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J. Biol. Chem., Vol. 276, Issue 10, 7266-7271, March 9, 2001
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From the Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720
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 in
Escherichia 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.
The atomic coordinates and the structure factors (code 1G15) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Co-crystal of Escherichia coli RNase HI with
Mn2+ Ions Reveals Two Divalent Metals Bound in the
Active Site*
*
This work was supported by National Institutes of Health
Grant 50945.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, University of California, 229 Stanley Hall, Berkeley, CA
94720. Tel.: 510-642-7678; Fax: 510-643-9290; E-mail: marqusee@zebra.berkeley.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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