Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site.
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 Mg(2+) ion-binding site in Escherichia coli RNase HI. In the crystal structure of the related RNase H domain of human immunodeficiency virus reverse transcriptase, however, two Mn(2+) ions were observed suggesting a different mode of metal binding. E. coli RNase HI shows catalytic activity in the presence of Mg(2+) or Mn(2+) ions, but these two metals show strikingly different optimal concentrations. Mg(2+) ions are required in millimolar concentrations, but Mn(2+) 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 Mn(2+) ions at 1.9-A resolution. Two octahedrally coordinated Mn(2+) 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.
Goedken
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ER
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Marqusee
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D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D008954:Models, Biological; D016914:Ribonuclease H / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology
D002412:Cations; D005973:Glutamine; D001224:Aspartic Acid; D008345:Manganese; D012965:Sodium Chloride; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium