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Crystal structure of Escherichia coli RNase HI in complex with Mg 2+ at 2.8 Å resolution: Proof for a single Mg 2+ ‐binding site
Author(s) -
Katayanagi Katsuo,
Okumura Mika,
Morikawa Kosuke
Publication year - 1993
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340170402
Subject(s) - rnase p , crystallography , crystal structure , chemistry , stereochemistry , rnase h , metal , active site , binding site , resolution (logic) , crystal (programming language) , rna , enzyme , biochemistry , organic chemistry , gene , artificial intelligence , computer science , programming language
To obtain more precise insight into the Mg 2+ ‐binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg 2+ . The analyzed cocrystal, which is not isomorphous with the Mg 2+ ‐free crystal previously refined at 1.48 Å resolution, was grown at a high MgSO 4 concentration more than 100 mM so that even weakly bound Mg 2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg 2+ ‐free crystal structure as a search model, and was refined to give a final R ‐value of 0.190 for intensity data from 10 to 2.8 Å, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg 2+ ‐bound and the metal‐free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg 2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal‐ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398‐1405, 1990) and partially supported by the crystallographic study of inactive HIV‐1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88‐95, 1991), the present result excludes the possibility that RNase HI requires two metal‐binding sites for activity. In contrast to the features in the metal‐free enzyme, the side chains of Asn‐44 and Glu‐48 are found to form coordinate bonds with Mg 2+ in the metal‐bound crystal. © 1993 Wiley‐Liss, Inc.