Dataset Information

Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling.

ABSTRACT: Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal transduction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simultaneous actions of multiple TCS activities. Here, we report a strategy that combines concentration-dependent phosphorylation profiling and mathematical modeling to characterize the cellular activities of the archetype Escherichia coli PhoR/PhoB system. Phosphorylation of the response regulator (RR) PhoB has been found to be dependent on the total concentrations of PhoB/PhoR and saturated at high concentrations. The relationship between RR phosphorylation and total concentrations has been defined by the modeling of the kinase and phosphatase reactions and quantified to derive the biochemical parameters of the PhoR/PhoB system in vivo. In a further test of this approach on a PhoB mutant, PhoB(F20D), it proved highly effective in exploring the mechanistic differences of TCSs that are not revealed by traditional reporter assays. Measurement of biochemical parameters for PhoB(F20D) led to the discovery that a weaker interaction between the histidine sensor kinase and RR could result in a higher and nonrobust phosphorylation due to diminished phosphatase activities.

SUBMITTER: Gao R

PROVIDER: S-EPMC3545780 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling.

Gao Rong R Stock Ann M AM

Proceedings of the National Academy of Sciences of the United States of America 20121224 2

Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal transduction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simu ...[more]

PMID: 23267085

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Project description:T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

Dataset Information

Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling.

Publications

Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling.

Similar Datasets

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