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 sigma(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 protein-protein 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.

Fig. 1.

Domain structure of A. vinelandii NifL and NifA showing the mutations analyzed in this study.

Fig. 2.

Response of NifA to oxidized N_his6NifL and N_his6NifL-R306C. NifA activity was measured by the formation of open-promoter complexes, as described in Methods, and plotted relative to the extent of NifA activity in the absence of NifL. Each data point is the mean of at least two independent experiments. All assays contained 4 mM GTP as hydrolyzable nucleotide and 250 nM NifA (calculated as a dimer). (A) Response of NifA to the indicated concentration of N_his6NifL (calculated as a tetramer) in the absence (▪) or presence (□) of 0.05 mM ADP. (B) Response of NifA to the indicated concentration of N_his6NifL-R306C (calculated as a tetramer) in the absence (▪) or presence (□) of 0.05 mM ADP.

Fig. 3.

Response of NifA proteins to N_his6NifL (147-519) and N_his6NifL-R306C (147-519). NifA activity was measured by the formation of open-promoter complexes and plotted relative to the extent of NifA activity in the absence of NifL. Each data point is the mean of at least two independent experiments. All assays contained 4 mM GTP as hydrolyzable nucleotide, 0.05 mM ADP, and 250 nM NifA (calculated as a dimer). (A) Response of NifA to the indicated concentration of N_his6NifL (147-519) and N_his6NifL-R306C (147-519) (calculated as a dimer). (B) Response of NifA to 2-oxoglutarate. NifA activity is plotted relative to the extent of NifA activity in the absence of NifL and 2-oxoglutarate. Reactions contained 750 nM N_his6NifL (147-519) (□) or 500 nM N_his6NifL-R306C (147-519) (▪), and the concentration of 2-oxoglutarate is indicated on the x axis. (C) Response of NifA-E356K to the indicated concentration of N_his6NifL (147-519) and N_his6NifL-R306C (147-519) (calculated as a dimer). (D) Response of NifA-E356K to the indicated N_his6NifL-R306C (147-519) concentration in the absence (□) or presence (▪) of 2 mM 2-oxoglutarate.

Fig. 4.

Limited trypsin proteolysis of N_his6NifL (147-519) and N_his6NifL-R306C (147-519) in the absence (A) or presence (B) of 2 mM ADP. We incubated 2 μM protein with trypsin (weight ratio, 100:1) for the times (in min) indicated above each lane. Arrowheads indicate the proteolysis products described in the text.

Fig. 5.

Model for regulation of NifA activity by NifL. The PAS-and ADP-binding (GHKL) domains of NifL are shown in gray, and the H-box region is represented by a black circle. The GAF and AAA+ domains of NifA are indicated by an open octagon and cube, respectively. For simplicity, the interacting partners are shown as monomers. (A) In the absence of 2-oxoglutarate, both the reduced and oxidized forms of NifL inhibit the activity of NifA, provided that adenosine nucleotide is bound to NifL (8, 15, 19). (B) Binding of 2-oxoglutarate to the GAF domain of NifA (indicated by stars) induces a conformational change that releases inhibition by the reduced form of NifL, enabling NifA to activate transcription (29, 30). (C) Oxidation of the flavin in the PAS domain of NifL (indicated by stippled ovals) causes a conformational change that enables NifL to inhibit NifA in the presence of 2-oxoglutarate. (D) Binding of the signal transduction protein GlnK (indicated by gray circles) to the C-terminal domain of NifL, enables the reduced form of NifL to interact with NifA, in the presence of 2-oxoglutarate. It is possible that the GlnK-NifL interaction promotes a conformational change similar to that induced by the oxidation of NifL. The GlnK-NifL-NifA ternary complex is formed under nitrogen-excess conditions when GlnK is primarily in the nonuridylylated form. Uridylylation of GlnK under nitrogen-limiting conditions prevents this interaction (17-19). We infer that the R306C substitution locks NifL in a conformation that is analogous to that shown in C and D, so that it is competent to inhibit NifA irrespective of other signals.