Mechanism and quantitation of cooperative interactions in the cyanobacterial circadian oscillator

Cyanobacteria are photosynthetic microbes that have shaped the very environment of the world we live in over several billion of years. This is, in large part, because of their ability to perform the chemically exclusive processes of photosynthesis and nitrogen fixation. To segregate these processes temporally, they have evolved an elegant and complex circadian clock that aligns their physiology with the solar day to maximize biological fitness. This clock keeps time through the action of a biochemical oscillator comprising just three proteins: KaiA, KaiB and KaiC, that, along with ATP, can recapitulate a 24-h pacemaking activity in vitro. This process is achieved by a negative feedback loop akin to a biochemical game of Rock, Paper, Scissors, whereby the phosphorylation state of the hexameric ATPase KaiC is controlled by the nucleotide exchange factor KaiA, the ability of KaiA to stimulate KaiC repression is controlled by the metamorphic protein KaiB, and the ability of KaiB to inactivate KaiA is controlled by KaiC phosphorylation state. This creates a repetitive biochemical cycle that takes just bout 24 hours to complete. Additionally, the output proteins SasA and CikA interact with the clock in various phases of the biochemical oscillation that influence their ability to activate the master circadian transcription factor RpaA and orchestrate circdain gene expression throughout the cyanobacterial cell.This thesis focuses on the relationship between KaiC and KaiB. In particular, on positive cooperativity that increases KaiB’s affinity for KaiC, or in other words, the process by which initial binding of KaiB to the KaiC hexamer enables more efficient recruitment of KaiB to the remaining 5 binding sites. While this effect is well documented when considering KaiB and KaiC on their own, in Chapter 2 we expand this concept in the in the context of the entire reconstituted clock system including output pathways that link biochemical oscillation to DNA binding by the transcription factor RpaA. In doing so, we uncovered the unexpected result that SasA expands the range of permissive KaiB concentrations for biochemical oscillation to occur. I showed that SasA does this by binding to KaiC analogously to how KaiB does, and then recruiting additional KaiB molecules through positive heterotropic cooperativity. Integrating a novel crystal structure of the interacting domains of KaiC and SasA with existing crystal structures of the KaiC hexamer, I identified SasA mutations that abrogate heterotropic cooperativity but have only minor effects on the output signaling function of SasA. Remarkably, cyanobacteria bearing these mutations have defective circadian rhythms, demonstrating that SasA’s ability to bolster KaiB recruitment is an evolved aspect of biochemical oscillation.Chapter 3 describes our structural analysis of phosphomimetic variants of KaiC that differ in their ability to bind KaiB. Because crystallography has failed to identify the structural basis of this discrimination previously, we employed cryo-electron microscopy to analyze KaiC particles as a structural ensemble frozen in ice. Each KaiC protomer is composed of two ATPase domains, termed CI and CII. The phase-determining phosphosites are located on CII, and KaiB binds to KaiC over 70 Å away to the ADP-bound form of CI. Our data corroborated previous studies that daytime KaiC, which does not bind KaiB, loses interactions amongst the CII domain protomers within the hexamer, while maintaining a hexameric CI domain. Additionally, we obtained a relatively high resolution (3.2 Å) structure of a compressed form of KaiC where the CII domain breaks into a split washer with 2-fold symmetry, causing two of the CII domain subunits to interact more tightly with their respective CI domains. Using mutagenesis and various functional assays, I identified an allosteric conduit that connects the ATPase domains of the CI and CII domains of KaiC in both intra-protomer and inter-protomer contexts. Importantly, I trace these interactions back to cooperativity in KaiB association, with mutants along the pathway disrupting both KaiB affinity and cooperativity. Furthermore, I link KaiB cooperativity to ATP hydrolysis in the CI domain by identifying a key residue that senses CI nucleotide state. This residue is dispensable for both ATP hydrolysis as well as KaiB association, but is critical for both KaiB cooperativity and in vivo circadian rhythms. This, along with additional nucleotide dependence studies reported in Chapter 4, suggests that allosteric control of cooperativity through the CI active site is the structural basis for restriction of KaiB association to the nighttime KaiC phosphostates.Finally, in the latter half of Chapter 4 I summarize experiments that used the solvatocromatic dye Sypro Orange to detect changes in KaiC structure as a function of temperature and mutagenesis. I describe our preliminary efforts to build on these results by identifying additional solvatochromatic dyes that can leverage this effect to report continuously on the phase of biochemical oscillation by discriminate binding to different KaiC phosphostates at constant temperature