Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis.
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 approximately 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 KD of approximately 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.
Lamb
Heather K
HK
School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK.
Ren
Jingshan
J
Park
Alison
A
Johnson
Christopher
C
Leslie
Kris
K
Cocklin
Simon
S
Thompson
Paul
P
Mee
Christopher
C
Cooper
Alan
A
Stammers
David K
DK
Hawkins
Alastair R
AR
D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
C079728:AreA protein, Aspergillus nidulans; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; C441635:NmrA protein, Aspergillus nidulans; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA