Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.

Significance The Kai system is a widely studied model in theoretical biology and systems biology. It is to date the only known circadian clock that can be reconstituted in vitro. Essential to the rhythmicity of the system is the formation of the KaiC–KaiB complex. Many aspects of this interaction, such as the mode of binding of KaiB, the stoichiometry of the interaction, and the exact binding interfaces have long remained ambiguous. We present a mass spectrometry-based structural model of the KaiC–KaiB interaction that answers many of these outstanding questions on the basis of direct experimental evidence. This structural model sheds light on the intricate workings of the in vitro oscillator.

BackgroundCircadian clocks are found in organisms of almost all domains including photosynthetic Cyanobacteria, whereby large diversity exists within the protein components involved. In the model cyanobacterium Synechococcus elongatus PCC 7942 circadian rhythms are driven by a unique KaiABC protein clock, which is embedded in a network of input and output factors. Homologous proteins to the KaiABC clock have been observed in Bacteria and Archaea, where evidence for circadian behavior in these domains is accumulating. However, interaction and function of non-cyanobacterial Kai-proteins as well as homologous input and output components remain mainly unclear.ResultsUsing a universal BLAST analyses, we identified putative KaiC-based timing systems in organisms outside as well as variations within Cyanobacteria. A systematic analyses of publicly available microarray data elucidated interesting variations in circadian gene expression between different cyanobacterial strains, which might be correlated to the diversity of genome encoded clock components. Based on statistical analyses of co-occurrences of the clock components homologous to Synechococcus elongatus PCC 7942, we propose putative networks of reduced and fully functional clock systems. Further, we studied KaiC sequence conservation to determine functionally important regions of diverged KaiC homologs. Biochemical characterization of exemplary cyanobacterial KaiC proteins as well as homologs from two thermophilic Archaea demonstrated that kinase activity is always present. However, a KaiA-mediated phosphorylation is only detectable in KaiC1 orthologs.ConclusionOur analysis of 11,264 genomes clearly demonstrates that components of the Synechococcus elongatus PCC 7942 circadian clock are present in Bacteria and Archaea. However, all components are less abundant in other organisms than Cyanobacteria and KaiA, Pex, LdpA, and CdpA are only present in the latter. Thus, only reduced KaiBC-based or even simpler, solely KaiC-based timing systems might exist outside of the cyanobacterial phylum, which might be capable of driving diurnal oscillations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0999-7) contains supplementary material, which is available to authorized users.