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Role of the amino‐terminal GAF domain of the NifA activator in controlling the response to the antiactivator protein NifL

The NifA protein from Azotobacter vinelandii belongs to a family of enhancer binding proteins (EBPs) that activate transcription by RNA polymerase containing the sigma factor σ54. These proteins have conserved AAA+ domains that catalyse ATP hydrolysis to drive conformational changes necessary for open complex formation by σ54‐RNA polymerase. The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino‐terminal GAF domain of NifA that binds 2‐oxoglutarate. Mutations conferring resistance to NifL are located in both the GAF and the AAA+ domains of NifA. To investigate the mechanism by which the GAF domain regulates the activity of the AAA+ domain, we screened for second‐site mutations that suppress the NifL‐resistant phenotype of mutations in the AAA+ domain. One suppressor mutation, F119S, in the GAF domain restores inhibition by NifL to an AAA+ domain mutation, E356K, in response to fixed nitrogen but not in response to oxygen. The biochemical properties of this mutant protein are consistent with the in vivo phenotype and demonstrate that interdomain suppression results in sensitivity to inhibition by NifL in the presence of the signal transduction protein GlnK, but not to the oxidized form of NifL. In the absence of an AAA+ domain mutation, the F119S mutation confers hypersensitivity to repression by NifL. Isothermal titration calorimetry demonstrates that this mutation prevents binding of 2‐oxoglutarate to the GAF domain. Our data support a model in which the GAF domain plays an essential role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. These observations are of general significance in considering how the activities of EBPs are controlled in response to environmental signals.