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 h ybrids in a divalent cation-dependent manner. Previous structural studies r evealed a single Mg2+ ion-binding site in Escherichia coli RNase HI. In: th e crystal structure of the related RNase H domain: of human immunodeficienc y virus reverse transcriptase, however, two Mn2+ ions were observed suggest ing a different mode of metal binding. E, coli RNase HI shows catalytic act ivity in the presence of Mg2+ or Mn2+ ions, but: these two metals show stri kingly different optimal concentrations. Mg2+ ions are required in millimol ar concentrations, but Mn2+ ions are only required-in micromolar quantities . Based upon the metal dependence off. coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysi s, and binding of a second metal is inhibitory. We have now solved the co-c rystal structure of E, coli RNase HI with Mn2+ ions at 1.9-Angstrom resolut ion. 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 As p-10 and Asp-134 make direct contacts to the second (attenuating) metal. Th is structure is consistent with biochemical evidence suggesting that two me tal ions may bind RNase H but liganding a second ion inhibits RNase H activ ity.