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J. Biol. Chem., Vol. 282, Issue 37, 26989-26996, September 14, 2007

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The Structure of the R184A Mutant of the Inositol Monophosphatase Encoded by suhB and Implications for Its Functional Interactions in Escherichia coli^*

Yanling Wang, Kimberly A. Stieglitz, Mikhail Bubunenko^¶, Donald L. Court^¶, Boguslaw Stec^||, and Mary F. Roberts¹

From the Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, the Basic Research Program, SAIC-Frederick, Inc. and the ^¶Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, and ^||The Burnham Institute for Medical Research, La Jolla, California 92037

The Escherichia coli product of the suhB gene, SuhB, is an inositol monophosphatase (IMPase) that is best known as a suppressor of temperature-sensitive growth phenotypes in E. coli. To gain insights into these biological diverse effects, we determined the structure of the SuhB R184A mutant protein. The structure showed a dimer organization similar to other IMPases, but with an altered interface suggesting that the presence of Arg-184 in the wild-type protein could shift the monomer-dimer equilibrium toward monomer. In parallel, a gel shift assay showed that SuhB forms a tight complex with RNA polymerase (RNA pol) that inhibits the IMPase catalytic activity of SuhB. A variety of SuhB mutant proteins designed to stabilize the dimer interface did not show a clear correlation with the ability of a specific mutant protein to complement the suhB mutation when introduced extragenically despite being active IMPases. However, the loss of sensitivity to RNA pol binding, i.e. in G173V, R184I, and L96F/R184I, did correlate strongly with loss of complementation of suhB. Because residue 184 forms the core of the SuhB dimer, it is likely that the interaction with RNA polymerase requires monomeric SuhB. The exposure of specific residues facilitates the interaction of SuhB with RNA pol (or another target with a similar binding surface) and it is this heterodimer formation that is critical to the ability of SuhB to rescue temperature-sensitive phenotypes in E. coli.

Received for publication, February 8, 2007 , and in revised form, July 5, 2007.

The atomic coordinates and structure factors (code 2QFL) 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 United States Department of Energy Biosciences Grant DE-FG02-91ER20025 (to M. F. R.), National Institutes of Health Grant GM64481 (to B. S.), a Trans National Institutes of Health/FDA Intramural Biodefense Program Grant from NIAID (to D. L. C.), the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research, and federal funds from the NCI, National Institutes of Health, under contract N01-CO-12400. 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.

¹ To whom correspondence should be addressed: Merkert Chemistry Center, Boston College, 2609 Beacon St., Chestnut Hill, MA 02467. Tel.: 617-552-3616; Fax: 617-552-2705; E-mail: mary.roberts{at}bc.edu.

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