The inner workings of an ancient biological clock.

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

Synchronization of the circadian clock to the environment tracked in real time.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

Resurrected Ancestors Reveal Origins of Metamorphism in XCL1.

In a recent study, Dishman et al. resurrected ancestors of the metamorphic chemokine, XCL1, inferred through phylogenetics, and found that metamorphism arose in the XCL1 lineage ~150 million years ago. A zigzagging evolutionary path suggests that the metamorphic properties are adaptive and reveals three design principles that could be used for technological applications.

Protocols for in vitro reconstitution of the cyanobacterial circadian clock.

Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

A Night-Time Edge Site Intermediate in the Cyanobacterial Circadian Clock Identified by EPR Spectroscopy.

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 KaiBiC6 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.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Bayesian modeling reveals metabolite-dependent ultrasensitivity in the cyanobacterial circadian clock.

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly detailed but mechanistically naive model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation likely plays an important role in metabolic compensation by suppressing premature phosphorylation at nighttime.

Identification and characterization of metamorphic proteins: Current and future perspectives.

Proteins that can reversibly alternate between distinctly different folds under native conditions are described as being metamorphic. The "metamorphome" is the collection of all metamorphic proteins in the proteome, but it remains unknown the extent to which the proteome is populated by this class of proteins. We propose that uncovering the metamorphome will require a synergy of computational screening of protein sequences to identify potential metamorphic behavior and validation through experimental techniques. This perspective discusses computational and experimental approaches that are currently used to predict and characterize metamorphic proteins as well as the need for developing improved methodologies. Since metamorphic proteins act as molecular switches, understanding their properties and behavior could lead to novel applications of these proteins as sensors in biological or environmental contexts.

Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA.

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.

Sequence-Based Prediction of Metamorphic Behavior in Proteins.

An increasing number of proteins have been demonstrated in recent years to adopt multiple three-dimensional folds with different functions. These metamorphic proteins are characterized by having two or more folds with significant differences in their secondary structure, in which each fold is stabilized by a distinct local environment. So far, ∼90 metamorphic proteins have been identified in the Protein Databank, but we and others hypothesize that a far greater number of metamorphic proteins remain undiscovered. In this work, we introduce a computational model to predict metamorphic behavior in proteins using only knowledge of the sequence. In this model, secondary structure prediction programs are used to calculate diversity indices, which are measures of uncertainty in predicted secondary structure at each position in the sequence; these are then used to assign protein sequences as likely to be metamorphic versus monomorphic (i.e., having just one fold). We constructed a reference data set to train our classification method, which includes a novel compilation of 136 likely monomorphic proteins and a set of 201 metamorphic protein structures taken from the literature. Our model is able to classify proteins as metamorphic versus monomorphic with a Matthews correlation coefficient of ∼0.36 and true positive/true negative rates of ∼65%/80%, suggesting that it is possible to predict metamorphic behavior in proteins using only sequence information.

Monitoring Protein-Protein Interactions in the Cyanobacterial Circadian Clock in Real Time via Electron Paramagnetic Resonance Spectroscopy.

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.

Structure, function, and mechanism of the core circadian clock in cyanobacteria.

Circadian rhythms enable cells and organisms to coordinate their physiology with the cyclic environmental changes that come as a result of Earth's light/dark cycles. Cyanobacteria make use of a post-translational oscillator to maintain circadian rhythms, and this elegant system has become an important model for circadian timekeeping mechanisms. Composed of three proteins, the KaiABC system undergoes an oscillatory biochemical cycle that provides timing cues to achieve a 24-h molecular clock. Together with the input/output proteins SasA, CikA, and RpaA, these six gene products account for the timekeeping, entrainment, and output signaling functions in cyanobacterial circadian rhythms. This Minireview summarizes the current structural, functional and mechanistic insights into the cyanobacterial circadian clock.

Rhythmic ring-ring stacking drives the circadian oscillator clockwise.

The oscillator of the circadian clock of cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, which together generate a self-sustained ∼24-h rhythm of phosphorylation of KaiC. The mechanism propelling this oscillator has remained elusive, however. We show that stacking interactions between the CI and CII rings of KaiC drive the transition from the phosphorylation-specific KaiC-KaiA interaction to the dephosphorylation-specific KaiC-KaiB interaction. We have identified the KaiB-binding site, which is on the CI domain. This site is hidden when CI domains are associated as a hexameric ring. However, stacking of the CI and CII rings exposes the KaiB-binding site. Because the clock output protein SasA also binds to CI and competes with KaiB for binding, ring stacking likely regulates clock output. We demonstrate that ADP can expose the KaiB-binding site in the absence of ring stacking, providing an explanation for how it can reset the clock.

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria.

In the cyanobacterial circadian oscillator, KaiA and KaiB alternately stimulate autophosphorylation and autodephosphorylation of KaiC with a periodicity of approximately 24 h. KaiA activates autophosphorylation by selectively capturing the A loops of KaiC in their exposed positions. The A loops and sites of phosphorylation, residues S431 and T432, are located in the CII ring of KaiC. We find that the flexibility of the CII ring governs the rhythm of KaiC autophosphorylation and autodephosphorylation and is an example of dynamics-driven protein allostery. KaiA-induced autophosphorylation requires flexibility of the CII ring. In contrast, rigidity is required for KaiC-KaiB binding, which induces a conformational change in KaiB that enables it to sequester KaiA by binding to KaiA's linker. Autophosphorylation of the S431 residues around the CII ring stabilizes the CII ring, making it rigid. In contrast, autophosphorylation of the T432 residues offsets phospho-S431-induced rigidity to some extent. In the presence of KaiA and KaiB, the dynamic states of the CII ring of KaiC executes the following circadian rhythm: CII STflexible → CIISpTflexible → CIIpSpTrigid → CIIpSTvery-rigid → CIISTflexible. Apparently, these dynamic states govern the pattern of phosphorylation, ST → SpT → pSpT → pST → ST. CII-CI ring-on-ring stacking is observed when the CII ring is rigid, suggesting a mechanism through which the ATPase activity of the CI ring is rhythmically controlled. SasA, a circadian clock-output protein, binds to the CI ring. Thus, rhythmic ring stacking may also control clock-output pathways.

Temperature-Dependent Fold-Switching Mechanism of the Circadian Clock Protein KaiB.

The oscillator of the cyanobacterial circadian clock relies on the ability of the KaiB protein to switch reversibly between a stable ground-state fold (gsKaiB) and an unstable fold-switched fold (fsKaiB). Rare fold-switching events by KaiB provide a critical delay in the negative feedback loop of this post-translational oscillator. In this study, we experimentally and computationally investigate the temperature dependence of fold switching and its mechanism. We demonstrate that the stability of gsKaiB increases with temperature compared to fsKaiB and that the Q10 value for the gsKaiB → fsKaiB transition is nearly three times smaller than that for the reverse transition. Simulations and native-state hydrogen-deuterium exchange NMR experiments suggest that fold switching can involve both subglobally and near-globally unfolded intermediates. The simulations predict that the transition state for fold switching coincides with isomerization of conserved prolines in the most rapidly exchanging region, and we confirm experimentally that proline isomerization is a rate-limiting step for fold switching. We explore the implications of our results for temperature compensation, a hallmark of circadian clocks, through a kinetic model.

The KaiA protein of the cyanobacterial circadian oscillator is modulated by a redox-active cofactor.

The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.

Site directed spin labeling to elucidating the mechanism of the cyanobacterial circadian clock.

Electron Paramagnetic Resonance (EPR) is a spectroscopic technique that provides structural and dynamic information on unpaired spins and their surrounding environments. Introduction of exogenous spin labels via site directed spin labeling (SDSL) enables characterization of systems of interests lacking intrinsic unpaired spins. This chapter describes the use of SDSL in quantifying KaiB-KaiC binding in the cyanobacterial circadian clock (Kai Clock), exploiting the changes in mobility of the local environment around the spin label on KaiB-KaiC interactions. While the Kai system serves as our model system to demonstrate SDSL-EPR utility in quantifying protein-protein interactions, this technique is readily amenable to other systems of interest whenever specific protein-protein interactions need to be isolated. We first present a protocol for spin labeling KaiB. Then, we detail the sample preparation and acquisition processes to maximize signal-to-noise for downstream analysis. We close this chapter by highlighting recent advances in SDSL technology to incorporate spin labels into proteins of interest and in EPR technology to improve detection sensitivity that may allow greater flexibilities to the types of experiments possible.

Coupling of distant ATPase domains in the circadian clock protein KaiC.

The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which, together with ATP, can generate a self-sustained approximately 24 h oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate, whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the "A-loops", are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. A dynamic equilibrium likely exists between the buried and exposed states, which determines the steady-state level of phosphorylation of KaiC. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state, KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation, and we show evidence in support of how this movement may be accomplished.