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

Lookup NU author(s): Dr Heather Lamb, Dr Alison Park, Dr Christopher Johnson, Dr Simon Cocklin, Paul Thompson, Christopher Mee, Professor Alastair Hawkins

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Abstract

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 KD 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.


Publication metadata

Author(s): Lamb HK, Ren J, Park A, Johnson C, Leslie K, Cocklin S, Thompson P, Mee C, Cooper A, Stammers DK, Hawkins AR

Publication type: Article

Publication status: Published

Journal: Protein Science

Year: 2004

Volume: 13

Issue: 12

Pages: 3127-3138

ISSN (print): 0961-8368

ISSN (electronic): 1469-896X

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1110/ps.04958904

DOI: 10.1110/ps.04958904

PubMed id: 15537757


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