Autophosphorylation and dephosphorylation by soluble forms of the nitrate-responsive sensors NarX and NarQ from Escherichia coli K-12

Abstract

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K(m)) for ADP were similar for both proteins, about 2 microM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 microM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 x 10(-4) s(-1)) was lower than that observed for MBP-NarQ (about 2.2 x 10(-4) s(-1)). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s(-1) for MBP-NarX and about 0.01 s(-1) for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 microM (KinA) to about 40 microM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.

FIG. 1.

Purification and autophosphorylation of MBP fusion proteins MBP-NarX₁₈₅ (MX) and MBP-NarQ₁₈₂ (MQ). (A) The purification of MBP fusion proteins was analyzed on a 10% Laemmli gel stained with Coomasie Blue (GelCode Blue Stain Reagent; Pierce) by FluorChem. The relative positions of molecular weight references are indicated. The amount of protein applied for each lane was 1.6 μg. (B) Radiolabeled proteins were prepared and visualized as stated in Materials and Methods.

FIG. 2.

Autophosphorylation of MBP-NarX₁₈₅ and MBP-NarQ₁₈₂ with or without an ATP-regenerating system. Labeled proteins were quantified from 5-μl time point samples as described in Materials and Methods. Quantification of phospho-MBP-NarX₁₈₅ (circles) and phospho-MBP-NarQ₁₈₂ (squares) with the addition of an ATP-regenerating system (solid symbols) or without (open symbols) in 100 μM [γ-³²P]ATP (MBP-NarX₁₈₅, 2,300 cpm pmol⁻¹, and MBP-NarQ₁₈₂, 1,800 cpm pmol⁻¹). Specific levels of phosphorylation are expressed as nmol of incorporated phosphate per total amount of protein used, expressed as μmol. Note the change in the x axis scale after 30 min. The data represent one of two independent measurements. Autophosphorylations of MBP-NarX₁₈₅ and MBP-NarQ₁₈₂ were conducted as separate experiments.

FIG. 3.

Effects of ADP on phosphorylated MBP-NarX₁₈₅ (MX) and MBP-NarQ₁₈₂ (MQ) monitored by TLC. Autophosphorylation was first performed for 6 min. The removal of 30 nM [γ-³²P]ATP and dephosphorylation reactions were performed as described in Materials and Methods, with the addition of 5 μM ADP. Where indicated, standard reaction buffer was modified by the absence of MgCl₂ and MnCl₂ and the addition of EDTA (5 mM); 2.5 μl of a 1:10⁴ dilution of 3.3 μM [γ-³²P]ATP (3,000 cpm fmol⁻¹) in stop solution was included as a reference (Ref).

FIG. 4.

Effects of unlabeled ATP on the accumulation of radiolabeled MBP-NarX₁₈₅ and MBP-NarQ₁₈₂ with the addition of an ATP-regenerating system (B and D) or without (A and C) in 30 nM [γ-³²P]ATP (MBP-NarX₁₈₅, 5,600 cpm fmol⁻¹, and MBP-NarQ₁₈₂, 5,000 cpm fmol⁻¹). At the time points shown, 5-μl samples of radiolabeled protein were quantified as described in Materials and Methods. Specific levels of phosphorylation are expressed as pmol of incorporated ³²P per total amount of protein used, expressed as μmol. Immediately following the removal of the 4-min sample, different concentrations of unlabeled ATP were added to the reaction mixtures (arrows) with the following final concentrations: 1× standard buffer (solid squares), 10 μM ATP (diamonds), 100 μM ATP (triangles), and 1,000 μM ATP (open squares). (A and B) MBP-NarX₁₈₅. (C and D) MBP-NarQ₁₈₂. The data represent one of two independent measurements.

FIG. 5.

Effects of ADP on the accumulation of radiolabeled MBP-NarX₁₈₅ (A) and MBP-NarQ₁₈₂ (B) in 30 nM [γ-³²P]ATP (MBP-NarX₁₈₅, 3,000 cpm fmol⁻¹, and MBP-NarQ₁₈₂, 7,000 cpm fmol⁻¹). At the time points shown, 5-μl samples of radiolabeled protein were quantified as described in Materials and Methods. Specific levels of phosphorylation are expressed as pmol of incorporated ³²P per total amount of protein used, expressed as μmol. Immediately following the removal of the 4-min sample, different concentrations of ADP were added to the reaction mixture (arrows) with the following final concentrations: 1× standard buffer (solid squares), 0.1 μM ADP (diamonds), 1 μM ADP (triangles), and 10 μM ADP (open squares). The data represent one of two independent measurements.

FIG. 6.

Kinetics of MBP-NarX₁₈₅ and MBP-NarQ₁₈₂ autophosphorylation (A and B) and dephosphorylation (C and D). Quantifications of autophosphorylation and dephosphorylation rates were performed as stated in Materials and Methods. The insets represent Lineweaver-Burk plots of the corresponding reaction velocities as 1/V versus 1/ATP or 1/ADP. Autophosphorylation kinetics were performed with an ATP-regenerating system in 30 nM [γ-³²P]ATP (∼7,000 cpm fmol⁻¹) with different concentrations of unlabeled ATP. (A) Autophosphorylation rates of MBP-NarX₁₈₅ incubated with increasing concentrations of ATP (0.5, 1, 2, 4, 8, 16, 32, and 64 μM). The x intercept (K_mATP) was 2.5 μM. The y intercept (k) was 0.59 × 10⁻⁴ s⁻¹. (B) Autophosphorylation rates of MBP-NarQ₁₈₂ incubated with increasing concentrations of ATP (2, 5, 10, 20, 40, 80, 160, and 320 μM). The x intercept (K_mATP) was 24.6 μM. The y intercept (k) was 3.0 × 10⁻⁴ s⁻¹. (C) Dephosphorylation rates of phospho-MBP-NarX₁₈₅ (∼3,000 cpm/fmol) incubated with increasing concentrations of ADP (0.25, 0.5, 1, 2.5, and 5 μM). The x intercept (K_mADP) was 2.9 μM. The y intercept (−k) was 0.033 s⁻¹. (D) Dephosphorylation rates of phospho-MBP-NarQ₁₈₂ (∼4,000 cpm/fmol) incubated with increasing concentrations of ADP (0.25, 0.5, 1, 2.5, and 5 μM). The x intercept (K_mADP) was 2.2 μM. The y intercept (−k) was 0.015 s⁻¹. The data shown are from one of three independent measurements from various protein purification preparations.