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W2048731211 abstract "Ribonuclease H (RNase H) selectively degrades the RNA strand of RNA·DNA hybrids in a divalent cation-dependent manner. Previous structural studies revealed a single Mg2+ ion-binding site inEscherichia coli RNase HI. In the crystal structure of the related RNase H domain of human immunodeficiency virus reverse transcriptase, however, two Mn2+ ions were observed suggesting a different mode of metal binding. E. coli RNase HI shows catalytic activity in the presence of Mg2+ or Mn2+ ions, but these two metals show strikingly different optimal concentrations. Mg2+ ions are required in millimolar concentrations, but Mn2+ ions are only required in micromolar quantities. Based upon the metal dependence of E. coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysis, and binding of a second metal is inhibitory. We have now solved the co-crystal structure of E. coli RNase HI with Mn2+ ions at 1.9-Å resolution. Two octahedrally coordinated Mn2+ ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.1G15 Ribonuclease H (RNase H) selectively degrades the RNA strand of RNA·DNA hybrids in a divalent cation-dependent manner. Previous structural studies revealed a single Mg2+ ion-binding site inEscherichia coli RNase HI. In the crystal structure of the related RNase H domain of human immunodeficiency virus reverse transcriptase, however, two Mn2+ ions were observed suggesting a different mode of metal binding. E. coli RNase HI shows catalytic activity in the presence of Mg2+ or Mn2+ ions, but these two metals show strikingly different optimal concentrations. Mg2+ ions are required in millimolar concentrations, but Mn2+ ions are only required in micromolar quantities. Based upon the metal dependence of E. coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysis, and binding of a second metal is inhibitory. We have now solved the co-crystal structure of E. coli RNase HI with Mn2+ ions at 1.9-Å resolution. Two octahedrally coordinated Mn2+ ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.1G15 human immunodeficiency virus The RNase H class of enzymes cleaves the RNA moiety of RNA·DNA hybrids in a divalent cation-dependent manner leaving 5′-phosphate and 3′-hydroxyl products. RNase H proteins are found in a wide variety of organisms ranging from bacteria to vertebrates (for review see Refs. 1Crouch R.J. New Biol. 1990; 2: 771-777PubMed Google Scholar and 2Hostomsky Z. Hostomska Z. Matthews D.A. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2 Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 341-376Google Scholar). From a medical perspective, the most significant function of RNase H is its critical action in the life cycle of retroviruses such as the human immunodeficiency virus (HIV).1 This activity arises from the C-terminal region of reverse transcriptase. The RNase H domain from HIV is homologous to other members of this class including RNase HI from Escherichia coli. Because inhibiting RNase H activity prohibits production of infectious virions (3Tisdale M. Schulze T. Larder B.A. Moelling K. J. Gen. Virol. 1991; : 59-66Crossref PubMed Scopus (142) Google Scholar), understanding the function and mechanism of RNase H is an important avenue toward the development of anti-retroviral compounds. RNase H requires divalent cations (either Mg2+ or Mn2+) for catalysis. Five conserved residues form the metal-binding active site of RNase H (Fig. 1 a). Two RNases H with metal ions bound have been studied by x-ray crystallography.E. coli RNase HI binds a single Mg2+ ion via three carboxylates (Asp-10, Glu-48, and Asp-70) that form Site 1 (4Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (308) Google Scholar,5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar), whereas the HIV RNase H domain shows two Mn2+ ions, one in a position similar to Site 1 and another (Site 2) liganded by the equivalents of Asp-10 and Asp-134 (6Davies J.D. Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (526) Google Scholar). Mutations in RNases H, which eliminate Mg2+-dependent activity, often allow retention of Mn2+-dependent activity (7Stahl S.J. Kaufman J.D. Vikic T.S. Crouch R.J. Wingfield P.T. Protein Eng. 1994; 7: 1103-1108Crossref PubMed Scopus (43) Google Scholar, 8Keck J.L. Marqusee S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2740-2744Crossref PubMed Scopus (49) Google Scholar, 9Keck J.L. Marqusee S. J. Biol. Chem. 1996; 271: 19883-19887Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Blain S.W. Goff S.P. J. Biol. Chem. 1996; 271: 1448-1454Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 11Goedken E.R. Raschke T.M. Marqusee S. Biochemistry. 1997; 36: 7256-7263Crossref PubMed Scopus (26) Google Scholar). This, together with the different stoichiometry observed via crystallography, raises the possibility of alternate binding modes and/or catalytic requirements for these metals. In E. coliRNase HI, conservative mutation of the residues comprising Site 1 (Asp-10, Glu-48, and Asp-70) eliminates activity (12Kanaya S. Kohara A. Miura Y. Sekiguchi A. Iwai S. Inoue H. Ohtsuka E. Ikehara M. J. Biol. Chem. 1990; 265: 4615-4621Abstract Full Text PDF PubMed Google Scholar), whereas mutations of the conserved histidine (His-124) (13Oda Y. Yoshida M. Kanaya S. J. Biol. Chem. 1993; 268: 88-92Abstract Full Text PDF PubMed Google Scholar) and aspartate (Asp-134) of Site 2 (14Haruki M. Noguchi E. Nakai C. Liu Y.Y. Oobatake M. Itaya M. Kanaya S. Eur. J. Biochem. 1994; 220: 623-631Crossref PubMed Scopus (47) Google Scholar) have smaller effects on catalysis. Despite structural efforts to identify additional weaker binding sites (5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar), no metal binding to Site 2 in E. coli RNase HI has been reported. Our laboratory has recently proposed a model for the metal dependence of E. coli RNase HI termed an “activation/attenuation mechanism” (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this model, a single metal bound at Site 1 is required for catalysis, and the binding of a second metal at Site 2 reduces catalysis ∼100-fold. In this report, we present a high resolution crystal structure of E. coli RNase HI in complex with Mn2+ ions. These data demonstrate that the active site of E. coli RNase HI can bind two Mn2+ ions in the sites predicted by the activation/attenuation hypothesis. Furthermore, we show that the surprising dependence of RNase H activity in the presence of Mn2+ upon ionic strength can also be explained by this model. Kunkel mutagenesis (16Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar) of pSM101 (carrying a synthetic gene directing the expression of RNase H*, a cysteine-free variant of E. coli RNase HI) was used to create pEG120 which encodes for K87A RNase H*. DNA sequencing confirmed the incorporation of the desired mutation. RNase H* and K87A proteins were overexpressed in E. coli and purified by cation-exchange chromatography as described previously (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Mass spectrometry confirmed the expected molecular mass of purified proteins within 1 Da (data not shown). Radiolabeled RNA·DNA hybrid was synthesized from M13K07 single-stranded DNA, and RNase H assays were conducted as described previously (8Keck J.L. Marqusee S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2740-2744Crossref PubMed Scopus (49) Google Scholar). RNase H reaction assays were carried out in standard conditions of 50 mmTris, pH 8.0, 1 μm (base pairs) RNA·DNA hybrid, 2.5% glycerol, 0.1 mg/ml linear polyacrylamide, 1.5 μm bovine serum albumin with the indicated concentrations of divalent cation (MnCl2 or MgCl2) and NaCl at 37 °C. The acid-soluble radioactivity (cleaved product) present in the supernatant was determined by liquid scintillation counting. Specific activity (units/mg) was determined from the slope of multiple assay time points in the linear range of enzymatic activity. One unit of RNase H activity is defined as the amount of enzyme needed to generate 1 μmol of acid-soluble product in 15 min under our reaction conditions. Crystals of K87A RNase H* were grown by the hanging-drop vapor diffusion method in 1 mmMnCl2, 20 mm Hepes, pH 8, 16% PEG-3350 with 6 mg/ml protein. Except for the metal ions, these conditions are similar to those used previously for the crystallization of wild-type E. coli RNase HI (17Yang W. Hendrickson W.A. Kalman E.T. Crouch R.J. J. Biol. Chem. 1990; 265: 13553-13559Abstract Full Text PDF PubMed Google Scholar) and for RNase H* (Goedken et al.(38Goedken E.R. Keck J.L. Berger J.M. Marqusee S. Protein Sci. 2000; 9: 1914-1921Crossref PubMed Scopus (45) Google Scholar)). Small needle-like crystals unsuitable for x-ray analysis frequently grew under these conditions. Occasionally, larger rod-shaped crystals suitable for x-ray diffraction appeared, growing to a maximum size of ∼600 × ∼15 × ∼15 μm within 4 weeks. Crystals were flash-frozen in liquid nitrogen directly from the drop conditions without the need for additional cryo-protectants. X-ray diffraction data were collected at the Advanced Light Source (Beamline 5.0.2) at Lawrence Berkeley National Laboratory. Diffraction data were integrated and scaled using MOSFLM and SCALA (18Collaborative Computational Project Number 4 Acta Crystallogr. Sect. A. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) under the control of Wedger Elves (kindly provided by J. Holton). The K87A RNase H*/Mn2+ co-crystal structure was solved by molecular replacement via AMoRe (19Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using the RNase H* coordinates (Protein Data Bank code 1F21) (38Goedken E.R. Keck J.L. Berger J.M. Marqusee S. Protein Sci. 2000; 9: 1914-1921Crossref PubMed Scopus (45) Google Scholar). Models were initially refined using REFMAC (20Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. A. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and later by the crystallography and NMR system (21Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse- Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Between refinement cycles, manual rebuilding into 2F o − F c andF o − F c maps was performed using O (22Jones T. Zou J.Y. Cowan S.W. Kjeldgarrd M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Immediately after molecular replacement, difference density attributable to two octahedrally coordinated Mn2+ions was observed in the enzyme-active site. Refinement statistics are summarized in Table I.Table IRNase H/Mn2+ crystallographic statisticsSpace groupP63Unit cell (Å)a = b= 71.65, c = 48.127(°)α = β = 90, γ = 120Resolution (Å)20.0–1.9R sym(%)aRsym = Σ ‖I hkl − 〈I hkl〉‖ / ΣI hkl, where I hkl is the intensity measurement for reflection with indices hkl and 〈I hkl〉 is the mean intensity for multiply recorded reflections.8.3 (42.0)bValues in parentheses are for highest-resolution bin (2.05–1.9 Å).Completeness (%)99.8 (99.3)I/ς4.2 (0.7)R work(%)cRwork, free = Σ ‖ ‖F obs‖ − ‖F calc‖ ‖ / Σ ‖F obs‖ where the R-factors are calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data held aside for unbiased cross-validation throughout refinement.22.3R free(%)cRwork, free = Σ ‖ ‖F obs‖ − ‖F calc‖ ‖ / Σ ‖F obs‖ where the R-factors are calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data held aside for unbiased cross-validation throughout refinement.26.1Total atoms included1227H2O included100Ramachandran distribution:Most favored regions (%)84.9Additionally allowed regions (%)15.1Generously allowed regions (%)0.0Disallowed regions (%)0.0Root mean square deviation, bonds (Å)0.006Root mean square deviation, angles (°)1.01a Rsym = Σ ‖I hkl − 〈I hkl〉‖ / ΣI hkl, where I hkl is the intensity measurement for reflection with indices hkl and 〈I hkl〉 is the mean intensity for multiply recorded reflections.b Values in parentheses are for highest-resolution bin (2.05–1.9 Å).c Rwork, free = Σ ‖ ‖F obs‖ − ‖F calc‖ ‖ / Σ ‖F obs‖ where the R-factors are calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data held aside for unbiased cross-validation throughout refinement. Open table in a new tab The coordinates of the RNase H*/Mn2+ structure have been deposited in the Protein Data Bank (1G15). Although E. coli RNase HI can use either Mn2+ or Mg2+ for activity, these divalent metals lead to very different activity profiles (9Keck J.L. Marqusee S. J. Biol. Chem. 1996; 271: 19883-19887Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 23Keck J.L. Marqusee S. Marshak D.R. Techniques in Protein Chemistry VIII. 8. Academic Press, New York1997: 409-416Google Scholar). The metal dependence of RNase H*, the cysteine-free version of E. coliRNase HI, is shown in Fig. 1 b. (This variant shows metal dependence that is identical to that of the wild-type enzyme.) In the presence of Mg2+ ions and moderate ionic strength (50 mm NaCl), the enzyme is activated in the low mm range (0.5–10 mm) and is repressed by very large concentrations of divalent metal (>10 mm). This activation profile is in contrast to that in the presence of Mn2+. This ion activates the enzyme at low metal concentrations over a narrow range (to ∼2 μm), and additional metal inhibits the protein. The activity at 1 mm Mn2+ is ∼100-fold decreased from its maximum at 2 μm Mn2+ (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Mutations in residue Asp-134 produce an enzyme that shares the activation profiles of the wild-type protein but shows a large increase in the Mn2+ concentrations required to inhibit the enzyme (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These observations led to the proposal of the activation/attenuation model for RNase H (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) in which a metal ion in Site 1 activates catalysis and the binding of another ion in Site 2 attenuates activity. To bolster the activation/attenuation model, we sought structural identification of the location and liganding geometry of Mn2+ ions at the activating and attenuating sites ofE. coli RNase HI. Cysteine-free E. coli RNase H* crystallizes in an orthorhombic lattice (38Goedken E.R. Keck J.L. Berger J.M. Marqusee S. Protein Sci. 2000; 9: 1914-1921Crossref PubMed Scopus (45) Google Scholar), but our attempts to identify bound metal ions in these crystal forms either by co-crystallization or by soaking into existing crystals were unsuccessful. That is, diffraction data to greater than 1.8-Å resolution were obtained for a crystal grown in the presence of 5 mm MgCl2 that was isomorphous to the apo-crystals. After molecular replacement with the apo-structure, however, no electron density attributable to metal ions was observed in the active site (data not shown). Similar results were found for 24-h soaks of RNase H* crystals in 20 mm MgCl2 or 8 mm MnCl2 (data not shown). Examination of lattice contacts in these orthorhombic crystals showed that the positively charged side chain of residue Lys-87 from a symmetry mate in the lattice protruded into the negatively charged enzyme-active site. We hypothesized that this crystal contact altered the electrostatic environment and accessibility of the metal-binding pocket precluding the entry of divalent cations. In a single enzyme monomer, Lys-87 is distant from the active site and is unlikely to influence metal binding. We therefore created a mutant of E. coli RNase H* lacking this residue (K87A) expecting that this variant would crystallize in the same lattice but allow liganding of divalent metals. K87A did not form the expected orthorhombic crystals in the presence or absence of divalent metal even when microseeded with RNase H* crystals; however, high quality hexagonal crystals did appear in conditions containing 1 mm MnCl2 at pH 8. The K87A hexagonal crystals diffract to 1.9 Å at a synchotron source and belong to the space group P63, having one protein molecule per asymmetric unit. We solved the structure of K87A RNase H* using the molecular replacement method with the metal-free RNase H* structure as the probe molecule. Data collection and refinement statistics are summarized in Table I. Overall the structure of K87A (Fig. 2) is highly similar to the RNase H* structure; comparison of the C-α coordinates (24Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3566) Google Scholar) of K87A to structures determined previously gave a root mean square deviation of 0.8 Å to the cysteine-free RNase H* and 1.0 Å to the wild-type protein. In addition to disordered N and C termini (residues 1–2 and 153–155), two loops, those between helices C and D (residues 92–96) and the loop between strand V and helix E containing a conserved histidine (residues 122–125, the “His-containing loop”), were disordered in the crystal. After molecular replacement, two difference density peaks lying 4.0 Å apart were easily identified in the electron density. Because they displayed canonical octahedral liganding geometry and no other divalent metals were present in the crystallization conditions, these peaks can be clearly identified as Mn2+ ions. Representative 2F o − F c maps calculated using the phases from the final model are shown in Fig. 2. At full occupancy both ions refined to low temperature factors (∼15 Å2) similar to the surrounding residues, suggesting these binding sites are fully occupied and that the data are well accounted for by the Mn2+ ions in the model. In comparison to the metal-free structure (Protein Data Bank accession code 1F21) (38Goedken E.R. Keck J.L. Berger J.M. Marqusee S. Protein Sci. 2000; 9: 1914-1921Crossref PubMed Scopus (45) Google Scholar), there are notable changes in the geometry of the active site. The side chain of Glu-48 flips dramatically toward the carboxylate pocket, and His-124 is no longer visible in the structure. The positions of the side chains Asp-10, Asp-70, and Asp-134, however, change very little in the co-crystal structure. Both metal ions have octahedral coordination with positions occupied either by protein atoms or solvent molecules. The first Mn2+ ion (Mn2+ 1) makes inner sphere (nonwater-mediated) contacts to the carboxylate oxygens of Asp-10, Glu-48, and Asp-70. The three remaining coordination sites on the ion are occupied by water molecules, one of which (Water 2) appears to interact with the other carboxylate oxygen of Asp-70. The second Mn2+ ion (Mn2+ 2) is coordinated by inner sphere contacts to Asp-10 and Asp-134 of the same molecule; an inner sphere contact to Glu-131 from an adjacent symmetry mate is also observed. Water molecules occupy the remaining coordination sites of the Mn2+ 2, one of which, Water 2, is shared between the two metal ions. Both metals are therefore “bridged” by having contacts both to Asp-10 and Water 2 suggesting that both metals can bind simultaneously. The water molecules making coordination contacts to the divalent metal ions may be replaced by contacts from the nucleic acid substrate during the RNase H reaction. How do these metal-binding sites compare with those previously observed? Fig.3 shows a comparison of crystallographic metal binding studies in RNases H. The position of the Mn2+ions and the conformation of liganding homologous residues in the HIV RNase H domain from reverse transcriptase (observed at 2.4 Å resolution) (6Davies J.D. Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (526) Google Scholar) are similar to that observed for K87A E. coli RNase HI in the presence of Mn2+. In contrast, the conformation of the active site residues Asp-10 and Glu-48 in the Mg2+ co-crystal with E. coli RNase HI (observed at 2.8 Å resolution (5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar)) is different from what we observed in our 1.9-Å structure with Mn2+ ions. In particular, the single Mg2+ ion is displaced away from Asp-70 such that it can no longer make direct contact with the metal ion as observed for Mn2+ 1 in our structure. The difference in precise positioning of the ion in Site 1 is interesting because solution studies on both E. coli RNase HI (25Kanaya S. Oobatake M. Liu Y. J. Biol. Chem. 1996; 271: 32729-32736Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and Moloney murine leukemia virus RNase H (26Goedken E.R. Marqusee S. Protein Eng. 1999; 12: 975-980Crossref PubMed Scopus (13) Google Scholar) suggest that Asp-70 (or its homologue) is more important for metal affinity than is Glu-48. Given the variable position of metal ions observed crystallographically, it is conceivable that in solution metals can adopt several positions in the active site. This may prove relevant for the enzymatic mechanism as first suggested by Kanaya et al. (25Kanaya S. Oobatake M. Liu Y. J. Biol. Chem. 1996; 271: 32729-32736Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Alternatively, the presence of a metal at Site 2 may induce conformational changes in the Site 1 causing movement of the ion toward Asp-70. Although x-ray crystallography demonstrates that a Mn2+ ion can bind at Site 2, proving that this metal is inhibitory as predicted by the activation/attenuation model is more difficult. Examination of the metal-dependent activity of RNase H* in the presence of Mn2+ or Mg2+ ions as a function of ionic strength, however, has proved informative (see Fig. 4 a). In particular, we noted that as NaCl concentrations were increased, the catalytic behavior of RNase H in the presence of 1 mmMnCl2 was very different from that in 1 mmMgCl2. The enzyme was strongly inhibited by increasing ionic strength in the presence of fixed amounts of Mg2+(where based upon crystallographic data we expect the involvement of one ion). In contrast, in the presence of Mn2+ (where we expect two ions to be bound by RNase H), activity levels increased as NaCl concentrations rose. This was a surprising result; a priori we had expected that NaCl would inhibit activity in the presence of either metal due to shielding of electrostatic effects that contribute to the affinity of the enzyme for its substrate and/or for its cofactor. Nonetheless, under conditions where the enzyme is attenuated by high Mn2+ concentrations, this attenuation appears to be partly relieved by increased ionic strength. Furthermore, it seems unlikely that the conformation of the RNA·DNA hybrid was altered by additional NaCl making it less susceptible to RNase H degradation because NaCl has the opposite effect (inhibition) in the presence of Mg2+. We subsequently found that addition of NaCl did indeed repress activity at other lower Mn2+ (0.5 and 2 μm) concentrations (Fig. 4 b). Hence, under metal concentrations where we expect at most one ion to be bound (the activation regime in Fig. 1 a), we detected strict inhibition by increasing NaCl concentrations. At 20 μm Mn2+ where the attenuating site becomes populated at low ionic strength (10–50 mm NaCl), we observed a maximum activity at intermediate (200 mm) concentrations of NaCl, followed by a decrease of activity at higher concentrations of salt. Fig. 4 c shows the Mn2+ dependence of RNase H activity at a series of different NaCl concentrations. This shows a displacement to higher divalent metal concentration on both the activation and attenuation regimes as the NaCl concentration increases. Furthermore, the maximum activity of the enzyme was also decreased at high ionic strengths, probably because of electrostatic shielding of RNase H from nucleic acid. We believe these data indicate that NaCl nonspecifically competes for the same sites as the Mn2+ions and thereby reduces the apparent binding affinity of each divalent metal. The unexpected increases in activity as the NaCl concentrations increase in 1 mm Mn2+ samples likely stem from the reduction in affinity for the second (attenuating) Mn2+ion. In this case, then, the overall effect of increased ionic strength was a loss of a Mn2+ ion in Site 2. The removal of the metal ion in Site 2 is expected to relieve the attenuation and overcome the decreased affinity for the substrate to give rise to a net increase in activity. Consistent with this idea, in the presence of Mg2+ concentrations from 0.1 to 50 mm (where we expect a single metal in Site 1), we found only inhibition as NaCl concentrations are increased (data not shown). RNase H catalyzes the disruption of the phosphodiester backbone of the RNA portion of RNA·DNA heteroduplexes in a divalent cation-dependent manner. This reaction requires a concerted deprotonation of water to form a nucleophilic hydroxide to attack the scissile phosphate of the RNA. Several different conserved active-site residues have been proposed to be the nucleophile-producing general base for this reaction including Glu-48, Asp-70, and His-124 (5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar, 13Oda Y. Yoshida M. Kanaya S. J. Biol. Chem. 1993; 268: 88-92Abstract Full Text PDF PubMed Google Scholar,25Kanaya S. Oobatake M. Liu Y. J. Biol. Chem. 1996; 271: 32729-32736Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26Goedken E.R. Marqusee S. Protein Eng. 1999; 12: 975-980Crossref PubMed Scopus (13) Google Scholar, 27Kanaya S. Kohara A. Miyagawa M. Matsuzaki T. Morikawa K. Ikehara M. J. Biol. Chem. 1989; 264: 11546-11549Abstract Full Text PDF PubMed Google Scholar, 28Kashiwagi T. Jeanteur D. Haruki M. Katayanagi K. Kanaya S. Morikawa K. Protein Eng. 1996; 9: 857-867Crossref PubMed Scopus (37) Google Scholar), but it remains an open question which (if any) serve in this role. RNase H is believed to require metal cofactors because divalent cations stabilize the negatively charged pentacovalent phosphorane intermediate of the nuclease reaction. The stoichiometry of metal ions to the RNase H active site has been evaluated by a number of methods. X-ray crystallography (5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar, 29Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Nakamura H. Ikehara M. Matsuzaki T. Morikawa K. J. Mol. Biol. 1992; 223: 1029-1052Crossref PubMed Scopus (197) Google Scholar) as well as a large battery of solution techniques (30Oda Y. Nakamura H. Kanaya S. Ikehara M. J. Biomol. NMR. 1991; 1: 247-255Crossref PubMed Scopus (40) Google Scholar, 31Black C.B. Cowan J.A. Inorg. Chem. 1994; 33: 5805-5808Crossref Scopus (38) Google Scholar, 32Casareno R.L.B. Cowan J.A. Chem. Commun. 1996; 15: 1813-1814Crossref Scopus (29) Google Scholar) have detected only a single Mg2+ion-binding site in E. coli RNase HI. In contrast, two Mn2+ ions were observed bound to the isolated HIV RNase H domain, and stoichiometries of one Mg2+ ion and two Mn2+ ions binding at the RNase H active site in full-length HIV-reverse transcriptase have been determined by calorimetric studies (33Cowan J.A. Ohyama T. Howard K. Rausch J.W. Cowan S.M. Le Grice S.F. J. Biol. Inorg. Chem. 2000; 5: 67-74Crossref PubMed Scopus (48) Google Scholar). Based upon these data and the metal dependence of RNase H activity (Fig. 1 b), our laboratory proposed an “activation/attenuation model.” This model posits that the first metal to bind RNase H is required for activity and occupies Site 1 (formed by the side chains of Asp-10, Glu-48, and Asp-70). A second metal with weaker affinity can occupy Site 2 (composed of the side chains of Asp-134 and possibly Asp-10 and His-124). This inhibitory metal accounts for the reduction of activity to ∼1% of maximal value at higher Mn2+ concentrations. By using x-ray crystallography, we have demonstrated that a Mn2+ ion can bind to Site 2. Mutagenesis of Asp-134 to less polar residues His and Asn results in an active enzyme with a decreased attenuation at high Mn2+ concentrations (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We observed that Asp-134 (along with Asp-10) makes direct inner sphere contacts to the second Mn2+ ion. Those results together with the observation that increasing ionic strength unexpectedly relieves the inhibition of RNase H at high Mn2+ concentration (Fig. 4) strongly suggest that the ion observed in Site 2 is inhibitory. E. coli RNase HI requires very different concentrations of Mg2+ ions than Mn2+ ions for optimal activity (Fig. 1 b). The enzyme shows a greater maximum activity in the presence of Mg2+ than in the presence of Mn2+ ions. Whether the inhibition observed at very high Mg2+ concentration (Fig. 1 b) results from weak binding by Mg2+ at Site 2 is unclear. However, given the large ionic strength of the reaction conditions of those samples, it seems equally likely that a reduction in the electrostatic affinity for the nucleic acid substrate gives rise to this inhibition. Although it is difficult to evaluate the free concentrations of divalent metal ionsin vivo, it should be noted that the concentration at which Mg2+ activates the enzyme (0.5–5 mm) approximates physiological values (34Snavely M.D. Sigel H. Metal Ions in Biological Systems. 20. Marcel Dekker, Inc., New York1990: 155-175Google Scholar, 35Williams R.J.P. Birch N.J. Magnesium and the Cell. Academic Press, Boston, MA1993: 15-29Google Scholar). Mn2+ ions, which are found at much smaller concentrations in vivo, bind the enzyme much more tightly (apparent K d ∼1 μm), and Mn2+ can inhibit RNase H activity in the presence of Mg2+ (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Therefore, we speculate that both ions may be relevant for the physiological regulation of RNase H activity. Notably, the attenuation of E. coli RNase HI via second metal binding does not eliminate all activity. Mutation in the conserved active-site histidine (His-124) eliminates attenuation at high Mn2+ concentrations but allows maximal activity that reaches only the attenuated rates of the wild-type protein (15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This led to the suggestion that attenuation at Site 2 occurs by removing the action of His-124. Two alternative ways metal binding at Site 2 could neutralize the histidine (presumably by eliminating a role in acid/base catalysis) were proposed: 1) that His-124 was directly involved in liganding the second metal-binding site or 2) that binding of a metal ion at Site 2 hinders activity because the His-containing loop is flipped out away from the active site. This loop has been seen in separate conformations in the apo-form (4Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (308) Google Scholar, 36Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Science. 1990; 249: 1398-1405Crossref PubMed Scopus (453) Google Scholar), one with the histidine side chain near the active site as in Fig. 1 a and the other where it is displaced away from the catalytic pocket. As the entire His-containing loop is disordered in the Mn2+co-crystal, it is unlikely that His-124 is directly involved in the liganding of the Site 2 metal. Thus, in light of the x-ray structure, the second possibility that His-124 is neutralized by second metal binding via increased flexibility of the His-containing loop seems more reasonable. The RNase H/Mn2+ co-crystal structure presented here provides direct structural evidence that E. coli RNase HI can bind two metal ions in the absence of substrate. Numerous conflicting proposals have been made regarding the number of metal ions necessary for activity and the precise catalytic mechanism of ribonuclease H (5Katayanagi K. Okumura M. Morikawa K. Proteins. 1993; 17: 337-346Crossref PubMed Scopus (141) Google Scholar, 6Davies J.D. Hostomska Z. Hostomsky Z. Jordan S.R. Matthews D.A. Science. 1991; 252: 88-95Crossref PubMed Scopus (526) Google Scholar, 13Oda Y. Yoshida M. Kanaya S. J. Biol. Chem. 1993; 268: 88-92Abstract Full Text PDF PubMed Google Scholar, 15Keck J. Goedken E.R. Marqusee S. J. Biol. Chem. 1998; 273: 34128-34133Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Kanaya S. Oobatake M. Liu Y. J. Biol. Chem. 1996; 271: 32729-32736Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 28Kashiwagi T. Jeanteur D. Haruki M. Katayanagi K. Kanaya S. Morikawa K. Protein Eng. 1996; 9: 857-867Crossref PubMed Scopus (37) Google Scholar, 37Nakamura H. Oda Y. Iwai S. Inoue H. Ohtsuka E. Kanaya S. Kimura S. Katsuda C. Katayanagi K. Morikawa K. Miyashito H. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11535-11539Crossref PubMed Scopus (196) Google Scholar), but a consensus is developing that a single metal is required for activity. It is unclear which co-crystal (Mg2+ or Mn2+) most closely approximates the position of the metal in Site 1 during catalysis on a nucleic acid. Our x-ray data indicate that all of the conserved carboxylates in the active site of RNase H can make direct contacts to divalent metals. Whether RNase H uses any of these side chains as a general base to deprotonate solvent molecule remains an open question. To evaluate which residues (if any) are the general base and acid of the nuclease reaction and how the RNA strand of RNA·DNA hybrids is recognized, a co-crystal of RNase H bound to substrate and/or substrate analogs will likely be required. We thank James Keck for assistance with x-ray crystallography and helpful discussions and the critical reading of this manuscript. We thank James Holton and Prof. James Berger for assistance with x-ray crystallography and the critical reading of this manuscript and David King for mass spectrometry." @default.
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W2048731211 title "Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site" @default.
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