A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Abstract

NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of 54 -dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of proteinprotein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.2-oxoglutarate ͉ signal transduction ͉ antiactivator ͉ redox control ͉ nitrogen regulation S tructural rearrangements of sensor proteins in response to environmental cues provide a fundamental mechanism for signal propagation within cells. However, although conformational changes have been well characterized in isolated signaling domains, mechanisms for signal transmission by means of interdomain interactions are frequently less well understood. For example, structural studies have identified ligand-induced conformational changes in the sensor domains of histidine protein kinases (HPKs), but it is not known how these changes are communicated to the kinase domain to control phosphoryl transfer.The Azotobacter vinelandii NifL regulatory protein is a histidine kinase-like protein that controls the expression of the genes required for nitrogen fixation in response to the redox, nitrogen, and carbon status. NifL is an antiactivator that tightly regulates the activity of its partner protein NifA, a member of the family of 54 -transcriptional activators (1, 2), by means of the formation of an inhibitory complex (3-5). The domain architecture of NifL is similar to that of some HPKs, with an N-terminal Per-ArntSim (PAS) domain (6, 7) containing a flavin adenine dinucleotide (FAD) cofactor that senses the redox status (8, 9) and a C-terminal domain containing conserved residues corresponding to the N, G1, F, and G2 boxes that constitute the ATPbinding domain of the GHKL superfamily of ATPases (10-14) (Fig. 1). Unlike the HPKs, the GHKL domain of NifL does not exhibit ATP hydrolysis or transphos...

Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion

“…steady-state level of nifH mRNA is not altered in the glnE mutant strain in the absence of added ammonium, we infer that there is sufficient 2-OG under nitrogen-limiting conditions in the mutant strain to saturate the 2-OG binding site on NifA, hence relieving inhibition by NifL (34,35). This sophisticated metabolic control of NifA activity facilitates the coupling of NH 4 ϩ production by nitrogenase to NH 4 ϩ assimilation, thus preventing the toxic consequences of glutamine overproduction observed when the glnE mutant strain is grown in the presence of external NH 4 ϩ .…”

Molecular Basis of Transcriptional Antiactivation

“…Inhibition of Ti plasmid conjugative transfer by TraM joins a growing list of prokaryotic regulatory systems involving an activator-antiactivator complex, including NifA-NifL regulation of nitrogen fixation in Azotobacter vinelandii (33) and Klebsiella pneumoniae (34), ComK-MecA-ClpC regulation of competence in Bacillus subtilis (35), GcvA-GcvR regulation of oxidative cleavage of glycine (36), and CRP-CytR regulation of many operons in E. coli (37). In the cases of NifA-NifL and ComKMecA, the antiactivator binds to and titrates the activator, thus preventing DNA binding and transcriptional activation (33)(34)(35).…”

“…In the cases of NifA-NifL and ComKMecA, the antiactivator binds to and titrates the activator, thus preventing DNA binding and transcriptional activation (33)(34)(35). However in the cases of GcvA-GcvR and CRP-CytR, the antiactivator and the activator bind promoter DNA in tandem, and the interactions between the antiactivator and the activator at the promoter inhibit transcriptional activation (36,37).…”

Role of the H Domain of the Histidine Kinase-like Protein NifL in Signal Transmission

“…Substitutions of Arg-306 cause NifL to constitutively inhibit NifA irrespective of environmental cues. We have demonstrated that the phenotype of one such mutation, R306C, is associated with a conformational change that apparently locks NifL in a form similar to the oxidized "on" state so that the mutant NifL protein inhibits NifA even under conditions appropriate for nitrogen fixation (20). We observe that some mutations in the GHKL domain of NifL that substantially reduce ADP binding suppress the phenotype of the R306C mutation.…”

“…Section 1734 solely to indicate this fact. 1 changes required to inhibit NifA (20). The key role of arginine 306 in signal transmission has prompted us to investigate the role of other residues of the H domain of NifL in signal propagation.…”