The cyanobacterial circadian clock in Synechococcus elongatus consists of three proteins, KaiA, KaiB, and KaiC. KaiA and KaiB rhythmically interact with KaiC to generate stable oscillations of KaiC ...phosphorylation with a period of 24 h. The observation of stable circadian oscillations when the three clock proteins are reconstituted and combined in vitro makes it an ideal system for understanding its underlying molecular mechanisms and circadian clocks in general. These oscillations were historically monitored in vitro by gel electrophoresis of reaction mixtures based on the differing electrophoretic mobilities between various phosphostates of KaiC. As the KaiC phospho-distribution represents only one facet of the oscillations, orthogonal tools are necessary to explore other interactions to generate a full description of the system. However, previous biochemical assays are discontinuous or qualitative. To circumvent these limitations, we developed a spin-labeled KaiB mutant that can differentiate KaiC-bound KaiB from free KaiB using continuous-wave electron paramagnetic resonance spectroscopy that is minimally sensitive to KaiA. Similar to wild-type (WT-KaiB), this labeled mutant, in combination with KaiA, sustains robust circadian rhythms of KaiC phosphorylation. This labeled mutant is hence a functional surrogate of WT-KaiB and thus participates in and reports on autonomous macroscopic circadian rhythms generated by mixtures that include KaiA, KaiC, and ATP. Quantitative kinetics could be extracted with improved precision and time resolution. We describe design principles, data analysis, and limitations of this quantitative binding assay and discuss future research necessary to overcome these challenges.
Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock ...consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC, KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.
Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central ...oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
As the only circadian oscillator that can be reconstituted in vitro with its constituent proteins KaiA, KaiB, and KaiC using ATP as an energy source, the cyanobacterial circadian oscillator serves as ...a model system for detailed mechanistic studies of day–night transitions of circadian clocks in general. The day-to-night transition occurs when KaiB forms a night-time complex with KaiC to sequester KaiA, the latter of which interacts with KaiC during the day to promote KaiC autophosphorylation. However, how KaiB forms the complex with KaiC remains poorly understood, despite the available structures of KaiB bound to hexameric KaiC. It has been postulated that KaiB-KaiC binding is regulated by inter-KaiB cooperativity. Here, using spin labeling continuous-wave electron paramagnetic resonance spectroscopy, we identified and quantified two subpopulations of KaiC-bound KaiB, corresponding to the “bulk” and “edge” KaiBC sites in stoichiometric and substoichiometric KaiB i C6 complexes (i = 1–5). We provide kinetic evidence to support the intermediacy of the “edge” KaiBC sites as bridges and nucleation sites between free KaiB and the “bulk” KaiBC sites. Furthermore, we show that the relative abundance of “edge” and “bulk” sites is dependent on both KaiC phosphostate and KaiA, supporting the notion of phosphorylation-state controlled inter-KaiB cooperativity. Finally, we demonstrate that the interconversion between the two subpopulations of KaiC-bound KaiB is intimately linked to the KaiC phosphorylation cycle. These findings enrich our mechanistic understanding of the cyanobacterial clock and demonstrate the utility of EPR in elucidating circadian clock mechanisms.
Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core ...oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.
Circadian rhythms are an ancient evolutionary adaptation found across the domains of life that synchronize behavior and physiology to the daily solar cycle for optimal fitness. In cyanobacteria, ...timekeeping is generated by three Kai proteins (KaiA, B and C) that form distinct protein complexes throughout the day, comprising a molecular clock that measures time in ~24‐hour increments. We recently showed that KaiB undergoes a rare transition from a tetrameric ground state to a monomeric, fold‐switched signaling state that binds to KaiC and plays a central organizing role in the evening phase of the clock. Using a version of KaiB locked into its active, fold‐switched state, we determined the structures of several complexes that illustrate how KaiB docks on the KaiC hexamer to recruit KaiA and the output protein CikA. A new structure of the output protein SasA bound to KaiC suggested that structural mimicry of the KaiB fold‐switched state is utilized to transition through distinct phases of the clock. Biochemical studies demonstrate that structural mimicry is used at low concentrations of SasA to cooperatively recruit KaiB and enhance oscillator function, while it leads to competition for KaiC at high concentrations. Collectively, this work reveals how mutually exclusive binding interfaces are used to regulate day/night transitions as well as both input and output signaling in the cyanobacterial circadian clock.
Support or Funding Information
National Institutes of Health R01 grants GM121507 (to C.L.P.) and GM107521 (to A.L.).
Stochastic diffusion of a solution of fluorophores after photoselection reduces the polarization of emission, or fluorescence anisotropy. Because this randomization process is slower for larger ...molecules, fluorescence anisotropy is effective for measuring the kinetics of protein-binding events. Here, we describe how to use the technique to carry out real-time observations in vitro of the cyanobacterial circadian clock.
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