Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100462
ISSN1083-351X
AutoresTomoshige Ando, Nujarin Jongruja, Nobuaki Okumura, Kosuke Morikawa, Shigenori Kanaya, Toshifumi Takao,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoRibonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme–substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization–mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate. Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme–substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization–mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate. Ribonuclease H (RNase H), a ubiquitous enzyme, is found in all organisms from bacteria to mammals (1Hostomsky Z. Zuzana H. Matthews D.A. Ribonucleases H.in: Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 341-376Google Scholar). RNase H is an endoribonuclease that catalyzes the hydrolysis of the RNA strand of RNA/DNA hybrids and produces 5'-phosphate and 3'-hydroxyl termini. RNase H requires divalent metal ions (Mg2+ or Mn2+) for its enzymatic activity (2Hans Stein P.H. Enzyme from calf thymus degrading the RNA moiety of DNA-RNA hybrids: Effect on DNA-dependent RNA polymerase.Science. 1969; 166: 393-395Crossref PubMed Scopus (176) Google Scholar, 3Hausen P. Stein H. Ribonuclease H: An enzyme degrading the RNA moiety of DNA-RNA hybrids.Eur. J. Biochem. 1970; 14: 278-283Crossref PubMed Scopus (144) Google Scholar, 4Miller H.I. Riggs A.D. Gill G.N. Ribonuclease H (hybrid) in Escherichia coli : Identification and characterization.J. Biol. Chem. 1973; 248: 2621-2624Abstract Full Text PDF PubMed Google Scholar). The important functions of RNases H are: 1) the removal of an RNA strand from an R-loop that is composed of two antiparallel DNA strands plus one RNA strand; 2) removal of RNA fragments (Okazaki fragments) that are produced during DNA replication (5Sugimoto K. Okazaki T. Okazaki R. Mechanism of DNA chain growth, II. Accumulation of newly synthesized short chains in E. coli infected with ligase-defective T4 phages.Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 1356-1362Crossref PubMed Scopus (60) Google Scholar, 6Zhao H. Zhu M. Limbo O. Russell P. RNase H eliminates R-loops that disrupt DNA replication but is nonessential for efficient DSB repair.EMBO Rep. 2018; 19: 1-10Crossref PubMed Scopus (37) Google Scholar, 7Lockhart A. Pires V.B. Bento F. Kellner V. Luke-Glaser S. Yakoub G. Ulrich H.D. Luke B. RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids.Cell Rep. 2019; 29: 2890-2900.e5Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). There are two types of RNase H enzymes; type 1 (RNase HI) and 2 (RNase HII and HIII), the amino acid sequences of which are not conserved except for the active sites, but their tertiary structures resemble each other and have the above activities (8Tadokoro T. Kanaya S. Ribonuclease H: Molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes.FEBS J. 2009; 276: 1482-1493Crossref PubMed Scopus (124) Google Scholar). It is also important to note that RNase HI from Escherichia coli (E. coli) is quite similar to the C-terminal domain of the reverse transcriptase (RT) of the human immunodeficiency virus type-1 (HIV-1), which actually also has RNase H activity and is considered to be a research target for future therapy (9Sarafianos S.G. Sarafianos S.G. Das K. Das K. Tantillo C. Tantillo C. Clark A.D.J. Clark A.D.J. Ding J. Ding J. Whitcomb J.M. Whitcomb J.M. Boyer P.L. Boyer P.L. Hughes S.H. et al.Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (353) Google Scholar, 10Budihas S.R. Gorshkova I. Gaidamakov S. Wamiru A. Bona M.K. Parniak M.A. Crouch R.J. McMahon J.B. Beutler J.A. Le Grice S.F.J. Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.Nucleic Acids Res. 2005; 33: 1249-1256Crossref PubMed Scopus (161) Google Scholar). The crystal structures of E. coli RNase HI were reported in 1990 by Katayanagi et al. (11Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Three-dimensional structure of ribonuclease H from E. coli.Nature. 1990; 347: 306-310Crossref PubMed Scopus (305) Google Scholar) and Yang et al. (12Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein.Science. 1990; 249: 1398-1405Crossref PubMed Scopus (450) Google Scholar). Soon afterward, two classes of catalytic mechanisms were proposed to explain the mechanism for the hydrolysis of RNase H, i.e., a one-metal ion mechanism (13Nakamura H. Oda Y. Kanaya S. Kimura S. Katsuda C. Katayanagi K. Morikawa K. Miyashiro H. Ikehara M. How does RNase H recognize a DNA-RNA hybrid?.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11535-11539Crossref PubMed Scopus (185) Google Scholar, 14Kashiwagi T. Jeanteur D. Haruki M. Katayanagi K. Kanaya S. Morikawa K. Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.Protein Eng. 1996; 9: 857-867Crossref PubMed Scopus (37) Google Scholar) and a two-metal ion mechanism, which was confirmed for the C-terminal domain of RT (15Davies 2nd, J.F. Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase.Science. 1991; 252: 88-95Crossref PubMed Scopus (521) Google Scholar). Despite the structural consistency between RNase HI and the C-terminal domain of RT, the need for more than one divalent metal ion has remained a controversial issue. In 2005, the crystal structure of Bacillus halodurans RNase H, which was mutated to an inactive form, complexed with an RNA/DNA hybrid and two Mg2+ ions, was reported (16Nowotny M. Gaidamakov S.A. Crouch R.J. Yang W. Crystal structures of RNase H bound to an RNA/DNA hybrid: Substrate specificity and metal-dependent catalysis.Cell. 2005; 121: 1005-1016Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). This finding, as well as other studies (17Goedken E.R. Marqusee S. Co-crystal of Escherichia coli RNase H with Mn2+ ions reveals two divalent metals bound in the active site.J. Biol. Chem. 2001; 276: 7266-7271Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Rosta E. Nowotny M. Yang W. Hummer G. Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations.J. Am. Chem. Soc. 2011; 133: 8934-8941Crossref PubMed Scopus (133) Google Scholar, 19Nowotny M. Gaidamakov S.A. Ghirlando R. Cerritelli S.M. Crouch R.J. Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription.Mol. Cell. 2007; 28: 264-276Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), provided support for the two-metal ion mechanism, although the structure of a complex composed of native RNase HI, RNA/DNA and a divalent metal ion has never been solved owing to the fact that the substrate promptly undergoes hydrolysis by its own activity, even in the crystalline form. This poses a problem for the structural analysis of a native complex such as an active enzyme and a natural substrate by X-ray crystallography or NMR. In this context, it should be noted that a recent study by time-resolved X-ray crystallography led to the proposal that the canonical two-metal binding mechanism should be revised to a considerable extent (20Samara N.L. Yang W. Cation trafficking propels RNA hydrolysis.Nat. Struct. Mol. Biol. 2018; 25: 715-721Crossref PubMed Scopus (16) Google Scholar). In addition, a higher concentration of metal ion, which is often used in preparing a crystal of a metal-requiring protein, makes the issue of the stoichiometry of the metal ion in the complex controversial (21Strange R.W. Antonyuk S.V. Hough M.A. Doucette P.A. Selverstone J. Hasnain S.S. Valentine J.S. Hasnain S.S. Variable metallation of human superoxide dismutase: Atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes.J. Mol. Biol. 2006; 356: 1152-1162Crossref PubMed Scopus (123) Google Scholar, 22Yamazaki Y. Takao T. Metalation states versus enzyme activities of Cu, Zn-superoxide dismutase probed by electrospray ionization mass spectrometry.Anal. Chem. 2008; 80: 8246-8252Crossref PubMed Scopus (30) Google Scholar). In the case of RNase HI, the crystals were prepared using a 100 mM MgSO4 solution (23Katayanagi K. Okumura M. Morikawa K. Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 Å resolution: Proof for a single Mg2+-binding site.Proteins. 1993; 17: 337-346Crossref PubMed Scopus (138) Google Scholar) or 1 mM MnCl2 (17Goedken E.R. Marqusee S. Co-crystal of Escherichia coli RNase H with Mn2+ ions reveals two divalent metals bound in the active site.J. Biol. Chem. 2001; 276: 7266-7271Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) solutions, the concentrations of which were 10 or 100 times higher than those for physiological conditions, respectively. Such high concentrations of metal ions might result in the formation of crystal structures with extra metal ions imbedded within them. Considering the concentrations of major divalent cations in E. coli cells (Mg2+: 10−2 M, Zn2+: 10−3 M, Mn2+: 10−5 M in total), (Mg2+: less than 2 mM, Zn2+: low nM, Mn2+: sub μM in the free form) (24Outten C. O'Halloran T. Femtomolar sensitivity of metallooregulatory protein controlling zinc homeostasis.Science. 2001; 292: 2488-2492Crossref PubMed Scopus (1096) Google Scholar, 25Daly M.J. Gaidamakova E.K. Matrosova V.Y. Kiang J.G. Fukumoto R. Lee D.Y. Wehr N.B. Viteri G.A. Berlett B.S. Levine R.L. Small-molecule antioxidant proteome-shields in Deinococcus radiodurans.PLoS One. 2010; 5: 10-15Crossref Scopus (203) Google Scholar), Mg2+ presumably functions as an integral cofactor in the case of RNase HI, which coordinates with the acidic catalytic residues in the active site as demonstrated in its structure (17Goedken E.R. Marqusee S. Co-crystal of Escherichia coli RNase H with Mn2+ ions reveals two divalent metals bound in the active site.J. Biol. Chem. 2001; 276: 7266-7271Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Rosta E. Nowotny M. Yang W. Hummer G. Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations.J. Am. Chem. Soc. 2011; 133: 8934-8941Crossref PubMed Scopus (133) Google Scholar). Meanwhile, regarding RT, it has been shown that other divalent cations (Zn2+ and Mn2+) also support the hydrolysis reaction at much lower concentrations, i.e., in the range of a few and several μM, respectively, while these two metal ions could also function as a potent inhibitor of RT in the presence of Mg2+ ion (26Fenstermacher K.J. DeStefano J.J. Mechanism of HIV reverse transcriptase inhibition by zinc: Formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.J. Biol. Chem. 2011; 286: 40433-40442Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The above investigators concluded that the Zn2+ inhibition is not due to the inhibition of catalysis but, rather, to the formation of a highly stable, kinetically diminished complex. In fact, RNase H activity is repressed under high concentrations of Mn2+ (1 mM) and Mg2+ (50 mM) (17Goedken E.R. Marqusee S. Co-crystal of Escherichia coli RNase H with Mn2+ ions reveals two divalent metals bound in the active site.J. Biol. Chem. 2001; 276: 7266-7271Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The unique coordination property of several divalent metal ions and the structural commonalty in the active domain of many nucleotide cleaving enzymes, including RT, prompted us to analyze the active complex of RNase HI, an RNA/DNA hybrid, and metal ions, with emphasis on the binding properties of Mn2+ and Zn2+ as a function of hydrolysis activity. In recent years, electrospray ionization (ESI) mass spectrometry (MS) has been used for the analysis of native proteins or proteins that are complexed with various compounds. Such measurements can be made on a water-based solvent system, which is close to physiological conditions. This technique has been widely used in the analyses of various types of noncovalent protein–protein and protein–ligand complexes (27Robinson C.V. Groß M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.U. Dobson C.M. Radford S.E. Conformation of GroEL-bound α-lactalbumin probed by mass spectrometry.Nature. 1994; 372: 646-651Crossref PubMed Scopus (190) Google Scholar, 28Loo J.A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry.Mass Spectrom. Rev. 1997; 16: 1-23Crossref PubMed Scopus (1144) Google Scholar, 29Van Berkel W.J.H. Van Den Heuvel R.H.H. Versluis C. Heck A.J.R. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry.Protein Sci. 2000; 9: 435-439Crossref PubMed Scopus (113) Google Scholar, 30Pinkse M.W.H. Heck A.J.R. Rumpel K. Pullen F. Probing noncovalent protein–ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry.J. Am. Soc. Mass Spectrom. 2004; 15: 1392-1399Crossref PubMed Scopus (34) Google Scholar, 31White H.D. Ashcroft A.E. Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry.Biophys. J. 2007; 93: 914-919Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). As a result, the stoichiometry of the components in a complex, even those that are transiently formed in solution, can be revealed. In this study, we report, for the first time, the detection of the ternary complex of native RNase HI: RNA/DNA hybrid: divalent metal ions (Mn2+ and Zn2+). It was possible to monitor a dynamic change in the complex within a few minutes. In addition, the stoichiometry of the Mn2+ ion to the RNase HI: substrate was measured by ESI-MS in parallel with an enzymatic activity assay, which allowed us to conclude that the ternary complex, the solution of which showed the most activity, predominantly involves two divalent cations but that a minor fraction is present with only one divalent cation, which essentially supports the canonical "two metal ions mechanism." Furthermore, the ternary complex containing, in fact, Zn2+ ions was found to hold the full-length substrate, but with a "nick" inside. This unexpected observation allowed us to conclude that Zn2+ ions are retained within a highly stable complex with an RNA strand, even after the cleavage of the phosphate backbone. This also explains why the properties of the Zn2+ ion are different from Mn2+ ions in terms of their ability to inhibit RNase H activity via the formation of a highly stable complex and also explains the essential role of the second divalent metal ion in hydrolysis activity. The ESI-MS of an equimolar mixture of RNase HI and 8-mer RNA/8-mer DNA gave multiply charged ion peaks (7+ to 9+), which corresponded to a 1:1 complex of RNase HI: RNA/DNA hybrid, implying that the complex was stable in solution and the gaseous phase after being sprayed, and its ion particles were maintained during the mass measurement (Fig. 1A). Note that the expanded view of the 8+ charged ion showed the adduction of ammonium or/and sodium ions, which are frequently observed in MS, especially, when an aqueous buffer is used. This result indicates that a 1:1 complex could be easily formed without metal ion(s). When the length of the RNA/DNA hybrid was increased to a 14-mer, traces of the 2 (RNase HI): 1 (RNA/DNA) complex were observed (Fig. 1B). This 2:1 complex was predominant when the protein concentration was increased to twice that of RNA/DNA hybrid (Fig. 1C), implying that a longer RNA/DNA chain would be capable of recruiting multiple enzyme molecules on it. In fact, the crystal structure of the human RNase H C-terminal domain: 14 mer of RNA/DNA hybrid showed that it was a 2:1 complex, in which each RNase H molecule was bound independently to the free space of the RNA/DNA strand (Fig. S1). It is noteworthy that the intensity of the complex with either the DNA and RNA strand was much less than that with the RNA/DNA hybrid (Fig. S2), which could be partly attributed to a lack of synergetic binding force between the strands or either of the strands and the enzyme (see Fig. S1A). It should also be noted that only traces of the complex with the single RNA strand were observed (Fig. S2A), which can be attributed to the repulsion between 2-OH group of the ribose skeleton and the binding site(s) of the enzyme (19Nowotny M. Gaidamakov S.A. Ghirlando R. Cerritelli S.M. Crouch R.J. Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV reverse transcription.Mol. Cell. 2007; 28: 264-276Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Mg2+ at a concentration of a few mM or Mn2+ at concentrations of several μM supports optimal enzyme activity (17Goedken E.R. Marqusee S. Co-crystal of Escherichia coli RNase H with Mn2+ ions reveals two divalent metals bound in the active site.J. Biol. Chem. 2001; 276: 7266-7271Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), while, at RT, Zn2+ inhibits the active enzyme with Mg2+ at a level of a few μM (26Fenstermacher K.J. DeStefano J.J. Mechanism of HIV reverse transcriptase inhibition by zinc: Formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.J. Biol. Chem. 2011; 286: 40433-40442Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Analogous to the human RNase H C-terminal domain, the former two metal ions could be coordinated with the carboxylate groups of Asp10, Asp70, Asp134, and Glu48 of RNase HI and a phosphate group of the RNA strand (see Fig. S1), but the structure of the Zn-coordinated enzyme has not yet been solved. Tsunaka et al. (32Tsunaka Y. Takano K. Matsumura H. Yamagata Y. Kanaya S. Identification of single Mn2+ binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography.J. Mol. Biol. 2005; 345: 1171-1183Crossref PubMed Scopus (29) Google Scholar) reported that the binding of the first Mn2+ ion eventually triggers the coordination of the two Mn2+ ions into the mutant RNase HI using a crystal prepared in the presence of 5 to 10 mM of MnCl2. Based on several reports regarding the metal-binding properties of RNase H, it would be interesting to determine whether those divalent metal ions are also specifically bound to the enzyme in solution. In the presence of each metal ion at a concentration of 4 μM, which is stoichiometrically equivalent to the RNase HI molecule, a single Zn2+ ion was found to bind to the enzyme in a stoichiometric manner, although this does not necessarily mean that it directly binds to a specific site (Fig. 2). At 16 and 200 μM concentrations of Mn2+, Mg2+, and Ca2+ ions, evidence of metal ion adduction was observed, which is frequently observed in MS of biological compounds and is evidenced by the progressive increase in the number of adduct ions along with increasing concentrations of metal ions. It is possible that these adduct ions could bind nonspecifically to the protein surface by electrostatic interactions. In order to detect the ternary complex with enzymatic activity being retained, the solution conditions and measurement parameters for ESI-MS were optimized (see Experimental procedures). Note that the pH was set at 6.0, which was lower than the optimal value (pH 7–8) for expressing activity, because the rate of substrate cleavage under the optimal condition was too fast to permit the enzyme: substrate complex to be detected in the current timescale of mass measurement (∼3.5 min). The enzymatic activity under the above conditions was measured using the 8-mer RNA/8-mer DNA as a substrate, which has a single cleavage site in the RNA molecule, with various concentrations of Mn2+ ion (0–20 μM) (Fig. S3). The value was found to be 0.84 pmol (RNA)/min・pmol (RNase HI), based on Equation 1, making it ca. 55 times less active than that obtained under the conditions that were used in Fig. S4. In order to avoid ion suppression in ESI-MS, the concentrations of protein, RNA/DNA hybrid, and divalent metal ion salts were minimized to 4 μM, 10 μM, and 4 to 20 μM, respectively, (Fig. 3). The mass measurement in parallel to the activity assay gave a correlation between complex formation and activity. When the concentration of Mn2+ was increased from 4 to 20 μM, which corresponds to 1 to 5 M equivalents to the enzyme, the ternary complex, RNase HI: RNA/DNA: two Mn2+, started to be observed at 4 μM of Mn2+ and became predominant at a concentration of around 16 μM (Fig. 3A), the concentration at which two Mn2+ ions could be readily associated with the complex, which was essentially negligible without the substrate (Fig. 2). The relative abundance of the ternary complex with two Mn2+ ions (blue bars in Fig. 3B) was in good agreement with an increase in enzyme activity. Note that around 70% "Percentage of product" in the activity can be attributed to the short incubation time (2 min) under the limited reaction conditions. When the mixture containing 16 μM Mn2+ was allowed to further stand for 3.5 min, the completely hydrolyzed products were obtained (see the inset of Fig. S3). Based on the above observations, it appears that RNase HI requires two Mn2+ ions for its activity to be fully expressed. The preparation with 16 μM Mn2+ (four equivalents to the enzyme) was subjected to ESI-MS measurement and continuously monitored from 1.5 to 3.5 min since after mixing all the components (Fig. 4A). From 1.5 to 2.0 min, the ternary complex with two Mn2+ ions was predominantly observed together with the substrate (Fig. 4B); from 2.0 to 3.0 min, the relative abundance of the ternary complex gradually decreased and those of the complexes of RNase HI: 5-mer RNA: 8-mer DNA (red), RNase HI: DNA (blue), and RNase HI (green) correspondingly increased; from 3.0 to 3.5 min, the ternary complex was barely observed, and instead, the latter three components became prominent (Fig. 4C). This result indicates that the reaction of the 2.5-fold substrate over the enzyme under the present conditions proceeded rapidly and reached a steady state in around 3 min, none of the resulting products that were comprised of the enzyme itself and that complexed with DNA or DNA/degraded RNA contained a Mn2+ ion. The observation of the complex with an intact DNA chain in the final solution provides support for the conclusion that the enzyme favorably binds to the ssDNA chain (Fig. S2). Notably, a minor portion of the RNase HI complex with a single Mn2+ was found along with the major complex that contained two Mn2+ ions (see Fig. 3). This may be related to the controversial issue of whether a single Mn2+ would be mobile within the active site during the progress of the reaction (14Kashiwagi T. Jeanteur D. Haruki M. Katayanagi K. Kanaya S. Morikawa K. Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.Protein Eng. 1996; 9: 857-867Crossref PubMed Scopus (37) Google Scholar). The ternary complex with a Zn2+ ion was tested following the same procedures as above. Unlike the results for the Mn complex, the MS profile remained unchanged throughout the measurement from 1.5 to 3.5 min after mixing the enzyme, substrate, and Zn2+ ion (Fig. 5, A and B). This result indicates that all of the constituents in the solution had already reached a steady state at the start of the measurement, which turned out to be comprised of five reaction products: RNase HI: Zn2+ (green), RNase HI: DNA: Zn2+ (blue), RNase HI: 5-mer RNA/DNA: Zn2+ (red), RNase HI: 6-mer RNA/DNA: Zn2+ (violet), and [RNase HI: nicked RNA/DNA hybrid: 2Zn2+] (orange). As expected from the results shown in Figure 2, all of the products contained a bound Zn2+ ion. The expanded view of the intact ternary complex at the 8+ charge state showed the presence of two ion species at m/z 2824.84 and 2835.40 as the major components, which was in agreement with the calculated values for RNase HI: RNA/DNA: Zn2+ (calcd. 2825.35 (8+)) and [RNase HI: RNA/DNA: 2Zn2+] +18 (calcd. 2835.53 (8+)), respectively (Fig. 5, C and D). The latter value was estimated to be the active ternary complex with two Zn2+ ions, but to retain the cleaved RNA chain, the +18 Da is due to H2O. Such a complex has never been observed for the Mn complex. The above reaction mixture, obtained at the starting point of the mass measurement, was also subjected to high-performance liquid chromatography (HPLC) (see the inset of Fig. S5), supporting the fact that the substrate was completely hydrolyzed at 1.5 min after mixing. This unexpected and surprising finding accounts for the Zn complex forming a stable complex with the RNA chain, even after cleavage had occurred, and further, for the role of the second divalent metal ion for the hydrolysis activity, although the disposition of the Zn2+ ions in the structure has not yet been elucidated. It should also be noted that the complex with the 6-mer RNA (colored in violet in Fig. 5A) was barely observed for the case of the Mn complex, but the complex with 5-mer RNA, which was the major cleavage product based on the HPLC profile (see Fig. S5), was observed in both cases. This difference in the cleavage products can be attributed to the slight change in the enzyme specificity via coordination with Zn2+ ions. The result is consistent with a previous report showing that Zn2+ ion supports enzyme activity at lower concentrations of Zn2+ ion (∼25 μM), and the observation of the complex, [RNase HI: nicked RNA/DNA hybrid: 2Zn2+], may account for the inhibitory property of the Zn complex at RT by forming a highly stable, kinetically diminished complex (26Fenstermacher K.J. DeStefano J.J. Mechanism of HIV reverse transcriptase inhibition by zinc: Formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.J. Biol. Chem. 2011; 286: 40433-40442Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). ESI-MS provided a snapshot of a ternary complex, RNase HI: RNA/DNA hybrid: divalent metal ions (Mn2+, Zn2+ in this study), which could be transiently formed in buffer solution, and the structure was maintained during the time required to make the MS measurement, which was carried out within a few minutes. The functional relevance of the observed complex was evidenced by the enzymatic activity, which was measured using the same solution that was used in the ESI-MS. With an incremental increase in the Mn2+ ion concentration from 0 to 5 mol per mole of RNase HI, the activity reached nearly the maximal level in the present assay conditions (see Experimental procedures), at which the complex comprised of RNase HI: RNA/DNA: Mn2+ (1: 1: 2) was observed to be the predominant component. In turn, the level of the RNase HI: RNA/DNA (1: 1) complex was decreased; the RNase HI: RNA/DNA: Mn2+ (1: 1: 1) complex was increased to around 8 μM of Mn2+, then decreased slightly, but remained in part, even at 20 μM of Mn2+ (Fig. 3). This is likely due to the turnover of Mn2+ ions during the enzyme reaction. However, considering the fact that the active enzyme with Mg2+ was inhibited by 50% in the presence of ∼500 μM MnCl2 (26Fenstermacher K.J. DeStefano J.J. Mechanism of HIV reverse transcriptase inhibition by zinc: Formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.J. Biol. Chem. 2011; 286: 40433-40442Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), the two-metal ion form might be product-inhibitory at higher concentrations of Mn2+ ion. While the specific affinity of the enzyme for Mn2+ ion was nearly negligible, two Mn2+ ions apparently coordinated at the same con
Referência(s)