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J. Biol. Chem., Vol. 278, Issue 28, 26094-26101, July 11, 2003

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Structures of Liganded and Unliganded RsrI N6-Adenine DNA Methyltransferase

A DISTINCT ORIENTATION FOR ACTIVE COFACTOR BINDING^*

Chad B. Thomas , Robert D. Scavetta  ¶, Richard I. Gumport  and Mair E. A. Churchill  ||

From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver Colorado 80262 and the Department of Biochemistry and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-L-methionine (AdoMet), the product S-adenosyl-L-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223–227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme.

Received for publication, April 10, 2003 , and in revised form, May 3, 2003.

The atomic coordinates and structure factors (code 1NW5, 1NW7, 1NW6, and 1NW8 (for M.RsrI-AdoMet, M.RsrI-AdoHcy, M.RsrI-sinefungin, and M.RsrI-L72P, respectively)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant RO1-GM59456 (to M. E. A. C.), National Institutes of Health Grant RO1-GM25621 and a grant from the University of Illinois at Urbana-Champaign Department of Biochemistry (to R. I. G.), and the a grant from the University of Illinois Research Board and National Institutes of Health Grant T32 GM07283 (to C. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ 85259.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver CO 80262. Tel.: 303-315-0427; Fax: 303-315-7097; E-mail: mair.churchill{at}uchsc.edu.

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