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Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis

¹ School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom
² Division of Structural Biology, University of Oxford, Oxford OX3 7BN, United Kingdom
³ Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom

(RECEIVED June 29, 2004; FINAL REVISION August 18, 2004; ACCEPTED August 21, 2004)

NmrA is a negative transcription-regulating protein that binds to the C-terminal region of the GATA transcription-activating protein AreA. The proposed molecular mechanism of action for NmrA is to inhibit AreA binding to its target promoters. In contrast to this proposal, we report that a C-terminal fragment of AreA can bind individually to GATA-containing DNA and NmrA and that in the presence of a mixture of GATA-containing DNA and NmrA, the AreA fragment binds preferentially to the GATA-containing DNA in vitro. These observations are consistent with NmrA acting by an indirect route, such as by controlling entry into the nucleus. Deletion of the final nine amino acids of a C-terminal fragment of AreA does not affect NmrA binding. Wild-type NmrA binds NAD⁺(P⁺) with much greater affinity than NAD(P)H, despite the lack of the consensus GXXGXXG dinucleotide-binding motif. However, introducing the GXXGXXG sequence into the NmrA double mutant N12G/A18G causes an ~13-fold increase in the K_D for NAD⁺ and a 2.3-fold increase for NADP⁺. An H37W mutant in NmrA designed to increase the interaction with the adenine ring of NAD⁺ has a decrease in K_D of ~4.5-fold for NAD⁺ and a marginal 24% increase for NADP⁺. The crystal structure of the N12G/A18G mutant protein shows changes in main chain position as well as repositioning of H37, which disrupts contacts with the adenine ring of NAD⁺, changes which are predicted to reduce the binding affinity for this dinucleotide. The substitutions E193Q/D195N or Q202E/F204Y in the C-terminal domain of NmrA reduced the affinity for a C-terminal fragment of AreA, implying that this region of the protein interacts with AreA.

Keywords: NmrA; site-directed mutagenesis; biocalorimetry; nitrogen metabolite repression

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04958904.

Reprint requests to: Alastair R. Hawkins, School of Cell and Molecular Biosciences, Catherine Cookson Building, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK; e-mail: a.r.hawkins{at}ncl.ac.uk; fax: +44-191-2227424.

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