Structure, function, and mechanism of the core circadian clock in cyanobacteria Jeffrey A. Swan1, Susan S. Golden2,3, Andy LiWang3,4, Carrie L. Partch1,3* 1Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064 2Department of Molecular Biology, University of California San Diego, La Jolla, CA 92093 3Center for Circadian Biology and Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093 4Chemistry and Chemical Biology, University of California Merced, Merced, CA 95343 *Correspondence to: [email protected] Running title: Structural review of the cyanobacterial clock Keywords: structural biology, circadian rhythm, circadian clock, cyanobacteria, protein dynamic, protein-protein interaction, post-translational modification, post-translational-oscillator, KaiABC Abbreviations: TTFL, transcription-translation feedback loop; PTO, post-translational oscillator; ATP, adenosine triphosphate; ADP, adenosine diphosphate; PsR, pseudo-receiver; HK, histidine kinase; RR, response regulator ______________________________________________________________________________ Abstract intertwined functions: timekeeping, entrainment Circadian rhythms enable cells and and output signaling. organisms to coordinate their physiology with the Timekeeping is achieved through a cycle of cyclic environmental changes that come as a result slow biochemical processes that together set the of Earth’s light/dark cycles. Cyanobacteria make period of oscillation close to 24 hours. For this use of a post-translational oscillator (PTO) to temporal information to be relevant, biological maintain circadian rhythms, and this elegant system clocks must appropriately synchronize with their has become an important model for the post- environment through entrainment. Temporal translational timing mechanisms. Comprised of information runs from the timekeeping apparatus to three proteins, the KaiABC system undergoes an the rest of the cell through output signaling oscillatory biochemical cycle that provides timing mechanisms that impart circadian changes in cues to achieve a 24-hour molecular clock. physiology. Together with the input/output proteins SasA, CikA Timekeeping mechanisms that have been and RpaA, these six gene products account for the observed in circadian clocks are broadly timekeeping, entrainment and output signaling categorized as transcription-translation feedback functions in cyanobacterial circadian rhythms. This loops (TTFLs) or post-translational oscillators review summarizes the current structural, (PTOs) (2). TTFLs achieve oscillation via delayed functional and mechanistic insights into the negative feedback, where a gene product represses cyanobacterial circadian clock. its own expression when its levels become high enough. This results in an oscillation in the Form and Function of Circadian Clocks expression level of the gene, with transitions Circadian rhythms are processes through between repression and derepression occurring at which cells and organisms predict and adapt to the critical points in the oscillatory cycle. By contrast, repetitive environmental changes incident to the 24- PTOs utilize timekeeping mechanisms that are hour solar day. These rhythms originate independent of transcription. Like TTFLs, PTOs intracellularly and persist in the absence of external involve a cycle of biochemical processes; but cues to orchestrate temporal organization of instead of being driven by changes in expression physiology (1). To achieve this, circadian clocks levels, their behavior is controlled by post- must perform three distinct but necessarily translational modifications, conformational Structural review of the cyanobacterial clock changes, protein-protein interactions and/or sustained PTO. In vivo, this PTO also functions subcellular localization. It is important to note that within a TTFL framework; i.e. circadian variation while we make a distinction here between TTFLs of clock protein expression resulting in a more and PTOs as general categories of negative robust rhythm (4,12-14). Together with three other feedback systems, many circadian clocks make use effector proteins, the KaiABC oscillator comprises of both transcription/translation as well post- a self-contained minimal system that is capable of translational timing steps to constitute a integrating timekeeping, entrainment and output biochemical oscillator. signaling processes into the cyanobacterial cell. Post-translational steps in circadian clocks Domain structures of the six core clock present unique biochemical challenges and proteins in cyanobacteria are shown in Figure 1. opportunities because they link dynamic molecular Genetic studies on cyanobacterial rhythms have processes to biological phenotype through covalent made extensive use of the mesophilic and conformational changes in protein structure. cyanobacterium Synechococcus elongatus, from PTOs can operate at constant protein which these genes were originally identified (10). concentrations, and are therefore amenable to in Biophysical studies have often taken advantage of vitro study. In the cyanobacterial Kai (Japanese, 回 proteins produced from homologous gene , “cycle”) system, an entire PTO consisting of only sequences in the thermophilic variant three protein gene products (KaiA, KaiB and KaiC; Thermosynechococcus elongatus for their or KaiABC) can reconstitute circadian biochemical improved solubility and in vitro behavior (15). The oscillation in vitro (3). As a result, this system has six proteins shown in Figure 1 are conserved become an important model for understanding how between S. and T. elongatus, and appear to discrete biochemical steps are integrated to give comprise a core unit capable of accounting for all rise to precise biological timing. of the required functions of a circadian clock. Several additional genes have been identified that Circadian Biology in Cyanobacteria play roles in the circadian rhythms of cyanobacteria Circadian rhythms are essentially (16-21), however this review will focus on the six ubiquitous in eukaryotes, but far fewer instances best-characterized gene products and the have been documented in prokaryotes (4). mechanisms through which they mediate Cyanobacteria temporally segregate photosynthesis timekeeping, entrainment and output signaling and nitrogen fixation, which require mutually functions in cyanobacterial circadian rhythms. For exclusive oxidative environments (5). Evolutionary a broader review on circadian biology in pressures such as this resulted in a cyanobacterial cyanobacteria, see references (4,22). transcriptome in which at least one third of transcripts undergo circadian variation in Structure and Function of KaiC accumulation levels (6) and active transcription of The core cyanobacterial clock is centered nearly all transcripts appears to be under circadian around the hexameric ATPase KaiC, which control (7). These changes in transcriptional output achieves timekeeping through circadian oscillations are linked to global changes in chromosome in phosphorylation that are intertwined with ultrastructure (8,9), but are controlled basally by a oscillations between the quaternary structures core biochemical oscillator (3,10). depicted in Figure 2. The primary sequence of KaiC The KaiABC gene cluster was originally is a concatenation of two P-loop ATPase domains identified in genetic screens aimed at finding (10), termed CI and CII. In solution, KaiC binds TTFLs involved in cyanobacterial circadian adenosine triphosphate (ATP) to form a hexamer regulation, with deletion resulting in an arrhythmic through interactions at the CI subunit interfaces phenotype (10). It was subsequently shown that the (23-25). Homologous ATPase active sites are products of this gene cluster can recapitulate present at the subunit interfaces of both CI and CII, circadian biochemical oscillation in vitro (3), and however, these active sites catalyze distinct that this circadian oscillation persists in vivo even chemical transformations. CI transfers phosphate when transcription and translation are inhibited from ATP to water, and CII catalyzes transfer of (11). Its ability to be reconstituted in vitro phosphate between ATP and the sidechain hydroxyl demonstrates that KaiABC can act as a self- groups of residues S431 and T432 (26,27). 2 Structural review of the cyanobacterial clock -4 -1 -1 Phosphorylation at these sites oscillates circadianly kcat/Km on the order of 10 M s , about six orders in concert with interactions between KaiC and the of magnitude slower than the structurally similar F1 other core clock proteins KaiA and KaiB (28,29). ATPase (38). High-resolution structures have been These interactions are regulated by the obtained for the isolated CI ring in the pre-ATP and phosphorylation state of KaiC, and take place on post-ATP hydrolysis states (38) resulting in the non-homologous regions of the CI and CII three specific structural changes depicted in Figure domains, known as the respective B and A-loops, 3A (38). Together with studies showing that where they in turn regulate the enzymatic activities catalytically dead KaiC is unable to interact with on KaiC in a negative feedback loop. KaiB (26), one emerging model is that CI ATPase During the day, KaiA is bound to the A- functions in timing the recruitment of KaiB and loops on CII where it promotes resultant formation of the nighttime repressive autophosphorylation (30-32). In the evening, KaiB complex, though no specific structural or binds to phosphorylated