Journal article
Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis
Protein science, v 13(12), pp 3127-3138
Dec 2004
PMID: 15537757
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 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.
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Details
- Title
- Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis
- Creators
- Heather K Lamb - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomJingshan Ren - Division of Structural Biology, University of Oxford, Oxford OX3 7BN, United KingdomAlison Park - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomChristopher Johnson - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomKris Leslie - Division of Structural Biology, University of Oxford, Oxford OX3 7BN, United KingdomSimon Cocklin - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomPaul Thompson - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomChristopher Mee - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United KingdomAlan Cooper - Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, United KingdomDavid K Stammers - Division of Structural Biology, University of Oxford, Oxford OX3 7BN, United KingdomAlastair R Hawkins - School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom
- Publication Details
- Protein science, v 13(12), pp 3127-3138
- Publisher
- Cold Spring Harbor Laboratory Press
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000225346100006
- Scopus ID
- 2-s2.0-9344220480
- Other Identifier
- 991014877913804721
InCites Highlights
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology