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Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.

ABSTRACT: The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using (32)P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from (32)P-labeled KaiC were examined. We detected the transient formation of [(32)P]ATP preceding the accumulation of (32)P(i). Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form P(i). We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity.

SUBMITTER: Nishiwaki T

PROVIDER: S-EPMC3365771 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.

Nishiwaki Taeko T Kondo Takao T

The Journal of biological chemistry 20120409 22

The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we perfo ...[more]

PMID: 22493509

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Project description:The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P(i) (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between α/β heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.

Dataset Information

Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.

Publications

Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.

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