poly(dT) and 32 P-labeled poly(rA) [poly(rA)/poly(dT)] [25], or 12-b DNA and 5P-32 P-labeled 12-b RNA (12-bp DNA/RNA hybrid) [26] as a substrate as previously described. The bu¡er was 10 mM Tris^ HCl (pH 8.0) or 10 mM MES^NaOH (pH 5.5) containing 50 mM KCl, 0.1 mM dithiothreitol (DTT), 0.05 mg ml31 bovine serum albumin, and 10 mM MgCl2 (+Mg2 ) or 10 mM EDTA (3Mg2 ). 2.5. Binding analysis with BIAcore The interaction between the protein and substrate was analyzed by the BIAcore1 instrument (Biacore), as described previously [27]. The protein (20 nM) was dissolved in 10 mM Tris^HCl (pH 8.0) containing 50 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.005% Tween P20 and injected at 25³C at a £ow rate of 100 Wl min31 onto the sensor chip surface, on which the 36-bp DNA/RNA hybrid was immobilized. The sequences of the 36-b RNA, which is biotinylated at the 5P-end, and the complementary 36-b DNA have previously been described [27]. The sensor chip, on which streptavidin had been immobilized and blocked with biotin, was used as a control surface. Binding surface was regenerated by
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washing with 2 M NaCl. The sensorgrams were analyzed by BIAsimulation software (Biacore) to estimate the association (ka ) and dissociation (kd ) rate constants. The association constant (KA ) was calculated from the equation KA = ka /kd . 2.6. Limited proteolytic digestion The mutant protein D10R/E48R was digested with chymotrypsin (Wako) at 25³C for 1 h in 10 mM sodium phosphate (pH 8.0) containing 50 mM NaCl and 2.5 mM DTT, in the presence or absence of excess molar amount of poly(rA)/poly(dT) at an enzyme:substrate ratio (by weight) of 1:30 or 1:10, respectively. The resultant peptides were separated by 20% SDS-PAGE, electroblotted onto a PVDF membrane, and determined for the N-terminal amino acid sequences with a model 491 protein sequencer (PE Applied Biosystems). Poly(rA)/poly(dT) was prepared by annealing poly(rA) with poly(dT) obtained from Pharmacia. 3. Results and discussion 3.1. Puri¢cation of the mutant protein D10R/E48R Upon induction for overproduction, the mutant protein D10R/E48R accumulated in the E. coli cells in a soluble form. Its production level was comparable to that of the wild-type protein. However, unlike the wild-type protein, the mutant protein did not adsorb to phosphocellulose (P-11), unless it was partially puri¢ed. Because DE-52 column chromatography and ammonium sulfate precipitation are e¡ective to remove nucleic acids associated nonspeci¢cally with the basic proteins, it seems likely that the mutant protein D10R/E48R interacts with nucleic acids much more strongly than does the wild-type protein. The mutant protein D10R/E48R was puri¢ed to give a single band on SDS-PAGE by three-step procedures (data not shown). The amount of the protein puri¢ed from 1 l of culture was approximately 7 mg. The UV spectrum of this mutant protein was nearly identical with that of the wild-type protein (A255 /A280 value of 0.67), indicating that the nucleic acids are not associated with the puri¢ed protein. The mutant protein D10R/E48R was eluted from
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the gel ¢ltration column as a single peak at the position where the wild-type protein was eluted, indicating that this mutant protein exists in a monomeric form. In the far-UV region, the CD spectrum of the mutant protein was nearly identical with that of the wild-type protein (data not shown), suggesting that the double mutation does not seriously a¡ect the main-chain folding. On the other hand, in the nearUV region, the CD spectrum of the mutant protein was slightly di¡erent from that of the wild-type protein (Fig. 1). The CD values of the former spectrum at 260^280 nm are higher than those of the latter one. However, the shapes of these spectra are similar to each other, suggesting that the double mutation causes a local conformational change, but only to a small extent. 3.2. Enzymatic activity The RNase H activity of the mutant protein D10R/E48R was determined by measuring the acidsoluble digestion products from the M13 DNA/RNA hybrid and poly(rA)/poly(dT), and also by analyzing the digestion products from the 12-bp DNA/RNA hybrid on gel electrophoresis. In all cases, the digestion of the substrate was carried out at pH 5.5 and 8.0, in the presence and absence of MgCl2 . However, this mutant protein hydrolyzed none of these sub-
Fig. 1. CD spectrum of the E. coli RNase HI mutant. The near-UV CD spectrum of the mutant protein D10R/E48R (thick line) is shown in comparison with that of the wild-type protein (thin line). The spectra were measured at pH 5.5 and 25³C.
strates at any condition examined. In addition, the E. coli MIC3001 transformants with pBR10R48R formed colonies at 30³C at pH 6.0, 6.5, and 7.0, but could not form them at 42³C at these pHs, indicating that the mutant protein cannot complement the ts growth phenotype of E. coli MIC3001 even at acidic pH. These results indicate that this mutant protein does not show any RNase H activity both in vitro and in vivo. The same result was obtained when the mutant protein was overproduced using the same system (pET system of Novagen) as that employed by Casareno et al. [16] (Tsunaka, Y., unpublished data). Thus, our result is inconsistent with the previous one [16]. This inconsistency may be result from the di¡erence in the assay method for the RNase H activity. Casareno et al. [16] have measured the RNase H activity by monitoring the change in the UV absorption at 260 nm upon hydrolysis of the DNA/ RNA hybrid using the stopped-£ow spectrophotometer. However, this change may also re£ect the conformational change of the substrate, instead of strand scission. Because the mutant protein D10R/ E48R forms a stable complex with the DNA/RNA hybrid (see below), the change in the absorption at 260 nm may re£ect the conformational change of the DNA/RNA hybrid upon binding to the protein. 3.3. Interaction with nucleic acids In order to examine whether the mutant protein D10R/E48R interacts with nucleic acids more strongly than does the wild-type protein, the interactions of these proteins with the 36-bp DNA/RNA hybrid were analyzed by BIAcore at pH 8.0 (Fig. 2). When the bu¡er containing the wild-type protein was passed over the surface of the sensor chip, on which the 36-bp DNA/RNA hybrid is immobilized, the RU value increased upon binding of the protein to the sensor chip. This value became constant due to an equilibrium between association of the protein to the sensor chip and dissociation of the protein from the sensor chip. Finally, this value decreased when the bu¡er alone was passed over the sensor chip, because the protein was dissociated from the sensor chip. In contrast, when the interaction between the mutant protein D10R/E48R and the 36bp DNA/RNA hybrid was analyzed equally, the RU
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Fig. 2. Interaction of the E. coli RNase HI mutant with the 36bp DNA/RNA hybrid. The sensorgram of the mutant protein D10R/E48R (thick line) obtained by surface plasmon resonance analysis is shown in comparison with that of the wild-type protein (thin line). The bu¡er containing the protein was replaced by the bu¡er alone at the time indicated by an arrow. A theoretical sensorgram (broken line) was drawn with BIAsimulation software, by using the ka and kd values of 1U105 M31 s31 and 5U1034 s31 , respectively. The resonance unit (RU) is an arbitrary unit used in the BIAcore system, which increases in proportion to the increase in the amount of the protein bound to the sensor chip.
value kept increasing upon binding of the protein to the sensor chip and did not reach a constant value. In addition, the protein was not clearly dissociated from the sensor chip when the bu¡er alone was passed over the sensor chip. The association (ka ) and dissociation (kd ) rate constants of the mutant protein D10R/E48R cannot be calculated from its sensorgram, because the interaction between this protein and the substrate does not reach an equilibrium between association and dissociation. However, ¢tting of the sensorgram experimentally determined to the theoretical ones allowed us to estimate the ka and kd values of this mutant protein as 1U105 M31 s31 and 5U1034 s31 , respectively (Fig. 2). The ka and kd values of the wild-type protein for the interaction with the 36-bp DNA/ RNA hybrid have been reported to be 1.5U106 M31 s31 and 3.2U1032 s31 , respectively [27]. Thus, the ka and kd values of the mutant protein are lower than those of the wild-type protein by 15- and 64fold, respectively. As a result, the association constant (KA ) of the mutant protein (2U108 M31 ) was higher than that of the wild-type protein (4.7U107
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M31 ) by 4.3-fold. These results suggest that the mutant protein is dissociated from the DNA/RNA hybrid much more slowly and thereby binds to it more strongly than the wild-type protein. When the interactions of these proteins with the 36-bp doublestranded (ds) DNA were examined by BIAcore using the sensor chip, on which the 36-bp ds DNA is immobilized, similar results were obtained (data not shown). In addition, the gel ¢ltration column chromatography indicated that the mutant protein forms a stable complex with the 21-b single-stranded DNA (5P-GTT CCG ATG CTA AGC TTT GCC-3P) both at pH 5.5 and 8.0, whereas the wild-type protein does not (data not shown). These results indicate that the mutant protein strongly binds to the nucleic acids in a nonspeci¢c manner. It is noted that the mutant protein gave an unusual sensorgram on BIAcore analysis at pH 5.5, probably because it strongly binds to the control sensor chip, on which the nucleic acid is not immobilized, as well. The wild-type protein did not give such an unusual sensorgram. The reason why the mutant protein strongly binds to the control sensor chip at pH 5.5 remained to be determined. 3.4. Binding site of nucleic acid Proteins are usually digested by protease at multiple sites. However, the susceptibilities to proteolytic digestion varied for di¡erent sites. For example, peptide bonds located within a loose conformation are usually more susceptible to proteolytic digestion than those located within a rigid conformation. If a given protein formed a stable complex with certain materials and the most susceptible sites to proteolytic digestion were located at the interface between these two molecules, this site would be protected from proteolytic digestion in the presence of these materials. Such a protein footprint analysis may be e¡ective to identify the nucleic-acid-binding site of the mutant protein D10R/E48R, because this mutant protein forms a stable complex with nucleic acids. Therefore, the mutant protein was digested with lysyl endopeptidase, trypsin, or chymotrypsin in a limited manner in the presence or absence of poly(rA)/poly(dT). When the proteolytic digests produced in the presence of poly(rA)/poly(dT) were compared with those produced in the absence of it by SDS-PAGE, a sig-
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ni¢cant di¡erence was observed only for chymotryptic digests. The peptide A, which migrated slightly faster than the intact protein on SDS-PAGE, was produced in the absence of poly(rA)/poly(dT), but was not produced in the presence of it (Fig. 3). The N-terminal amino acid sequence of this peptide was identical with that of the intact protein, indicating that this peptide represents a truncated form of the protein, in which a number of the C-terminal residues are removed. It is known that chymotrypsin preferably cleaves the peptide bonds at the C-termini of the amino acid residues with bulky hydrophobic side chains. Therefore, the peptide bonds at the Ctermini of Leu146 and Tyr151 are the candidates that were cleaved by chymotrypsin. Because the molecular weight of the peptide A is estimated to be reduced
Fig. 3. SDS-PAGE of the peptides generated by limited digestion with chymotrypsin. The mutant protein D10R/E48R was digested with chymotrypsin in a limited manner in the absence (lane 3) or presence (lane 4) of excess amount of poly(rA)/poly(dT). The enzyme:substrate ratios (by weight) were 1:30 and 1:10 for the digestions in the absence and presence of poly(rA)/ poly(dT), respectively. Chymotrypsin alone was also incubated at the same concentration as that used to digest the mutant protein in the presence of poly(rA)/poly(dT) (lane 5). Samples were subjected to electrophoresis on a 20% polyacrylamide gel in the presence of SDS. After electrophoresis, the gel was stained with Coomassie brilliant blue. Lane 1, a low molecular weight marker kit (Pharmacia Biotech) containing phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and K-lactalbumin (14 kDa). Lane 2, undigested protein. The positions of the peptides A and B are indicated by arrows.
by V500 on SDS-PAGE as compared to that of the intact protein, and because the sizes of the peptides truncated at the C-termini of Leu146 and Tyr151 are 1020 and 455, respectively, it is highly likely that the peptide A represents the truncated protein with Tyr151 at the C-terminus. No additional peptides were produced upon further digestion, suggesting that chymotrypsin primarily cleaves the speci¢c peptide bond between Tyr151 and Glu152 , and then cleaves the second sites in a rather nonspeci¢c manner. The peptide B was a fragment of chymotrypsin which was probably produced by self-digestion, because the N-terminal amino acid sequence of this peptide was determined to be IVNGE, which is corresponding to the peptide starting from Ile16 of chymotrypsin. The peptide which migrated faster than the peptide A and slower than the peptide B on SDSPAGE (Fig. 3, lane 4) remained to be identi¢ed, because the yield of this peptide was too low to determine its N-terminal amino acid sequence. The protein footprint analysis indicated that the speci¢c peptide bond probably between Tyr151 and Glu152 of the mutant protein was protected from the chymotryptic digestion in the presence of poly(rA)/poly(dT). Tyr151 is located in the groove-like depression of the protein molecule, which extends from the active site to the basic protrusion and has been suggested to be involved in substrate binding (Fig. 4) [28]. According to a model for the complex between E. coli RNase HI and a DNA/RNA hybrid [29], the DNA/RNA hybrid interacts with this groove-like depression, such that the RNA moiety approximately one turn removed from that interacting with the active site interacts with the basic protrusion (Fig. 4). Therefore, it seems likely that the DNA/RNA hybrid binds to the mutant protein D10R/E48R in a similar manner and this interaction is dramatically strengthened by a decrease in the negative charge density at the active site. 3.5. Catalytic mechanism E. coli RNase HI exhibits little enzymatic activity in the presence of the Ba2 or Ca2 ion [30]. Nevertheless, these divalent cations can also bind to the position where the Mg2 ion binds [29]. This may indicate that the divalent cation does not simply provide su¤cient positive charge density in the active
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Controversy still remains as to the metal-binding site of the enzyme. The crystal structure of the enzyme complexed with the Mg2 ion has shown that Asp10 and Glu48 provide ligands for Mg2 binding [11]. However, determination of the pka values of the individual acidic residues [31] and analysis for the interactions between the active site mutants and the Mg2 ion [15] have strongly suggested that Asp10 and Asp70 provide ligands for Mg2 binding and Glu48 is involved in the catalytic function. The observation that the mutant protein D10R/E48R is inactive both in vivo and in vitro is consistent with a proposal that Glu48 is involved in the catalytic function. Acknowledgements
Fig. 4. A model for the complex between E. coli RNase HI and DNA/RNA hybrid. The crystal structure of E. coli RNase HI (PDB: 2RN2) was drawn with the program RasMol. N and C represent the N- and C-termini of this protein, respectively. The side chains of the active site residues (Asp10 , Glu48 , Asp70 , His124 , and Asp134 ), as well as that of Tyr151 are shown. The solid region represents the basic protrusion, which has been shown to be important for substrate binding [32]. The solid and broken lines represent RNA and DNA strands of DNA/RNA hybrid, respectively. Possible orientations of these strands are also shown. This model was originally constructed by Katayanagi et al. [29].
site but ¢nely tunes the geometry of the active site residues to make them functional. Therefore, it would not be surprising if we could not reproduce the result of Casareno et al. [16], which shows that the mutations of the acidic residues to basic ones at the active site mimic the role of the divalent cation. Instead, we showed that the resultant mutant protein D10R/E48R formed a stable complex with nucleic acids. The active site of the enzyme is in sharp contrast to the basic protrusion in charge distribution. Because the DNA/RNA hybrid binds to the region, which includes both the active site and basic protrusion, and because the mutations reduce the negative charge density in the active site, such a sharp contrast in charge distribution may be required to allow the enzyme to perform multiple turnovers.
We thank M. Itaya, Mitsubishi Kasei Institute of Life Sciences, for providing E. coli MIC3001 and MIC3009. We also thank N. Hirano and N. Ohtani for helpful discussions.
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Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site Yasuo Tsunaka, Mitsuru Haruki, Masaaki Morikawa, Shigenori Kanaya * Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Received 29 November 2000; received in revised form 14 February 2001; accepted 28 February 2001
Abstract The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp10 and Glu48 are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011^11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu48 is not responsible for Mg2 binding but is involved in the catalytic function. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: RNase H; Catalytic mechanism; Active site; Site-directed mutagenesis; Substrate binding ; DNA/RNA hybrid
1. Introduction RNase HI from Escherichia coli endonucleolytically hydrolyzes only the RNA strand of DNA/ RNA hybrid at alkaline pH [1]. The enzyme is a basic protein with a pI value of 9.0 and is composed of a single polypeptide chain with 155 amino acid residues. The enzyme has been structurally and functionally well studied [2,3]. The physiological role of the enzyme has also been extensively studied using E. coli rnhA mutants, and the involvement of the enAbbreviations: DTT, dithiothreitol; CD, circular dichroism; ts, temperature-sensitive; ds DNA, double-stranded DNA * Corresponding author. Fax: +81-6-6879-7938; E-mail: [email protected]
zyme in replication and transcription has been proposed [4]. E. coli RNase HI is a member of the polynucleotidyl transferase family [5], which includes RuvC resolvase from E. coli [6], HIV-1 integrase [7], ASV integrase [8], and bacteriophage Mu transposase [9]. A characteristic common to the enzymes of this family is a high negative charge density at the active site, in which divalent cation(s) bind. Two alternative mechanisms have been proposed for the catalytic function of E. coli RNase HI. One is a two-metalion mechanism [10] and the other is a general acid^ base mechanism [2]. However, crystallographic [11], NMR [12], metallobiochemical [13], and enzymatic [14] studies have made the general acid^base mechanism more plausible than the two-metal-ion mecha-
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nism. According to the general acid^base mechanism, one metal ion, instead of two, is required for activity, and the hydroxyl ion, which attacks the phosphate group for the RNA cleavage, is activated by an amino acid residue, instead of the metal ion. According to the latest general acid^base mechanism [15], Asp10 and Asp70 provide ligands for Mg2 binding; His124 accepts a proton from the attacking water molecule which acts as a general base; Asp134 holds this water molecule; Glu48 anchors the water molecule that acts as a general acid. The observation that the carboxyl group of Glu48 directly coordinates with the Mg2 ion [11] is not inconsistent with this mechanism, because the carboxyl group of Glu48 may coincidentally shift the position upon Mg2 binding due to electrostatic attraction. However, the observation that the mutant protein D10R/ E48R, in which Asp10 and Glu48 are both replaced by Arg, exhibited the metal-independent RNase H activity only at acidic pH [16] does not support this mechanism, because it shows that Glu48 is not related to the catalytic function. However, either the digestion products of this unusual RNase H activity or the catalytic residues involved in this unusual RNase H activity remained to be determined. Therefore, it would be informative to analyze the structures and functions of this mutant protein in more detail. Here we report that the mutant protein D10R/ E48R does not show any RNase H activity in our assay system both in vivo and in vitro. Instead, it exhibits high-nucleic-acid-binding a¤nity. These results are consistent with the current model for the catalytic mechanism of E. coli RNase HI, in which Glu48 is related to catalytic function, instead of Mg2 binding. 2. Materials and methods 2.1. Preparation of the mutant protein D10R/E48R The mutant rnhA gene encoding the mutant protein D10R/E48R, in which the codons for Asp10 (GAT) and Glu48 (GAG) are changed to CGT and CCG for Arg, respectively, was constructed by PCR as described previously [17]. This mutant rnhA gene was substituted for the wild-type rnhA gene in plas-
mid pJAL600, which was previously constructed [17], to generate plasmid pJAL10R48R. For overproduction of the mutant protein D10R/E48R, the rnhA mutant strain E. coli MIC3009, which was kindly donated by M. Itaya [18], was transformed with plasmid pJAL10R48R. Overproduction and puri¢cation were performed as described previously [17] with a slight modi¢cation. The cell lysates were subjected to anion-exchange column chromatography, followed by fractionation with ammonium sulfate precipitation, prior to P-11 column chromatography. Anionexchange column chromatography was carried out using a column (3 ml) of DE-52 (Whatman) equilibrated with 10 mM Tris^HCl (pH 7.5) containing 1 mM EDTA (TE bu¡er). The £ow-through fraction, which contains the mutant protein D10R/E48R, was precipitated at 80% saturation level with ammonium sulfate to remove nucleic acid. This latter process was repeated twice. The protein concentration was determined from the UV absorption, assuming that the mutant protein D10R/E48R has the same absorption coe¤cient, A0:1% 280 = 2.0, as that of the wild-type protein [19]. The cellular production level and the purity of the mutant protein were estimated by 15% SDS-PAGE [20], followed by staining with Coomassie brilliant blue. The wild-type protein was previously puri¢ed [17]. 2.2. Biochemical characterizations The molecular weight of the protein was estimated by applying an aliquot of the puri¢ed sample to a column (1.0U60 cm) of Sephacryl S-300 (Pharmacia) equilibrated with 10 mM Tris^HCl (pH 8.0) or 10 mM MES^NaOH (pH 5.5) containing 50 mM KCl. The £ow rate was 0.1 ml min31 , and 1-ml fractions were collected. Bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa) were individually applied to the column as standard proteins. The far-UV (200^260 nm) and near-UV (250^320 nm) circular dichroism (CD) spectra were measured in 20 mM MES^NaOH (pH 5.5) containing 50 mM NaCl at 25³C on a J-725 spectropolarimeter (Japan Spectroscopic Co.), as described previously [21]. The mean residue ellipticity (a, deg cm2 dmol31 ) was calculated by using an average amino acid molecular weight of 110.
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2.3. Complementation assay The rnhA mutant strain E. coli MIC3001, which shows RNase H-dependent temperature-sensitive (ts) growth phenotype and forms colonies at 30³C but not at 42³C, was kindly donated by M. Itaya [18]. Plasmid pBR10R48R was constructed by replacing the wild-type rnhA gene in plasmid pBR860, which was previously constructed [22], with the mutant rnhA gene encoding the mutant protein D10R/ E48R. E. coli MIC3001 was transformed with plasmid pBR10R48R by electroporation with Bio-Rad Gene pulser as described previously [23] and spread on two Luria^Bertani-medium agar plates with 5 g l31 NaCl and 50 mg l31 ampicillin. One plate was incubated at 30³C and the other was incubated at 42³C. 2.4. RNase H activity in vitro The RNase H activity was determined at 30³C for 15 min by using a hybrid between M13 DNA and 3 H-labeled RNA (M13 DNA/RNA hybrid) [24], poly(dT) and 32 P-labeled poly(rA) [poly(rA)/poly(dT)] [25], or 12-b DNA and 5P-32 P-labeled 12-b RNA (12-bp DNA/RNA hybrid) [26] as a substrate as previously described. The bu¡er was 10 mM Tris^ HCl (pH 8.0) or 10 mM MES^NaOH (pH 5.5) containing 50 mM KCl, 0.1 mM dithiothreitol (DTT), 0.05 mg ml31 bovine serum albumin, and 10 mM MgCl2 (+Mg2 ) or 10 mM EDTA (3Mg2 ). 2.5. Binding analysis with BIAcore The interaction between the protein and substrate was analyzed by the BIAcore1 instrument (Biacore), as described previously [27]. The protein (20 nM) was dissolved in 10 mM Tris^HCl (pH 8.0) containing 50 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.005% Tween P20 and injected at 25³C at a £ow rate of 100 Wl min31 onto the sensor chip surface, on which the 36-bp DNA/RNA hybrid was immobilized. The sequences of the 36-b RNA, which is biotinylated at the 5P-end, and the complementary 36-b DNA have previously been described [27]. The sensor chip, on which streptavidin had been immobilized and blocked with biotin, was used as a control surface. Binding surface was regenerated by
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washing with 2 M NaCl. The sensorgrams were analyzed by BIAsimulation software (Biacore) to estimate the association (ka ) and dissociation (kd ) rate constants. The association constant (KA ) was calculated from the equation KA = ka /kd . 2.6. Limited proteolytic digestion The mutant protein D10R/E48R was digested with chymotrypsin (Wako) at 25³C for 1 h in 10 mM sodium phosphate (pH 8.0) containing 50 mM NaCl and 2.5 mM DTT, in the presence or absence of excess molar amount of poly(rA)/poly(dT) at an enzyme:substrate ratio (by weight) of 1:30 or 1:10, respectively. The resultant peptides were separated by 20% SDS-PAGE, electroblotted onto a PVDF membrane, and determined for the N-terminal amino acid sequences with a model 491 protein sequencer (PE Applied Biosystems). Poly(rA)/poly(dT) was prepared by annealing poly(rA) with poly(dT) obtained from Pharmacia. 3. Results and discussion 3.1. Puri¢cation of the mutant protein D10R/E48R Upon induction for overproduction, the mutant protein D10R/E48R accumulated in the E. coli cells in a soluble form. Its production level was comparable to that of the wild-type protein. However, unlike the wild-type protein, the mutant protein did not adsorb to phosphocellulose (P-11), unless it was partially puri¢ed. Because DE-52 column chromatography and ammonium sulfate precipitation are e¡ective to remove nucleic acids associated nonspeci¢cally with the basic proteins, it seems likely that the mutant protein D10R/E48R interacts with nucleic acids much more strongly than does the wild-type protein. The mutant protein D10R/E48R was puri¢ed to give a single band on SDS-PAGE by three-step procedures (data not shown). The amount of the protein puri¢ed from 1 l of culture was approximately 7 mg. The UV spectrum of this mutant protein was nearly identical with that of the wild-type protein (A255 /A280 value of 0.67), indicating that the nucleic acids are not associated with the puri¢ed protein. The mutant protein D10R/E48R was eluted from
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the gel ¢ltration column as a single peak at the position where the wild-type protein was eluted, indicating that this mutant protein exists in a monomeric form. In the far-UV region, the CD spectrum of the mutant protein was nearly identical with that of the wild-type protein (data not shown), suggesting that the double mutation does not seriously a¡ect the main-chain folding. On the other hand, in the nearUV region, the CD spectrum of the mutant protein was slightly di¡erent from that of the wild-type protein (Fig. 1). The CD values of the former spectrum at 260^280 nm are higher than those of the latter one. However, the shapes of these spectra are similar to each other, suggesting that the double mutation causes a local conformational change, but only to a small extent. 3.2. Enzymatic activity The RNase H activity of the mutant protein D10R/E48R was determined by measuring the acidsoluble digestion products from the M13 DNA/RNA hybrid and poly(rA)/poly(dT), and also by analyzing the digestion products from the 12-bp DNA/RNA hybrid on gel electrophoresis. In all cases, the digestion of the substrate was carried out at pH 5.5 and 8.0, in the presence and absence of MgCl2 . However, this mutant protein hydrolyzed none of these sub-
Fig. 1. CD spectrum of the E. coli RNase HI mutant. The near-UV CD spectrum of the mutant protein D10R/E48R (thick line) is shown in comparison with that of the wild-type protein (thin line). The spectra were measured at pH 5.5 and 25³C.
strates at any condition examined. In addition, the E. coli MIC3001 transformants with pBR10R48R formed colonies at 30³C at pH 6.0, 6.5, and 7.0, but could not form them at 42³C at these pHs, indicating that the mutant protein cannot complement the ts growth phenotype of E. coli MIC3001 even at acidic pH. These results indicate that this mutant protein does not show any RNase H activity both in vitro and in vivo. The same result was obtained when the mutant protein was overproduced using the same system (pET system of Novagen) as that employed by Casareno et al. [16] (Tsunaka, Y., unpublished data). Thus, our result is inconsistent with the previous one [16]. This inconsistency may be result from the di¡erence in the assay method for the RNase H activity. Casareno et al. [16] have measured the RNase H activity by monitoring the change in the UV absorption at 260 nm upon hydrolysis of the DNA/ RNA hybrid using the stopped-£ow spectrophotometer. However, this change may also re£ect the conformational change of the substrate, instead of strand scission. Because the mutant protein D10R/ E48R forms a stable complex with the DNA/RNA hybrid (see below), the change in the absorption at 260 nm may re£ect the conformational change of the DNA/RNA hybrid upon binding to the protein. 3.3. Interaction with nucleic acids In order to examine whether the mutant protein D10R/E48R interacts with nucleic acids more strongly than does the wild-type protein, the interactions of these proteins with the 36-bp DNA/RNA hybrid were analyzed by BIAcore at pH 8.0 (Fig. 2). When the bu¡er containing the wild-type protein was passed over the surface of the sensor chip, on which the 36-bp DNA/RNA hybrid is immobilized, the RU value increased upon binding of the protein to the sensor chip. This value became constant due to an equilibrium between association of the protein to the sensor chip and dissociation of the protein from the sensor chip. Finally, this value decreased when the bu¡er alone was passed over the sensor chip, because the protein was dissociated from the sensor chip. In contrast, when the interaction between the mutant protein D10R/E48R and the 36bp DNA/RNA hybrid was analyzed equally, the RU
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Fig. 2. Interaction of the E. coli RNase HI mutant with the 36bp DNA/RNA hybrid. The sensorgram of the mutant protein D10R/E48R (thick line) obtained by surface plasmon resonance analysis is shown in comparison with that of the wild-type protein (thin line). The bu¡er containing the protein was replaced by the bu¡er alone at the time indicated by an arrow. A theoretical sensorgram (broken line) was drawn with BIAsimulation software, by using the ka and kd values of 1U105 M31 s31 and 5U1034 s31 , respectively. The resonance unit (RU) is an arbitrary unit used in the BIAcore system, which increases in proportion to the increase in the amount of the protein bound to the sensor chip.
value kept increasing upon binding of the protein to the sensor chip and did not reach a constant value. In addition, the protein was not clearly dissociated from the sensor chip when the bu¡er alone was passed over the sensor chip. The association (ka ) and dissociation (kd ) rate constants of the mutant protein D10R/E48R cannot be calculated from its sensorgram, because the interaction between this protein and the substrate does not reach an equilibrium between association and dissociation. However, ¢tting of the sensorgram experimentally determined to the theoretical ones allowed us to estimate the ka and kd values of this mutant protein as 1U105 M31 s31 and 5U1034 s31 , respectively (Fig. 2). The ka and kd values of the wild-type protein for the interaction with the 36-bp DNA/ RNA hybrid have been reported to be 1.5U106 M31 s31 and 3.2U1032 s31 , respectively [27]. Thus, the ka and kd values of the mutant protein are lower than those of the wild-type protein by 15- and 64fold, respectively. As a result, the association constant (KA ) of the mutant protein (2U108 M31 ) was higher than that of the wild-type protein (4.7U107
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M31 ) by 4.3-fold. These results suggest that the mutant protein is dissociated from the DNA/RNA hybrid much more slowly and thereby binds to it more strongly than the wild-type protein. When the interactions of these proteins with the 36-bp doublestranded (ds) DNA were examined by BIAcore using the sensor chip, on which the 36-bp ds DNA is immobilized, similar results were obtained (data not shown). In addition, the gel ¢ltration column chromatography indicated that the mutant protein forms a stable complex with the 21-b single-stranded DNA (5P-GTT CCG ATG CTA AGC TTT GCC-3P) both at pH 5.5 and 8.0, whereas the wild-type protein does not (data not shown). These results indicate that the mutant protein strongly binds to the nucleic acids in a nonspeci¢c manner. It is noted that the mutant protein gave an unusual sensorgram on BIAcore analysis at pH 5.5, probably because it strongly binds to the control sensor chip, on which the nucleic acid is not immobilized, as well. The wild-type protein did not give such an unusual sensorgram. The reason why the mutant protein strongly binds to the control sensor chip at pH 5.5 remained to be determined. 3.4. Binding site of nucleic acid Proteins are usually digested by protease at multiple sites. However, the susceptibilities to proteolytic digestion varied for di¡erent sites. For example, peptide bonds located within a loose conformation are usually more susceptible to proteolytic digestion than those located within a rigid conformation. If a given protein formed a stable complex with certain materials and the most susceptible sites to proteolytic digestion were located at the interface between these two molecules, this site would be protected from proteolytic digestion in the presence of these materials. Such a protein footprint analysis may be e¡ective to identify the nucleic-acid-binding site of the mutant protein D10R/E48R, because this mutant protein forms a stable complex with nucleic acids. Therefore, the mutant protein was digested with lysyl endopeptidase, trypsin, or chymotrypsin in a limited manner in the presence or absence of poly(rA)/poly(dT). When the proteolytic digests produced in the presence of poly(rA)/poly(dT) were compared with those produced in the absence of it by SDS-PAGE, a sig-
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ni¢cant di¡erence was observed only for chymotryptic digests. The peptide A, which migrated slightly faster than the intact protein on SDS-PAGE, was produced in the absence of poly(rA)/poly(dT), but was not produced in the presence of it (Fig. 3). The N-terminal amino acid sequence of this peptide was identical with that of the intact protein, indicating that this peptide represents a truncated form of the protein, in which a number of the C-terminal residues are removed. It is known that chymotrypsin preferably cleaves the peptide bonds at the C-termini of the amino acid residues with bulky hydrophobic side chains. Therefore, the peptide bonds at the Ctermini of Leu146 and Tyr151 are the candidates that were cleaved by chymotrypsin. Because the molecular weight of the peptide A is estimated to be reduced
Fig. 3. SDS-PAGE of the peptides generated by limited digestion with chymotrypsin. The mutant protein D10R/E48R was digested with chymotrypsin in a limited manner in the absence (lane 3) or presence (lane 4) of excess amount of poly(rA)/poly(dT). The enzyme:substrate ratios (by weight) were 1:30 and 1:10 for the digestions in the absence and presence of poly(rA)/ poly(dT), respectively. Chymotrypsin alone was also incubated at the same concentration as that used to digest the mutant protein in the presence of poly(rA)/poly(dT) (lane 5). Samples were subjected to electrophoresis on a 20% polyacrylamide gel in the presence of SDS. After electrophoresis, the gel was stained with Coomassie brilliant blue. Lane 1, a low molecular weight marker kit (Pharmacia Biotech) containing phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and K-lactalbumin (14 kDa). Lane 2, undigested protein. The positions of the peptides A and B are indicated by arrows.
by V500 on SDS-PAGE as compared to that of the intact protein, and because the sizes of the peptides truncated at the C-termini of Leu146 and Tyr151 are 1020 and 455, respectively, it is highly likely that the peptide A represents the truncated protein with Tyr151 at the C-terminus. No additional peptides were produced upon further digestion, suggesting that chymotrypsin primarily cleaves the speci¢c peptide bond between Tyr151 and Glu152 , and then cleaves the second sites in a rather nonspeci¢c manner. The peptide B was a fragment of chymotrypsin which was probably produced by self-digestion, because the N-terminal amino acid sequence of this peptide was determined to be IVNGE, which is corresponding to the peptide starting from Ile16 of chymotrypsin. The peptide which migrated faster than the peptide A and slower than the peptide B on SDSPAGE (Fig. 3, lane 4) remained to be identi¢ed, because the yield of this peptide was too low to determine its N-terminal amino acid sequence. The protein footprint analysis indicated that the speci¢c peptide bond probably between Tyr151 and Glu152 of the mutant protein was protected from the chymotryptic digestion in the presence of poly(rA)/poly(dT). Tyr151 is located in the groove-like depression of the protein molecule, which extends from the active site to the basic protrusion and has been suggested to be involved in substrate binding (Fig. 4) [28]. According to a model for the complex between E. coli RNase HI and a DNA/RNA hybrid [29], the DNA/RNA hybrid interacts with this groove-like depression, such that the RNA moiety approximately one turn removed from that interacting with the active site interacts with the basic protrusion (Fig. 4). Therefore, it seems likely that the DNA/RNA hybrid binds to the mutant protein D10R/E48R in a similar manner and this interaction is dramatically strengthened by a decrease in the negative charge density at the active site. 3.5. Catalytic mechanism E. coli RNase HI exhibits little enzymatic activity in the presence of the Ba2 or Ca2 ion [30]. Nevertheless, these divalent cations can also bind to the position where the Mg2 ion binds [29]. This may indicate that the divalent cation does not simply provide su¤cient positive charge density in the active
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Controversy still remains as to the metal-binding site of the enzyme. The crystal structure of the enzyme complexed with the Mg2 ion has shown that Asp10 and Glu48 provide ligands for Mg2 binding [11]. However, determination of the pka values of the individual acidic residues [31] and analysis for the interactions between the active site mutants and the Mg2 ion [15] have strongly suggested that Asp10 and Asp70 provide ligands for Mg2 binding and Glu48 is involved in the catalytic function. The observation that the mutant protein D10R/E48R is inactive both in vivo and in vitro is consistent with a proposal that Glu48 is involved in the catalytic function. Acknowledgements
Fig. 4. A model for the complex between E. coli RNase HI and DNA/RNA hybrid. The crystal structure of E. coli RNase HI (PDB: 2RN2) was drawn with the program RasMol. N and C represent the N- and C-termini of this protein, respectively. The side chains of the active site residues (Asp10 , Glu48 , Asp70 , His124 , and Asp134 ), as well as that of Tyr151 are shown. The solid region represents the basic protrusion, which has been shown to be important for substrate binding [32]. The solid and broken lines represent RNA and DNA strands of DNA/RNA hybrid, respectively. Possible orientations of these strands are also shown. This model was originally constructed by Katayanagi et al. [29].
site but ¢nely tunes the geometry of the active site residues to make them functional. Therefore, it would not be surprising if we could not reproduce the result of Casareno et al. [16], which shows that the mutations of the acidic residues to basic ones at the active site mimic the role of the divalent cation. Instead, we showed that the resultant mutant protein D10R/E48R formed a stable complex with nucleic acids. The active site of the enzyme is in sharp contrast to the basic protrusion in charge distribution. Because the DNA/RNA hybrid binds to the region, which includes both the active site and basic protrusion, and because the mutations reduce the negative charge density in the active site, such a sharp contrast in charge distribution may be required to allow the enzyme to perform multiple turnovers.
We thank M. Itaya, Mitsubishi Kasei Institute of Life Sciences, for providing E. coli MIC3001 and MIC3009. We also thank N. Hirano and N. Ohtani for helpful discussions.
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