Robust and tunable circadian rhythms from differentially sensitive catalytic domains Connie Phonga, Joseph S. Marksonb, Crystal M. Wilhoitea, and Michael J. Rusta,1 aDepartment of Molecular Genetics and Cell Biology, Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637; and bGraduate Committee on Higher Degrees in Biophysics, Departments of Molecular and Cellular Biology and Chemistry and Chemical Biology, Harvard University Center for Systems Biology, Cambridge, MA 02138 Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 27, 2012 (received for review July 15, 2012) Circadian clocks are ubiquitous biological oscillators that coordi- reaction buffer. This manipulation simulates the metabolic nate an organism’s behavior with the daily cycling of the external changes that occur in vivo in response to a dark pulse and results environment. To ensure synchronization with the environment, in a modulation of KaiC phosphorylation (6). the period of the clock must be maintained near 24 h even as amplitude and phase are altered by input signaling. We show that, Results in a reconstituted circadian system from cyanobacteria, these con- Period of the KaiABC Oscillator Is Robust Against Changes in ATP/ADP flicting requirements are satisfied by distinct functions for two Signaling. To dissect the mechanism of the oscillator’s response fi domains of the central clock protein KaiC: the C-terminal autoki- to input signaling through the ATP/ADP ratio, we rst isolated nase domain integrates input signals through the ATP/ADP ratio, its effect on KaiC’s kinase activity by studying nonoscillatory and the slow N-terminal ATPase acts as an input-independent KaiA-KaiC reactions, where the absence of KaiB removes the timer. We find that phosphorylation in the C-terminal domain fol- negative feedback on phosphorylation that permits oscillations. Decreasing the ATP/ADP ratio caused a marked slowing of the lowed by an ATPase cycle in the N-terminal domain is required to phosphorylation rate, a phenomenon that we observed at various form the inhibitory KaiB•KaiC complexes that drive the dynamics absolute concentrations of ATP (Fig. 1B, Fig. S1A, and Table of the clock. We present a mathematical model in which this S1), while leaving the phosphatase activity unaffected (6). ATPase-mediated delay in negative feedback gives rise to a com- If the ∼24-h period of the circadian oscillator is determined by pensatory mechanism that allows a tunable phase and amplitude a balance between the KaiC kinase and phosphatase rates, then SYSTEMS BIOLOGY while ensuring a robust circadian period. inhibition of kinase activity through the ATP/ADP ratio would be expected to upset that balance and change the oscillator period. biological clock | in vitro | robustness | metabolic input processing To experimentally test this prediction, we subjected oscillating KaiA-KaiB-KaiC reactions to a sustained rather than transient ircadian clocks are endogenous oscillatory systems that allow drop in the ATP/ADP ratio and measured oscillator performance Can organism to anticipate daily rhythmic variations in the (Fig. 1C and Fig. S1 B–E). Surprisingly, we found that the core external environment. Despite having apparently diverse mo- oscillator continued to measure time robustly across a range of lecular origins, the functional properties of circadian systems are ATP/ADP ratios, encompassing metabolic conditions measured remarkably conserved across most organisms that have been in living cells under various light levels (6, 7). Rhythms persist studied. Even when deprived of a rhythmic input, circadian with a period that remains close to 24 h (within 5%), although the clocks continue to generate self-sustaining oscillations, and these attenuation of kinase activity caused by lower ATP/ADP ratios oscillations have a period that is close to 24 h over a wide range does change the amplitude of the oscillator (Fig. 1 D and E). of conditions, including varying light levels, nutrient abundance, Thus, the KaiABC protein oscillator is intrinsically capable and temperature (1). The robust period of the oscillator seems of robustly adapting to input signaling to maintain circadian to be critical for its physiological role: in multiple organisms, rhythmicity. The robustness of the period in the face of pertur- mutants with clocks whose free-running periods are markedly bations to kinase activity suggests that there may be slow steps in different from 24 h are associated with decreased lifespan and the oscillator dynamics that are critical for appropriate timing fitness defects (2–4). In contrast, the phase and amplitude of but that do not involve phosphorylation. To identify these steps circadian rhythms are generally tunable, and input signals can and elucidate the mechanistic basis of how the circadian oscil- reset the oscillator to efficiently bring the clock into synchrony lator maintains a robust period, we undertook a combined ex- with a rhythmic environment. perimental and mathematical modeling study of the ability of the The competing demands for both a sensitive response to input biochemical circuit driving the KaiABC in vitro oscillator to signaling and a robustly invariant oscillator period place strong process nucleotide input signals. constraints on the possible mechanisms underlying the circadian clock. It is currently unclear how the biochemical circuitry that N-Terminal Domain ATPase Activity Is Required for KaiB•KaiC Complex generates circadian rhythms satisfies these constraints in any Assembly and Negative Feedback. KaiC, the only enzyme in the core organism. To study the molecular origins of robust periodicity, oscillator, consists of two homologous catalytic domains that are we analyzed the biochemically tractable circadian oscillator from members of the RecA/DnaB superfamily of P-loop ATPases. Both the cyanobacterium Synechococcus elongatus. domains have conserved nucleotide binding motifs, and thus, each The Synechococcus core oscillator can be reconstituted in vitro is a potential target for the ATP/ADP signals that can reset the using three purified proteins, KaiA, KaiB, and KaiC, and ATP oscillator (Fig. 2A). The N-terminal domain (CI) binds ATP with (Fig. 1A). KaiC is a multifunctional enzyme with slow ATPase, high affinity and plays an important structural role in assembling autokinase, and autophosphatase activities. The effector proteins KaiA and KaiB work together to modulate KaiC’s enzymatic activity in a phosphorylation-dependent manner, switching the system between kinase- and phosphatase-dominated modes. The Author contributions: C.P., J.S.M., and M.J.R. designed research; C.P. and C.M.W. per- fi formed research; C.P. and M.J.R. analyzed data; and C.P. and M.J.R. wrote the paper. puri ed oscillator generates coherent, self-sustaining rhythms in fl the phosphorylation state of KaiC and exhibits many of the The authors declare no con ict of interest. conserved properties of the in vivo circadian clock (5). The This article is a PNAS Direct Submission. KaiABC in vitro oscillator is also capable of processing input 1To whom correspondence should be addressed. E-mail: [email protected]. signals, and the phase of the oscillation can be reset by tran- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. siently altering the ratio of ATP to ADP nucleotides in the 1073/pnas.1212113110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212113110 PNAS Early Edition | 1of6 A KaiC monomer hexamer CII CI U-KaiCU-KaiC (top(top view) KaiAKaiA SS-KaiC-KaiC TT-KaiC-KaiC P P P P Input: ATP/ADPTP/ADP KaKaiBiB SST-KaiCT-KaiC P P P P Fig. 1. The period of the circadian oscillator is robust BCto varying input signals. (A)SchematicoftheKaiABC oscillator. A purified posttranslational protein circuit generates circadian rhythms in KaiC phosphorylation in the presence of ATP. Active KaiA promotes auto- phosphorylation on initially unphosphorylated KaiC (U-KaiC) first on Thr432 and then, on Ser431. Ser431- phosphorylated KaiC (S-KaiC) forms KaiB•KaiC com- plexes that inhibit KaiA and promote system-wide dephosphorylation, driving stable oscillations. The phase of the oscillation can be shifted by metabolic signaling through the ATP/ADP ratio. (B)KaiCauto- phosphorylation in KaiA-KaiC reactions at various ATP/ADP conditions (colored symbols); all buffers have 10 mM total nucleotide (similar data previously DEpublished in ref. 6). (C) KaiABC in vitro reactions in buffers at various ATP/ADP conditions; 10 mM total nucleotide. Oscillations with a robust period persist under the conditions tested. (D) Period of the oscil- lations shown in C determined by fitting the data after the first trough to a sinusoidal function. Error bars indicate the SE of the least squares fit. Dashed lines indicate linear regressions of the data to show trend. (E) Peak and trough heights of the oscillations shown in C determined by averaging the peaks and troughs after the first trough. Error bars indicate SDs. Dashed lines indicate linear regressions of the data to show trend. hexameric KaiC particles (8). Both domains also have conserved We, therefore, sought to define separate roles for catalysis in putative catalytic glutamates. The C-terminal domain (CII) has each domain by selectively mutating the conserved catalytic res- both kinase and phosphatase activities that are modulated by idues. We found that, in a mutant where the conserved catalytic KaiA (9, 10). Both