Abstract
Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 Å. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.
Main Text
Circadian clocks are self-sustained biological oscillators that are found ubiquitously in prokaryotic and eukaryotic organisms. In eukaryotes these systems are complex and very sophisticated, whereas in prokaryotes the core mechanism is regulated by a posttranslational oscillator that can be reconstituted in vitro with three proteins (kaiA, kaiB, and kaiC)1 and ATP. Seminal work on the KaiABC system resulted in a comprehensive understanding of its circadian clock: KaiC is the central component that auto-phosphorylates through binding of KaiA and auto-dephosphorylates upon association with KaiB2-5. The interplay between these three proteins has been shown in vitro to constitute a true circadian oscillator characterized by persistence, resetting, and temperature compensation. Consequently, the KaiABC system is considered an elegant and the simplest implementation of a circadian rhythm. The evolutionary history of kai genes established kaiC as the oldest member dating back ∼3.5 bya, with subsequent additions of kaiB and most recently kaiA to form the extant kaiBC and kaiABC clusters, respectively6,7. Interestingly, a few studies of more primitive organisms that lack kaiA hinted that the kaiBC-based systems might provide already a basic, hourglass-like timekeeping mechanism8-10. Contrary to the self-sustained oscillators found in cyanobacteria, such a timer requires an environmental cue to drive the clock and for the daily “flip” of the hourglass. The central role of circadian rhythms in many biological processes, controlled by the day and night cycle on earth, makes their evolution a fascinating topic.
Here, we investigate such a primitive circadian clock by biochemical and structural studies of the KaiBC system of the purple, non-sulfur photosynthetic proteobacterium Rhodobacter sphaeroides KD131 (RS; hereafter referred to as KaiBRS and KaiCRS). Surprisingly, the organism shows sustained rhythms of gene expression in vivo, but whether kaiBC is responsible for this observation remains inconclusive in the absence of a kaiC knockout11. A more recent study of the closely-related Rhodopseudomonas palustris demonstrated causality between the proto-circadian rhythm of nitrogen fixation and expression of the kaiC gene using a KO strain10. We discover using in vitro experiments that KaiBCRS is indeed a primordial circadian clock with a mechanism that is different from the widely studied circadian oscillator in Synechococcus elongatus PCC 7942 (hereafter referred to as KaiABCSE)2-5. We identify an environmental cue that regulates the phosphorylation state and consequently produces a 24-hour clock in vivo as the switch in ATP-to-ADP ratio between day and night. Our kinetic results combined with X-ray and cryo-EM structures of the relevant states unravel a long-range allosteric pathway that is crucial for function of the hourglass and sheds light on the evolution of self-sustained oscillators. Notably, we find a novel protein fold for KaiCRS and uncover a register shift in the coiled-coil domain spanning ∼115 Å as the key regulator in this system, which shows intriguing structural similarities to dynein signaling12.
C-terminal tail as a primitive regulatory moiety
To gain insight into the evolution of the kaiBC cluster, we constructed a phylogenetic tree of kaiC after the emergence of the kaiB gene (Fig. 1a, Extended Data Fig. 1a). The first obvious question is how KaiCRS and other members in the clade can auto-phosphorylate despite having no KaiA, which is known to be crucial for this function in the canonical KaiABC system at its optimum temperature. We observe a large clade that exhibits a C-terminal tail about 50 amino acids longer compared to kaiC in other clades (Extended Data Fig. 1b). This C-terminal extension near the A-loop is predominantly found in the kaiC2 subgroup, which was previously annotated as having two serine phosphorylation sites instead of the Thr/Ser pair found in kaiC1 and kaiC3 subgroups (Extended Data Fig. 1b)13-15. In Synechococcus elongatus, the binding of KaiASE to the A-loop of KaiCSE tethers them in an exposed conformation16 that activates both auto-phosphorylation and nucleotide exchange17. Given the proximity of the extended C-terminal tail to the A-loop we conjectured that it could serve as the “primitive” regulatory moiety made redundant concomitantly with the appearance of KaiA.
To test our hypothesis, we first measured the auto-phosphorylation and nucleotide exchange rates in KaiCRS that both depend heavily on the presence of KaiA in the KaiABCSE system. We observe an auto-phosphorylation rate for KaiCRS that is ∼16-fold higher than for KaiCSE activated by KaiASE (6.5 ± 1.0 h-1 versus 0.40 ± 0.02 h-1, respectively; Fig. 1b and Extended Data Figs. 2a-e). Similarly, the nucleotide exchange is faster in KaiCRS compared to KaiCSE even in the presence of KaiASE (18.0 ± 1.5 h-1 compared to 4.7 ± 0.3 h-1, respectively; Fig. 1c and Extended Data Fig. 2f). Our data clearly show that KaiCRS can perform both auto-phosphorylation and nucleotide exchange on its own and, in fact, does so faster than its more recently evolved counterparts.
Coiled-coil interaction assembles KaiCRS dodecamer
To assess mechanistically how KaiC in the kaiA-lacking systems accomplishes auto-phosphorylation we turned to structural biology. The crystal structure of KaiCRS, unlike KaiC from cyanobacteria, reveals a homododecamer consisting of two homohexameric domains joined by a twelve-helical coiled-coil domain that is formed by the extended C-terminal tail (PDB 8dba; Fig. 1d and Extended Data Table 1). A closer inspection of the CII domains in KaiCRS and KaiCSE/TE showed an obvious difference in A-loop orientations: an extended conformation in KaiCRS versus a buried orientation in KaiCSE/TE (Fig. 1e). The existence of such an extended conformation upon binding of KaiA was conjectured earlier based on the perceived hyper- and hypo-phosphorylation upon removing the A-loop or disrupting KaiA binding, respectively18. Importantly, a recent cryo-EM structure of the nighttime phosphomimetic KaiCSE S431E/T432A in its compressed state was solved and directly showed a disordered A-loop that no longer interacts with the 422-loop19, similar to the extended A-loop conformation we observe in KaiCRS (Fig. 1e). The loss of interaction between the A-loop and 422-loop (just 10 residues apart from the phosphorylation sites), results in closer proximity between the hydroxyl group of Ser431/Thr432 and the Ψ-phosphate of ATP, thereby, facilitating the phosphoryl-transfer step20. Furthermore, the sequence similarity between KaiCRS and KaiCSE is less than 30% for the A-loop and residues considered important for stabilization of this loop in its buried orientation (i.e., 422-loop and residues 438-444; Fig. 1e). Taken together, our structural and kinetic data support the idea that an exposed A-loop is key for KaiA-independent enhancement of nucleotide exchange and hence auto-phosphorylation in KaiCRS and perhaps other KaiBC-based systems.
Is the purpose of the coiled-coil domain merely to “pull up” the A-loop or does it actively participate in nucleotide exchange and auto-phosphorylation of KaiC? To further understand its role, we generated a truncation at residue Glu490 based on the phylogenetic tree and crystallographic information (KaiCRS-Δcoil; Extended Data Fig. 1b) to disrupt the coiled-coil interaction between the two hexamers. Indeed, the crystal structure of KaiCRS-Δcoil (PDB 8db3; Fig. 2a,b and Extended Data Table 1) and its size-exclusion chromatogram and analytical ultracentrifugation profile (Extended Data Fig. 3a-c) show a hexameric structure with no coiled-coil interaction. Nucleotide exchange rates in the CII domain for KaiCRS-Δcoil and wild type are comparable (19.1 ± 0.8 h-1 and 18.0 ± 1.5 h-1, respectively; Extended Data Fig. 3d) as are the phosphorylation rates (5.5 ± 0.4 h-1 and 7.4 ± 0.3 h-1 for KaiCRS-Δcoil and wild type, respectively; Extended Data Fig. 3e,f). These results indicate that the extended A-loop and not the coiled-coil interaction plays a pivotal role in nucleotide exchange and auto-phosphorylation in KaiCRS, potentially explaining auto-phosphorylation in other KaiBC-based systems lacking a coiled-coil bundle. Notably, the coiled-coil bundle provides additional hexameric stability: the KaiCRS dodecamer is stable for extended periods of time in the presence of only ADP (Extended Data Fig. 3g,h), whereas for KaiCSE oligomers are not observed under these conditions21.
Long-range allosteric network in KaiCRS
The change in phosphorylation state of KaiC has been well established to be the central feature for the circadian rhythm22,23. Interestingly, when comparing the unphosphorylated form of full-length KaiCRS (PDB 8dba) and its phosphomimetic mutant (S413E/S414E, PDB xxxx, Extended Data Fig. 4, and Extended Data Table 2) we observe two distinct coiled-coil interactions. Upon phosphorylation, the coiled-coil pairs swap partners by interacting with the other neighboring chain from the opposite hexamer resulting in a register shift that propagates ∼115 Å along the entire coiled-coil (Fig. 2c and Extended Data Fig. 5). In the phosphomimetic state, the register comprises bulkier hydrophobic residues thus resulting in a more stable interaction than for the dephosphorylated form (Fig. 2d and Extended Data Fig. 3g). Furthermore, the C-terminal residues of KaiCRS-S413E/S414E interact with the CII domain of the opposite hexamer, whereas the lack of electron density for the last 30 residues in the wild-type structure indicates more flexibility in the dephosphorylated state. Importantly, we discover that these conformational changes in the coiled-coil domain seem to be coupled through a long-range allosteric network to the phosphorylation sites. The rotameric states of residues Ser413, Ser414, Trp419, Val421, Tyr436, Leu438, Val449, and Arg450 move concertedly and point towards the nucleotide-binding site when the protein is phosphorylated or away in the absence of a phosphate group (Fig. 2e, Extended Data Fig. 5d). We hypothesize that the proximity of the nucleotide to the phosphorylated residue allows for a more efficient phosphoryl transfer and, therefore, determined experimentally the impact of the coiled-coil domain on the auto-dephosphorylation of KaiCRS. Indeed, we observe a noticeable effect: wild-type protein dephosphorylates comparatively quickly (kobs = 11.5 ± 0.8 h-1) in the presence of only ADP, whereas little dephosphorylation was observed for KaiCRS-Δcoil (Fig. 2f and Extended Data Fig. 3i) where the allosteric propagation is disrupted (Extended Data Fig. 5d). Consistent with this accelerated dephosphorylation due to the coiled-coil domain, our crystallographic data show a phosphate-group on Ser414 for KaiCRS-Δcoil but not for the wild-type protein (Fig. 2a and Extended Data Fig. 5d).
ATP-to-ADP ratio to reset the clock
The surprising result here is that KaiCRS can auto-dephosphorylate on its own despite being constitutively active for phosphorylation due to its extended A-loop conformation. In the canonical kaiABC system, the interaction between KaiB and KaiC is required to provide a new binding interface which sequesters KaiA from its “activating” binding site, thereby promoting auto-dephosphorylation at the organism’s optimum temperature24-26. The obvious next questions are whether the KaiCRS system can oscillate and secondly if there is a regulatory role for KaiBRS in this process. Comparing the in vitro phosphorylation states of KaiCRS in the absence and presence of KaiBRS shows an initial, fast phosphorylation followed by an oscillatory-like pattern in the presence of KaiBRS (hereafter referred to as KaiBCRS), whereas KaiCRS alone remains phosphorylated (Fig. 3a,b). Interestingly, the ATP consumption during the reaction with KaiBRS is significantly higher than without (Fig. 3a) and we have seen earlier that KaiCRS will dephosphorylate completely in the presence of only ADP (cf. Fig. 2f). These results suggest that the phosphorylation state of KaiCRS and thus the observed oscillatory half-cycle (Fig. 3a,b), is likely related to a change in ATP-to-ADP ratio and we conjectured this could constitute the environmental cue to reset the timer. To test our hypothesis, an ATP-recycling system was added after complete dephosphorylation of KaiBCRS and, as predicted, KaiCRS was able to restart the cycle and phosphorylate again (Extended Data Fig. 6a). Naturally, in vivo the ATP-to-ADP ratio will not vary as drastically as in this in vitro experiment since nucleotide homeostasis is tightly regulated. To mimic the day- and night-period for Rhodobacter sphaeroides we repeated the experiments while keeping the ATP-to-ADP ratio constant (mostly ATP at daytime due to photosynthesis versus 25:75% ATP:ADP during nighttime, respectively)27. In the presence of high ATP (i.e., mimicking daytime) KaiCRS remains singly or doubly phosphorylated (Fig. 3c and Extended Data Fig. 6b) irrespective of KaiBRS, whereas a constant 25:75% ATP-to-ADP ratio (i.e., mimicking nighttime) results in a much higher fraction of dephosphorylated KaiCRS in the presence of KaiBRS (Fig. 3c). Moreover, when the ATP-to-ADP ratio is flipped to mimic the daytime, KaiCRS is able to phosphorylate again (cf. Fig. 3c; around the 28-hour mark). Our data support the notion that the phosphorylation behavior depends strongly on the ATP-to-ADP ratio and, more importantly, demonstrate that the physical binding of KaiBRS results in a higher level of KaiCRS dephosphorylation at nighttime.
Next, we investigated the accelerated ATPase activity in KaiCRS upon complex formation. The ATPase activity reported for KaiCSE is very low (∼15 ATP molecules day-1 molecule-1 KaiCSE) and was proposed as a reason for the “slowness”28. KaiCRS alone already shows a significantly faster ATPase rate that gets further enhanced by binding of KaiBRS (208 ± 19 and 1,557 ± 172 ATP molecules day-1 KaiCRS-1, respectively; left two bars in Fig. 3d and Extended Data Fig. 6c-g). Furthermore, KaiCRS exhibits no temperature compensation for its ATPase activity (Q10 ∼1.9; Extended Data Fig. 6c), a feature that is present in KaiCSE and proposed to be a prerequisite for self-sustained rhythms28. The deviation from unity for Q10 is consistent with our earlier observation that the KaiBCRS system is not a true circadian oscillator but rather an hourglass-timer (cf. Fig. 3b).
Regulatory role of KaiBRS
The mechanistic details of how KaiB binding in the CI domain allosterically affects the auto-dephosphorylation of KaiCRS in the CII domain remain unclear. Intuitively, there are three plausible scenarios to explain this, namely that KaiBRS binding (i) stimulates the phosphoryl-transfer from pSer back to ADP (Extended Data Fig. 7a), (ii) increases the hydrolysis rate of the active-site ATP (Extended Data Fig. 8a), or (iii) accelerates the nucleotide exchange in the CII domain (Extended Data Fig. 8e). To differentiate between these possibilities, we performed radioactivity experiments to follow nucleotide interconversion, measured ATPase activity for wild-type KaiCRS and mutant forms that are incapable of ATPase activity in the CI or CII domain, and quantified nucleotide-exchange rates by fluorescence using mant-ATP. First, we detected fast, transient 32P-ATP formation in our radioactivity experiments when starting from 32P-phosphorylated KaiCRS due to its ATP-synthase activity in the CII domain (Fig. 3e and Extended Data Fig. 7b-d). The observed phosphoryl-transfer rate is independent of KaiBRS (kobs = 12.0 ± 1.7 h-1 and 15.4 ± 1.7 h-1 in its absence and presence, respectively; Fig. 3f) and agrees well with the rates determined from our gel electrophoresis experiments (11.0 ± 0.8 h-1 and 11.5 ± 0.8 h-1 with/without KaiBRS, respectively; Extended Data Fig. 7e,f). Our experimental data confirm that KaiCRS undergoes dephosphorylation via an ATP-synthase mechanism similarly to what was observed for KaiCSE29; KaiB does not expedite the actual phosphoryl-transfer reaction, which is never the rate-limiting step. Since we were unable to stabilize the first phosphorylation site (Ser414) in the presence of ADP, the rates reported here correspond exclusively to dephosphorylation of Ser413. Secondly, to deconvolute the contributions of the CI and CII domains to the observed ATPase activity, we measured ADP production by KaiCRS mutants that abolish hydrolysis in either the CI (KaiCRS-E62Q/E63Q) or CII (KaiCRS-E302Q/E303Q) domain. For wild-type KaiCRS, the binding of KaiBRS results in a 7.5-fold increase in ATPase activity, and we show that both domains are affected and contribute additively (3-fold for CI and at least 1.7-fold for CII, respectively) to the overall effect (Fig. 3d and Extended Data Fig. 8b-d). Of note, the fold increase in the CII domain represents a lower limit since the KaiCRS-E62Q/E63Q mutations interfere with KaiBRS binding as reported before for KaiCSE30. Thirdly, our measurements of the nucleotide exchange rate show that it is also unaffected by KaiBRS binding (19.8 ± 1.8 h-1 and 18.0 ± 1.5 h-1 with/without, respectively; Fig. 3g); since there is no Trp residue near the nucleotide binding site in the CI domain, only the exchange rate in the CII domain could be determined. Interestingly, the change in fluorescence amplitude is much smaller in the presence of KaiBRS, demonstrating that even though the binding of KaiBRS does not accelerate nucleotide exchange, it appears to induce a conformational rearrangement in the CII domain especially at higher temperature (Fig. 3g and Extended Data Fig. 8f-h).
Structure of KaiBCRS complex
To elucidate the structural underpinning of the faster ATPase activity upon KaiBRS binding, we solved the cryo-EM structures of KaiCRS alone (PDB xxxx) and in complex with KaiBRS (PDB xxxx) (Extended Data Table 2). Twelve KaiBRS molecules (monomeric in solution, Extended Data Fig. 9a) bind to the CI domain of the KaiCRS-S413E/S414E dodecamer (Fig. 4a-c and Extended Data Fig. 9b). The bound state of KaiBRS adopts the same “fold-switch” conformation as observed for KaiBTE25 and suggests that this is the canonical binding-competent state (Fig. 4b). Upon binding of KaiBRS, the CI-CI interfaces loosen up (Fig. 4c), which allows for the formation of a tunnel that connects bulk solvent to the position of the hydrolytic water in the active sites (Fig. 4d and Extended Data Fig. 9c). There are other lines of evidence for the weakened interactions within the CI domains. First, KaiBRS binding to either KaiCRS-CI domain (Extended Data Fig. 10a) or KaiCRS-Δcoil (i.e., missing the C-terminal extensions; Extended Data Fig. 10b) results in disassembly of the hexameric KaiCRS structure into its monomers. In contrast, full-length KaiCRS maintains its oligomeric state upon binding of KaiBRS likely due to the stabilization provided by the coiled-coil interaction. Secondly, a decrease in melting temperature (TM) of KaiCRS is observed with increasing KaiBRS concentration (Extended Data Fig. 10c). There is no interaction between neighboring KaiBRS molecules within the complex (Extended Data Fig. 9b), suggesting a non-cooperative assembly of KaiBRS to KaiCRS contrary to what is observed for the KaiBCSE/TE complex31,32.
Furthermore, we noticed that KaiB-bound structures in phosphomimetic variants of KaiCRS (Fig. 4c,d) and KaiCSE26 have ADP bound in their CI domain, demonstrating that the post-hydrolysis state is also the binding-competent state for KaiBRS. To test this hypothesis, a His-tagged KaiBRS protein was used in pull-down assays to detect its physical interaction with wild-type and mutant forms of KaiCRS bound with either ADP or ATP. Nearly all KaiBRS is complexed to ADP-bound KaiCRS, whereas less than 30% co-elutes in the ATP-bound form regardless of the phosphorylation state (Fig. 4e and Extended Data Fig. 10d,e). The complex formation depends inversely on the ATP-to-ADP ratio (Extended Data Fig. 10f). We performed fluorescence anisotropy competition experiments for a more quantitative description of the binding interaction between KaiCRS and KaiBRS: very similar KD values were obtained for unphosphorylated, wild-type KaiCRS (Fig. 4f) and its phosphomimetic form (Extended Data Fig. 10g) bound with ADP (0.42 ± 0.03 μM and 0.79 ± 0.06 μM, respectively). No measurable binding curves were obtained for ATP-bound phosphorylated wild-type KaiCRS (Fig. 4f) or KaiCRS-S413E/S414E (Extended Data Fig. 10g) with ATP-recycling system, likely due to the small fraction of complex present. Our data show that the post-hydrolysis state in the CI domain is key for KaiBRS binding, whereas the phosphorylation state of KaiCRS has only a marginal effect.
In summary, we unequivocally demonstrate that binding of KaiBRS at the CI domain in the post-hydrolysis state facilitates the hydrolysis of transiently formed ATP after dephosphorylation of KaiCRS in the CII domain (Fig. 4g). Our fluorescence experiments (Fig. 3g and Extended Data Fig. 8f) detect a conformational change in the CII domain upon KaiBRS binding, but we do not observe major structural changes in the cryo-EM structures. Based on the temperature dependence of the fluorescence amplitudes (Extended Data Fig. 8f) we conjecture that the inability to detect conformational differences is likely because of the low temperature. Since the CII domain prefers to bind ATP over ADP (Extended Data Fig. 10h), ATP hydrolysis in the CII domain stimulated by KaiBRS is particularly important to keep KaiCRS in its dephosphorylated state at nighttime, where the exogenous ATP-to-ADP ratio remains sufficiently high to otherwise result in ATP-binding in the CII active site (cf. Fig. 3c and Extended Data Fig. 6b).
Discussion
The KaiBCRS system studied here represents a primordial, hourglass timekeeping machinery and its mechanism provides insight into more evolved circadian oscillators like KaiABC. The dodecameric KaiCRS shows constitutive kinase-activity due to its extended C-terminal tail that forms a coiled-coil bundle with the opposing hexamer and elicits a conformation akin to the exposed A-loop conformation in KaiACSE, and auto-phosphorylation occurs within half an hour. In the KaiABCSE system, the transition from unphosphorylated to doubly phosphorylated KaiC takes place over about twelve hours and the fine-tuning of this first half of the circadian rhythm is accomplished by the emergence of KaiASE during evolution. The second clock protein, KaiB, binds at the CI domain with the same “fold-switched” state in both systems. The interaction is controlled by the phosphorylation state in the KaiABCSE system, and its sole function is to sequester KaiASE from the “activating” binding site, whereas KaiB binding directly accelerates the ATPase activity in the KaiBCRS system regardless of the phosphorylation state. The KaiBCRS system requires an environmental switch in ATP-to-ADP concentration to reset the clock and thus follows the day-night schedule when nucleotide concentrations inherently fluctuate in the organism. By contrast, the self-sustained oscillator KaiABCSE remains functional over a wide range of nucleotide concentrations and responds to changes in the ATP-to-ADP ratio by changing its phosphorylation period and amplitude to remain entrained with the day and night cycle33.
The novel structural fold of KaiC utilizes the versatile coiled-coil architecture as part of a long-range allosteric network that regulates KaiCRS dephosphorylation. Nature uses conformational changes in coiled-coil domains for a variety of regulatory functions34, including the activity of the motor protein dynein in cellular transport of cargo along the actin filament12. A similar register shift, although in a coiled-coil interaction formed by only two helices is used in dynein motility. Given that this simple heptad repeat sequence emerged multiple times and is found throughout all kingdoms of life35 it is an example of convergent evolution.
Data availability
Structure factors and refined models are deposited in the Protein Data Bank (PDB) under accession codes 8dba (wild-type KaiCRS, x-ray), 8db3 (KaiCRS-Δcoil, x-ray), xxxx (KaiCRS-S413E/S414E, cryo-EM), and xxxx (KaiCRS-S413E/S414E:KaiBRS, cryo-EM), respectively. Cryo-EM maps are deposited in the Electron Microscopy Data Bank (EMDB) under accession codes xxxx (KaiCRS-S413E/S414E) and xxxx (KaiCRS-S413E/S414E:KaiBRS), respectively.
Author contributions
W.P., R.A.P.P., and D.K. conceived the project and designed experiments. W.P. performed and analyzed all biochemical data. W.P. and R.A.P.P. set up the crystal trays and R.A.P.P. collected and analyzed the X-ray crystallographic data. W.P. prepared the samples for the cryo-EM studies and collected negative stain images to screen for optimal sample conditions; T.G. collected and processed all cryo-EM data, and reconstructed the cryo-EM maps under supervision of N.G.; R.A.P.P. built and interpreted the structural models. W.P. and N.B. performed and analyzed experiments with radioactively labeled KaiC. M.H. built the KaiC phylogeny. W.P., R.O., and D.K. wrote the paper; and all authors commented on the manuscript and contributed to data interpretation.
Competing interests
D.K. is co-founder of Relay Therapeutics and MOMA Therapeutics. The remaining authors declare no competing interests.
Acknowledgements
D.K. and N.G. are supported by the Howard Hughes Medical Institute (HHMI). We would like to thank Mike Rigney for assistance with negative-stain data collection at the Brandeis University Electron Microscopy Facility, and Zhiheng Yu and the staff of the Janelia Research Campus cryo-EM facility for advice and assistance with data collection. The Berkeley Center for Structural Biology is supported in part by HHMI. Beamline 8.2.1 of the Advanced Light Source, a U.S. DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01. Mass spectral data were obtained at the University of Massachusetts Mass Spectrometry Core Facility, RRID:SCR_019063.
Footnotes
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