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, 284 (16), 10353-60

The Sensor Kinase TodS Operates by a Multiple Step Phosphorelay Mechanism Involving Two Autokinase Domains

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The Sensor Kinase TodS Operates by a Multiple Step Phosphorelay Mechanism Involving Two Autokinase Domains

Andreas Busch et al. J Biol Chem.

Abstract

Expression of the Pseudomonas putida tod operon, which encodes enzymes for toluene metabolism, takes place from the P(todX) promoter and is mediated by the TodS/TodT two component system. The sensor kinase TodS has a complex domain arrangement containing two functional modules, each harboring a sensor- and an autokinase domain and separated by a receiver domain. Based on site-directed mutagenesis of phosphoaccepting His-190, Asp-500, and His-760 and in vitro transphosphorylation experiments with recombinant TodS fragments, we show that TodS uses a multiple step phosphorelay mechanism to activate TodT. Toluene binding stimulates exclusively phosphorylation of His-190, which is followed by phosphotransfer to Asp-500 and subsequently to His-760 prior to phosphorylation of TodT Asp-57. Mutation of His-190, Asp-500, and H760A prevented up-regulation of toluene-mediated stimulation of TodT transphosphorylation in vitro and reduced in vivo expression of P(todX) to the basal level. Calorimetric studies support that TodT binds to the C-terminal kinase module with a K(D) of approximately 200 nm and 1:1 stoichiometry. This is the first report of a multiple step phosphorelay mechanism of a sensor kinase that involves two autokinase domains.

Figures

FIGURE 1.
FIGURE 1.
A, schematic drawing of the domain arrangement of tripartite sensor kinases and TodS. Tm, transmembrane region; RRR, response regulator receiver domain. The domain arrangement for tripartite sensor kinases is taken from ArcB, the best studied family member, and the figure is adapted from Kwon et al. (8). The phosphorelay is indicated. The TodS domains were predicted by SMART (30). B, summary of truncated TodS versions used in this study. The ends of recombinant fragments were determined according to the domain annotation by SMART (30) and secondary structure prediction of TodS using the consensus method (38). Fragment ends were placed in regions for which a turn or coil secondary structure was predicted. All fragments were produced as His tag fusion proteins and purified as detailed for TodS in Lacal et al. (15). For further information see Table 1 and “Experimental Procedures.”
FIGURE 2.
FIGURE 2.
Autophosphorylation of site-directed mutants and recombinant fragments of TodS. Protein at 20 μm was incubated with 200 μm ATP (containing 4 μCi of [γ-32P]ATP) for 20 min prior to analysis by SDS-PAGE. A, autophosphorylation of TodSH190A and TodSH760A in the presence and absence of 100 μm toluene. Both mutants have similar basal activities but toluene stimulates only TodSH760A. B, autophosphorylation of NTodS-long and CTodS-long in the absence of toluene.
FIGURE 3.
FIGURE 3.
Transphosphorylation between recombinant fragments of TodS. NTodS was phosphorylated with 200 μm ATP (containing 4 μCi of [γ-32P]ATP), and the solution was applied to NAP-5 gel filtration columns (GE Healthcare) to separate protein from ATP. An equimolar amount of MTodS was added to the protein fraction. At the time intervals indicated, samples were removed for SDS-PAGE analysis.
FIGURE 4.
FIGURE 4.
Transphosphorylation between recombinant fragments of TodS and TodT. TodS fragments NTodS-short, N-TodS-long, and CTodS-long at 20 μm where incubated with 200 μm ATP containing 4 μCi of [γ-32P]ATP for 20 min. An equimolar amount of MTodS was added to the sample in lane 5, and the corresponding amount of buffer to the remaining samples. After 10 min of incubation, an equimolar amount of TodT was added to the samples in lanes 1, 2, 4, and 5, and the corresponding amount of buffer was added to the sample in lane 3. Samples were taken after another 10 min of incubation.
FIGURE 5.
FIGURE 5.
Transphosphorylation between TodT and TodS or its mutants TodSH190A, TodSD500A, and TodSH760A. Wild-type and mutant TodS at 20 μm were incubated with 200 μm ATP (containing 4μCi of [γ-32P]ATP) and 100 μm toluene for 20 min. An equimolar amount of TodT was added, and samples were removed for SDS-PAGE analysis after 15 min of incubation.
FIGURE 6.
FIGURE 6.
ITC study of the interaction of TodS with TodT. Upper panel, raw titration data of 1.6 μm TodS with aliquots of 23 μm TodT in the presence of 1 mm ATP. For further experimental details see “Experimental Procedures” and Krell (27). Lower panel, integrated and dilution-corrected peak areas from the raw data. The curves were fitted with the “one-binding-site” model of the MicroCal version of ORIGIN.
FIGURE 7.
FIGURE 7.
Transphosphorylation between TodT or TodTD57A and TodS. 20 μm TodS was incubated with 200 μm ATP (containing 4 μCi of [γ-32P]ATP) and 100 μm toluene for 20 min. TodT or TodTD57A was then added to a final concentration of 30 μm, and cultures were incubated for 15 min and analyzed by SDS-PAGE.
FIGURE 8.
FIGURE 8.
Mechanistic model for the function of TodS. The binding of effectors, such as toluene, occurs at the N-terminal PAS sensor domain, which stimulates phosphorylation of its neighboring kinase domain. The phosphoryl group is then passed on to Asp-500 of the internal response regulator receiver domain and subsequently to His-760 of the C-terminal kinase domain, which is the site for transphosphorylation of TodT. The C-terminal kinase domain has autokinase activity, and it remains to be elucidated whether this is modulated by potential recognition of a signal by the PAS 2 sensor domain.

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