Mutational analysis of the nucleotide-binding domain of the anti-activator NifL.
The NifL regulatory protein controls transcription of nitrogen fixation genes in Azotobacter vinelandii by modulating the activity of the transcriptional activator NifA through direct protein-protein interactions. The ability of NifL to integrate the antagonistic signals of redox and nitrogen status is achieved via the involvement of discrete domains in signalling specific environmental cues. NifL senses the redox status via an FAD co-factor located within the amino-terminal PAS domain and responds to the fixed nitrogen status by interaction with the signal transduction protein GlnK, which binds to the C-terminal GHKL domain of NifL. The GHKL domain binds adenosine nucleotides and is similar to the core catalytic domain of the histidine protein kinases. Binding of ADP to this domain increases the inhibitory activity of NifL and the formation of protein complexes with NifA. This inhibition is antagonised by the binding of 2-oxoglutarate, a key metabolic signal of the carbon status, to the amino-terminal GAF domain of NifA. In this study we have examined the properties of three mutations within conserved residues in the GHKL domain of NifL that impair signal transduction. All three mutations decrease the affinity of NifL for ADP significantly, but the mutant proteins exhibit discrete properties. The N419D mutation prevents inhibition of NifA activity by NifL both in vivo and in vitro. In contrast, the G455A and G480A mutations eliminate the redox response, but the mutant proteins retain some sensitivity to the fixed nitrogen status and the ability to interact with the GlnK signal transduction protein. Our data suggest that the absence of the redox switch in the G455A and G480A mutants is a consequence of their inability to override the allosteric effect of 2-oxoglutarate on NifA activity. Overall, these results demonstrate that the binding of adenosine nucleotides to the GHKL domain of NifL plays an important role in counteracting the response of NifA to 2-oxoglutarate, under conditions that are inappropriate for nitrogen fixation.
Perry
Susan
S
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UK, UK.
Shearer
Neil
N
Little
Richard
R
Dixon
Ray
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D009711:Nucleotides; D014157:Transcription Factors; C084296:nifL protein, Bacteria; D005182:Flavin-Adenine Dinucleotide; D000244:Adenosine Diphosphate