The KaiA protein of the cyanobacterial circadian oscillator is modulated by a redox-active cofactor.
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ABSTRACT:
SUBMITTER: Wood TL
PROVIDER: S-EPMC2851934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The core circadian oscillator of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. This circadian oscillator could be functionally reconstituted in vitro with these three proteins, and therefore has been a very important model in circadian rhythm research. KaiA can bind to KaiC and then stimulate its phosphorylation, but their interaction mechanism remains elusive. In this study, we followed the "second-site suppressor" strategy to investigate the interaction mechanism of KaiA and KaiC. Using protein sequence analyses, we showed that there exist co-varying residues in the binding interface of KaiA and KaiC. The followed mutagenesis study verified that these residues are important to the functions of KaiA and KaiC, but their roles could not be fully explained by the reported complex structures of KaiA and KaiC derived peptides. Combining our data with previous reports, we suggested a dynamic interaction mechanism in KaiA-KaiC interaction, in which both KaiA and the intrinsically disordered tail of KaiC undergo significant structural changes through conformational selection and induced fit during the binding process. At last, we presented a mathematic model to support this hypothesis and explained the importance of this interaction mechanism for the KaiABC circadian oscillator.
| S-EPMC4844972 | biostudies-literature
Project description:KaiA protein that stimulates KaiC phosphorylation in the cyanobacterial circadian clock was recently shown to be destabilized by dibromothymoquinone (DBMIB), thus revealing KaiA as a sensor of the plastoquinone (PQ) redox state and suggesting an indirect control of the clock by light through PQ redox changes. Here we show using X-ray crystallography that several DBMIBs are bound to KaiA dimer. Some binding modes are consistent with oligomerization of N-terminal KaiA pseudoreceiver domains and/or reduced interdomain flexibility. DBMIB bound to the C-terminal KaiA (C-KaiA) domain and limited stimulation of KaiC kinase activity by C-KaiA in the presence of DBMIB demonstrate that the cofactor may weakly inhibit KaiA-KaiC binding.
| S-EPMC3561481 | biostudies-literature
Project description:In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.
| S-EPMC3610728 | biostudies-literature
Project description:The recurrent pattern of light and darkness generated by Earth's axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins, KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a nighttime metabolic program that is essential for S. elongatus to survive in the dark.
| S-EPMC6064986 | biostudies-literature
Project description:The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.
| S-EPMC3587310 | biostudies-literature
Project description:The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.
| S-EPMC6549140 | biostudies-literature
Project description:Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock.
| S-EPMC3309039 | biostudies-literature
Project description:Synchronization of the circadian clock in cyanobacteria with the day/night cycle proceeds without an obvious photoreceptor, leaving open the question of its specific mechanism. The circadian oscillator can be reconstituted in vitro, where the activities of two of its proteins, KaiA and KaiC, are affected by metabolites that reflect photosynthetic activity: KaiC phosphorylation is directly influenced by the ATP/ADP ratio, and KaiA stimulation of KaiC phosphorylation is blocked by oxidized, but not reduced, quinones. Manipulation of the ATP/ADP ratio can reset the timing of KaiC phosphorylation peaks in the reconstituted in vitro oscillator. Here, we show that pulses of oxidized quinones reset the cyanobacterial circadian clock both in vitro and in vivo. Onset of darkness causes an abrupt oxidation of the plastoquinone pool in vivo, which is in contrast to a gradual decrease in the ATP/ADP ratio that falls over the course of hours until the onset of light. Thus, these two metabolic measures of photosynthetic activity act in concert to signal both the onset and duration of darkness to the cyanobacterial clock.
| S-EPMC3497819 | biostudies-literature
Project description:The mechanisms by which cellular oscillators keep time and transmit temporal information are poorly understood. In cyanobacteria, the timekeeping aspect of the circadian oscillator, composed of the KaiA, KaiB, and KaiC proteins, involves a cyclic progression of phosphorylation states at Ser431 and Thr432 of KaiC. Elucidating the mechanism that uses this temporal information to modulate gene expression is complicated by unknowns regarding the number, structure, and regulatory effects of output components. To identify oscillator signaling states without a complete description of the output machinery, we defined a simple metric, Kai-complex output activity (KOA), that represents the difference in expression of reporter genes between strains that carry specific variants of KaiC and baseline strains that lack KaiC. In the absence of the oscillator, expression of the class 1 paradigm promoter P(kaiBC) was locked at its usual peak level; conversely, that of the class 2 paradigm promoter P(purF) was locked at its trough level. However, for both classes of promoters, peak KOA in wild-type strains coincided late in the circadian cycle near subjective dawn, when KaiC-pST becomes most prevalent (Ser431 is phosphorylated and Thr432 is not). Analogously, peak KOA was detected specifically for the phosphomimetic of KaiC-pST (KaiC-ET). Notably, peak KOA required KaiB, indicating that a KaiBC complex is involved in the output activity. We also found evidence that phosphorylated RpaA (regulator of phycobilisome associated) represses an RpaA-independent output of KOA. A simple mathematical expression successfully simulated two key features of the oscillator-the time of peak KOA and the peak-to-trough amplitude changes.
| S-EPMC3791705 | biostudies-literature
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 ?/? 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.
| S-EPMC3293397 | biostudies-literature