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. 2006 Mar 10;281(10):6768-75.
doi: 10.1074/jbc.M509687200. Epub 2006 Jan 5.

Identification of a ubiquinone-binding site that affects autophosphorylation of the sensor kinase RegB

Lee R Swem  1 , Xing Gong, Chang-An Yu, Carl E Bauer
Affiliations

Identification of a ubiquinone-binding site that affects autophosphorylation of the sensor kinase RegB

Lee R Swem et al. J Biol Chem. .

Abstract

Rhodobacter capsulatus regulates many metabolic processes in response to the level of environmental oxygen and the energy state of the cell. One of the key global redox regulators of the cell's metabolic physiology is the sensor kinase RegB that controls the synthesis of numerous energy generation and utilization processes. In this study, we have succeeded in purifying full-length RegB containing six transmembrane-spanning elements. Exogenous addition of excess oxidized coenzyme Q1 is capable of inhibiting RegB autophosphorylation approximately 6-fold. However, the addition of reduced coenzyme Q1 exhibits no inhibitory effect on kinase activity. A ubiquinone-binding site, as defined by azidoquinone photo affinity cross-linking, was determined to lie within a periplasmic loop between transmembrane helices 3 and 4 that contains a fully conserved heptapeptide sequence of GGXXNPF. Mutation of the phenylalanine in this heptapeptide renders RegB constitutively active in vivo, indicating that this domain is responsible for sensing the redox state of the ubiquinone pool and subsequently controlling RegB autophosphorylation.

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Figures

FIGURE 1
FIGURE 1. SDS-PAGE analysis and autophosphorylation assays of RegB
A, solubilized and purified RegB separated by SDS-PAGE. Lane 1 contains the molecular weight mass followed by the wash fraction and the elution fraction after the addition of imidazole. B, autophosphorylation of purified monomer RegB using γ-32P-labeled ATP as a tracer. The aliquots were removed at 0.25, 0.5, 0.75, 1, 2, 4, 10, and 15 min after the addition of ATP, and the reaction was quenched with SDS-PAGE loading buffer before being separated by SDS-PAGE. The graph below the autoradiograph represents the arbitrary units of 32P incorporation derived from phosphorimaging data analysis.
FIGURE 2
FIGURE 2. RegB autophosphorylation assays in the presence of ubiquinone
A, autophosphorylation assays of RegB in the presence and absence of ubiquinone (Q1) at a 50-fold molar excess to RegB. Ethanol was added to the RegB sample lacking ubiquinone to account for the ethanol concentration of the ubiquinone treated sample. The two reactions were incubated for 20 min at 37 °C before the addition of [γ-32P]ATP. Aliquots were removed at 0.5, 1, 2, 4, and 10 min and quenched with SDS-PAGE loading buffer before being separated by SDS-PAGE. B, autophosphorylation assays of RegB in the presence of either oxidized ubiquinone or reduced ubiquinol (Q1). RegB was incubated in the presence of a 50-fold molar excess of ubiquinone or ubiquinol at 37 °C for 20 min prior to the addition of [γ-32P]ATP. Aliquots were removed at 1, 2, 4, and 8 min and quenched with SDS-PAGE loading buffer before being separated by SDS-PAGE. C, autophosphorylation assays of C265SA mutant RegB in the presence and absence of ubiquinone (Q1) at a 50-fold molar excess to RegB. Ethanol was added to the RegB sample lacking ubiquinone to account for the ethanol concentration of the ubiquinone treated sample. The two reactions were incubated for 20 min at 37 °C before the addition of [γ-32P]ATP. Aliquots were removed at 0.5, 1, 2, 4, and 10 min and quenched with SDS-PAGE loading buffer before being separated by SDS-PAGE. All of the autophosphorylation assays were visualized and quantitated using the Typhoon phosphorimaging system (Amersham Biosciences).
FIGURE 3
FIGURE 3. Azido-Q uptake of RegB and the HPLC profile of digested RegB
A, effect of illumination time on azido-Q uptake and inactivation of the sensor kinase RegB. Purified RegB in 20 mm Tris-Cl, pH 8.0, containing 150 mm NaCl and 30% glycerol was incubated with azido-Q in ethanol (open circles and open squares) or ethanol only (solid circles) for 1 h at 4 °C in the dark. The samples were then illuminated with long wavelength UV light for the indicated times at 0 °C. The determination of activity (open circles and solid circles) and radioactivity (open squares) were performed as described under “Experimental Procedures.” B, 3H radioactivity distribution in an HPLC chromatogram of a protease K-digest of [3H]azido-Q-labeled RegB. The labeled protein was digested, fractioned, and assayed for radioactivity as described under “Experimental Procedures.”
FIGURE 4
FIGURE 4. Topological representation and homolog pile up of the transmembrane-spanning domains of RegB
A, RegB has six transmembrane-spanning domains that anchor it to the bacterial membrane. The boxed red letters represent the 100% conserved pentapeptide ubiquinone-binding motif. The amino acids of the peptide fragment identified by HPLC and azido-Q cross-linking experiments are colored black. The site of phosphorylation is colored green, and the redox active Cys265 is colored blue. Amino acid residues past 270 were omitted for simplicity. B, RegB homologs were selected from Brucella suis, Brucella melitensis, Mesorhizobium loti, Rhizobium leguminosarum, Agrobacterium tumefaciens, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Roseobacter denitrificans, Rhodovulum sulfidophilum, R. sphaeroides, R. capsulatus, Caulobacter crescentus, and Pseudomonas syringae. The asterisks denote the 100% conserved residues in the newly identified ubiquinone-binding site. The alignment was created using ClustalW and the ALN file was imported into BoxShade to color code the conserved residues in black, similar residues in gray, and nonconserved residues in white. The line above the alignment denotes the identified ubiquinone-binding peptide.
FIGURE 5
FIGURE 5. Spectral scans of wild type and RegB F112A R. capsulatus cells
R. capsulatus strains SB1003 and RegB F-A were grown to the same cell density under aerobic, semi-aerobic, and photosynthetic conditions. The cells were then lysed, clarified and spectrally scanned from 400 to 900 nm. A, aerobically grown cells. B, semi-aerobically gown cells. C, photosynthetically grown cells. The solid lines denote SB1003, whereas the dashed lines denote the RegB F112A mutant strain.
FIGURE 6
FIGURE 6. Northern blot analysis of puf and puc operon expression
The puf (A) and puc (B) Northern blot autoradiograph of RNA isolated from aerobic and anaerobic grown wild type (WT) and the F112A mutant (FA). The RNA was probed using a digoxigenin-labeled probe and visualized using x-ray film. The data were then plotted as a bar graph. RNA transcript for puf and puc were normalized using 16 S ribosomal RNA levels as a control. Northern blots were carried out three times independent times with three different RNA preparations. The data were consistent between all three replicas.
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