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Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock*

  • Martin Egli
    Correspondence
    To whom correspondence should be addressed. Tel.: 615-343-8070
    Affiliations
    Department of Biochemistry and Center for Structural Biology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-0146
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM073845 (to M. E.).
Open AccessPublished:June 16, 2014DOI:https://doi.org/10.1074/jbc.R114.579607
      The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism.

      Introduction

      The circadian clock in the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus consists of a three-protein biochemical oscillator that is coupled to a transcription-translation feedback loop (TTFL)
      The abbreviations used are: TTFL
      transcription translation feedback loop
      PTO
      post-translational oscillator
      ESP
      electrostatic surface potential
      HDX
      hydrogen-deuterium exchange
      SAXS
      small angle x-ray scattering
      p
      phosphorylated form.
      (
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      The cyanobacterial circadian system: from biophysics to bioevolution.
      ,
      • Mackey S.R.
      • Golden S.S.
      • Ditty J.L.
      The itty-bitty time machine genetics of the cyanobacterial circadian clock.
      ,
      • Egli M.
      • Johnson C.H.
      A circadian clock nanomachine that runs without transcription or translation.
      ,
      • Johnson C.H.
      • Egli M.
      Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.
      ). The KaiA, KaiB, and KaiC proteins in the presence of ATP reconstitute a temperature-compensated timer in vitro (
      • Tomita J.
      • Nakajima M.
      • Kondo T.
      • Iwasaki H.
      No transcription-translation feedback in circadian rhythm of KaiC phosphorylation.
      ,
      • Nakajima M.
      • Imai K.
      • Ito H.
      • Nishiwaki T.
      • Murayama Y.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.
      ,
      • Murakami R.
      • Mutoh R.
      • Iwase R.
      • Furukawa Y.
      • Imada K.
      • Onai K.
      • Morishita M.
      • Yasui S.
      • Ishii K.
      • Valencia Swain J.O.
      • Uzumaki T.
      • Namba K.
      • Ishiura M.
      The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.
      ) and function as a post-translational oscillator (PTO) in vivo, whereby the latter constitutes the master clock (
      • Qin X.
      • Byrne M.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      Coupling of a core post-translational pacemaker to a slave transcription/translation feedback loop in a circadian system.
      ). The cyanobacterial clock contradicts the widely held belief from studies of eukaryotic cells that the core mechanism of the circadian clock is based on a TTFL (
      • Hardin P.E.
      • Hall J.C.
      • Rosbash M.
      Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels.
      ,
      • Aronson B.D.
      • Johnson K.A.
      • Loros J.J.
      • Dunlap J.C.
      Negative feedback defining a circadian clock: autoregulation of the clock gene frequency.
      ).
      KaiC is the only enzyme among the three proteins and exhibits rhythmic phosphorylation and dephosphorylation with an ∼24-h period (
      • Tomita J.
      • Nakajima M.
      • Kondo T.
      • Iwasaki H.
      No transcription-translation feedback in circadian rhythm of KaiC phosphorylation.
      ,
      • Nakajima M.
      • Imai K.
      • Ito H.
      • Nishiwaki T.
      • Murayama Y.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.
      ,
      • Murakami R.
      • Mutoh R.
      • Iwase R.
      • Furukawa Y.
      • Imada K.
      • Onai K.
      • Morishita M.
      • Yasui S.
      • Ishii K.
      • Valencia Swain J.O.
      • Uzumaki T.
      • Namba K.
      • Ishiura M.
      The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.
      ). Of the two phosphorylation sites observed in KaiC (Thr-432 and Ser-431 (
      • Xu Y.
      • Mori T.
      • Pattanayek R.
      • Pattanayek S.
      • Egli M.
      • Johnson C.H.
      Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.
      ,
      • Nishiwaki T.
      • Satomi Y.
      • Nakajima M.
      • Lee C.
      • Kiyohara R.
      • Kageyama H.
      • Kitayama Y.
      • Temamoto M.
      • Yamaguchi A.
      • Hijikata A.
      • Go M.
      • Iwasaki H.
      • Takao T.
      • Kondo T.
      Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.
      )), Thr-432 becomes phosphorylated first followed by Ser-431, and dephosphorylation then proceeds in the same order: TS → pTS → pTpS → TpS → TS (
      • Nishiwaki T.
      • Satomi Y.
      • Kitayama Y.
      • Terauchi K.
      • Kiyohara R.
      • Takao T.
      • Kondo T.
      A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.
      ,
      • Rust M.J.
      • Markson J.S.
      • Lane W.S.
      • Fisher D.S.
      • O'Shea E.K.
      Ordered phosphorylation governs oscillation of a three-protein circadian clock.
      ). KaiC autophosphorylates and autodephosphorylates, but the other two proteins are needed to confer rhythmicity; early research demonstrated that KaiA stimulates KaiC phosphorylation (
      • Williams S.B.
      • Vakonakis I.
      • Golden S.S.
      • LiWang A.C.
      Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.
      ,
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      Cyanobacterial circadian clockwork: roles of KaiA, KaiB, and the kaiBC promoter in regulating KaiC.
      ) and KaiB antagonizes KaiA action (
      • Kitayama Y.
      • Iwasaki H.
      • Nishiwaki T.
      • Kondo T.
      KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.
      ). Dephosphorylation is also accompanied by exchange of KaiC subunits among hexamers (
      • Kageyama H.
      • Nishiwaki T.
      • Nakajima M.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
      ,
      • Ito H.
      • Kageyama H.
      • Mutsuda M.
      • Nakajima M.
      • Oyama T.
      • Kondo T.
      Autonomous synchronization of the circadian KaiC phosphorylation rhythm.
      ,
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ), a process that is believed to be important for synchronization of the clock (
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      The cyanobacterial circadian system: from biophysics to bioevolution.
      ). However, swapping of KaiC subunits also proceeds in the absence of either KaiA or KaiB (
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ), and sequestration of KaiA by the KaiBC complex may be more important for properly synchronizing the clock (
      • van Zon J.S.
      • Lubensky D.K.
      • Altena P.R.
      • ten Wolde P.R.
      An allosteric model of circadian KaiC phosphorylation.
      ,
      • Clodong S.
      • Dühring U.
      • Kronk L.
      • Wilde A.
      • Axmann I.
      • Herzel H.
      • Kollmann M.
      Functioning and robustness of a bacterial circadian clock.
      ,
      • Qin X.
      • Byrne M.
      • Mori T.
      • Zou P.
      • Williams D.R.
      • McHaourab H.
      • Johnson C.H.
      Intermolecular associations determine the dynamics of the circadian KaiABC oscillator.
      ,
      • Brettschneider C.
      • Rose R.J.
      • Hertel S.
      • Axmann I.M.
      • Heck A.J.
      • Kollmann M.
      A sequestration feedback determines dynamics and temperature entrainment of the KaiABC circadian clock.
      ).
      Crystal structures of Kai proteins revealed a domain-swapped dimer for KaiA (
      • Ye S.
      • Vakonakis I.
      • Ioerger T.R.
      • LiWang A.C.
      • Sacchettini J.C.
      Crystal structure of circadian clock protein KaiA from Synechococcus elongatus.
      ) (Fig. 1A), a dimer of dimers for KaiB (
      • Garces R.G.
      • Wu N.
      • Gillon W.
      • Pai E.F.
      Anabaena circadian clock proteins KaiA and KaiB reveal potential common binding site to their partner KaiC.
      ,
      • Hitomi K.
      • Oyama T.
      • Han S.
      • Arvai A.S.
      • Getzoff E.D.
      Tetrameric architecture of the circadian clock protein KaiB: a novel interface for intermolecular interactions and its impact on the circadian rhythm.
      ,
      • Iwase R.
      • Imada K.
      • Hayashi F.
      • Uzumaki T.
      • Morishita M.
      • Onai K.
      • Furukawa Y.
      • Namba K.
      • Ishiura M.
      Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ,
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ) (Fig. 1B), and a homohexamer for KaiC (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      ) (Fig. 1C) (reviewed in Ref.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      Structural insights into a circadian oscillator.
      ). The quaternary structures in solution as established by small angle x-ray scattering (SAXS) remain the same as in the solid state (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). The 360-kDa KaiC hexamer resembles two stacked doughnuts with overall dimensions of ∼100 × 100 × 100 Å (Fig. 1D). Twelve ATP molecules are lodged between subunits, six each in the N-terminal CI and C-terminal CII rings (Fig. 1C). The N-terminal region of CII forms a linker that tethers together the CI and CII domains of individual subunits (Fig. 1C). The C-terminal ends of CII domains include an S-shaped loop (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ), termed A-loop (
      • Kim Y.I.
      • Dong G.
      • Carruthers Jr., C.W.
      • Golden S.S.
      • LiWang A.
      The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.
      ), and the last 20 residues protrude from the dome-shaped surface of the upper ring (Fig. 1C).
      Figure thumbnail gr1
      FIGURE 1Architecture of S. elongatus Kai proteins. A, crystal structure of domain-swapped KaiA dimer with subunits colored in light and dark gray, respectively (
      • Pattanayek R.
      • Sidiqi S.K.
      • Egli M.
      Crystal structure of the redox-active cofactor DBMIB bound to circadian clock protein KaiA and structural basis for DBMIB's ability to prevent stimulation of KaiC phosphorylation by KaiA.
      ). B, crystal structure of KaiB tetramer (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ). Structures of KaiC at high and low resolution are shown. C, crystal structure of S. elongatus KaiC with the N-terminal CI and C-terminal CII domains colored in light and dark gray, respectively (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      ). Note the C-terminal tentacles (colored in magenta) constituting the KaiA binding site that are resolved to different degrees at 2.8 Å resolution. ATP molecules are drawn in space-filling mode with carbon atoms highlighted in green. In all depictions of KaiC included here, the original orientation has been retained, i.e. the N-terminal CI ring is at the bottom and the C-terminal CII ring is at the top. D, negative stain EM structure of KaiC viewed from the side (center) and rotated by +45° (bottom) and −45° (top) around the horizontal axis (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      ). E, SAXS structure of KaiC (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). The protrusion distinguishes the CII from the CI end. ESPs of the CI (F) and CII (G) rings in KaiC hexamer are shown. The minimum and maximum values of the electrostatic potential are −5 (red) and +5 kt/e (blue), respectively. KaiC rings are viewed from the N terminus (left), the side (center), and the C terminus (right). ATP molecules (green) can be seen inside positively polarized clefts in the views from the C-terminal side.
      KaiA binds to the C-terminal KaiC peptide and unravels the A-loop (
      • Vakonakis I.
      • LiWang A.C.
      Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: implications for KaiC regulation.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      ). Molecular dynamics simulations provided evidence that unfolding of an A-loop increases the mobility of the P-loop region, ATP, and the loop region harboring Thr-432 and Ser-431 (
      • Egli M.
      • Pattanayek R.
      • Sheehan J.H.
      • Xu Y.
      • Mori T.
      • Smith J.A.
      • Johnson C.H.
      Loop-Loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
      ). Removal of an A-loop destabilizes adjacent loops and points to a concerted allosteric mechanism of the stimulation of KaiC phosphorylation by KaiA (
      • Egli M.
      • Pattanayek R.
      • Sheehan J.H.
      • Xu Y.
      • Mori T.
      • Smith J.A.
      • Johnson C.H.
      Loop-Loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
      ). This is also supported by the observation that a single KaiA dimer can push KaiC to the hyperphosphorylated state (
      • Hayashi F.
      • Ito H.
      • Fujita M.
      • Iwase R.
      • Uzumaki T.
      • Ishiura M.
      Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC.
      ). The order of phosphorylation (Thr-432 first) is kinetically controlled as Thr-432 lies closer to the ATP γ-phosphate than Ser-431 (
      • Xu Y.
      • Mori T.
      • Pattanayek R.
      • Pattanayek S.
      • Egli M.
      • Johnson C.H.
      Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.
      ,
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      ). Overall, the KaiA-KaiC interaction is better understood at this time than the KaiB-KaiC interaction or the interactions underlying the ternary KaiABC complex. This is because the binding site of KaiA on KaiC can be established without ambiguity and a plausible mechanism of the increase in KaiC phosphorylation in the presence of KaiA has been formulated.

      Open Questions Regarding the Structure and Function of KaiB in the PTO

      KaiB does not bind to the C-terminal KaiC tentacle, and the possibility of KaiB simply acting as a competitive inhibitor and displacing KaiA from KaiC can therefore be ruled out (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ), but what triggers KaiB binding in the first place? Does KaiB bind to the same ring, CII, and therefore in the vicinity of KaiA (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ), or at the opposite end, to CI (
      • Chang Y.G.
      • Tseng R.
      • Kuo N.W.
      • LiWang A.
      Rhythmic ring-ring stacking drives the circadian oscillator clockwise.
      ,
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ), and does KaiB bind as a tetramer (
      • Akiyama S.
      • Nohara A.
      • Ito K.
      • Maéda Y.
      Assembly and disassembly dynamics of the cyanobacterial periodosoome.
      ), as a dimer (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ), or as recent evidence suggests, in the monomeric form (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ,
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      )? The challenges that the KaiB-KaiC interaction has had in store for structural biology are matched by those at the functional level. Does KaiB sense the phosphorylation level of KaiC (
      • Kageyama H.
      • Nishiwaki T.
      • Nakajima M.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ) or the kinase state of KaiC, i.e. by preferential binding to a particular conformational or dynamic state of the latter (
      • Chang Y.G.
      • Kuo N.W.
      • Tseng R.
      • LiWang A.
      Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria.
      ), does it actively stimulate dephosphorylation (
      • Williams S.B.
      • Vakonakis I.
      • Golden S.S.
      • LiWang A.C.
      Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.
      ,
      • Kitayama Y.
      • Iwasaki H.
      • Nishiwaki T.
      • Kondo T.
      KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.
      ), or is its function in the PTO limited to sequestration of KaiA? The CI ATPase activity is required for KaiB binding (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ,
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      ), but it is unclear whether KaiB binds there or whether the activity is sensed allosterically, e.g. at some distance from the CI ring?
      Simplifying somewhat, one could attribute the difficulties to track down function and KaiC interactions of KaiB to the complexity of KaiC activities and architecture. The former are composed of kinase, phospho-transferase (ATP synthase (
      • Egli M.
      • Mori T.
      • Pattanayek R.
      • Xu Y.
      • Qin X.
      • Johnson C.H.
      Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
      ,
      • Nishiwaki T.
      • Kondo T.
      Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.
      )), and ATPases in CII and CI. KaiB may modulate phosphorylation, dephosphorylation, or ATP hydrolysis, or perhaps influence the ratio between bound ADP and ATP (
      • Nishiwaki-Ohkawa T.
      • Kitayama Y.
      • Ochiai E.
      • Kondo T.
      Exchange of ADP with ATP in the CII ATPase domain promotes auto-phosphorylation of cyanobacterial clock protein KaiC.
      ). Because of the rotational symmetry of the KaiC particle (Fig. 1D), in the absence of high-resolution structural information, it is difficult to determine which ring is which and therefore the location of KaiB on KaiC. Moreover, the intrinsically dynamic nature of the KaiABC clock poses hurdles for structural studies of protein-protein interactions. Rather than moving in lockstep, individual Kai proteins coexist with binary and ternary complexes such that the concentrations of individual species in the PTO oscillate over the daily cycle both in vitro and in vivo (
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ).

      KaiC Architecture

      The two fused hexameric rings of KaiC that display similar widths and heights (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      ) are consistent with the earlier notion that the protein is the result of a gene duplication (
      • Leipe D.D.
      • Aravind L.
      • Grishin N.V.
      • Koonin E.V.
      The bacterial replicative helicase DnaB evolved from a RecA duplication.
      ). In the crystal structures of KaiC, with resolutions of up to 2.8 Å, the symmetry is broken by the C termini that protrude from the CII ring (Fig. 1C). In three-dimensional reconstructions of KaiC based on negative stain or cryo-EM (at 16 Å resolution or less), these C termini are invisible (Fig. 1D). Averaging typically wipes out electron density that could be attributed to the tails (
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ,
      • Mori T.
      • Saveliev S.V.
      • Xu Y.
      • Stafford W.F.
      • Cox M.M.
      • Inman R.B.
      • Johnson C.H.
      Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.
      ,
      • Hayashi F.
      • Suzuki H.
      • Iwase R.
      • Uzumaki T.
      • Miyake A.
      • Shen J.-R.
      • Imada K.
      • Furukawa Y.
      • Yonekura K.
      • Namba K.
      • Ishiura M.
      ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.
      ), and any attempt to distinguish between the CI and CII ends in raw negative stain images or class sums is guesswork at best. However, the KaiA dimer in EM images of KaiA-KaiC binary complexes marks the CII end because this protein binds to a C-terminal KaiC tail (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      ). Also SAXS studies of KaiC alone allow a distinction of the CI and CII ends, although the resolutions achieved with this technique and negative stain EM are probably not too different. Thus, the CII tails appear as a large protrusion in molecular envelopes computed to simulate scattering curves (Fig. 1E) (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). A comparison of the shapes of the KaiC hexamer based on crystallography, EM, or SAXS offers no evidence of large scale conformational changes, consistent with SAXS studies that established variations in KaiC volume of no more than 4% over the daily cycle (
      • Akiyama S.
      • Nohara A.
      • Ito K.
      • Maéda Y.
      Assembly and disassembly dynamics of the cyanobacterial periodosoome.
      ).
      At the level of the CI and CII domain sequences and folding, there is some similarity to replicative helicases (e.g. DnaB) and recombinases (e.g. RecA) (
      • Leipe D.D.
      • Aravind L.
      • Grishin N.V.
      • Koonin E.V.
      The bacterial replicative helicase DnaB evolved from a RecA duplication.
      ), but the KaiC hexameric rings show less likeness to helicases. In terms of both the hexameric ring conformation and the active site configuration, KaiC shows the closest resemblance to F1-ATPase, although the similarity is not apparent at the sequence level (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Egli M.
      • Mori T.
      • Pattanayek R.
      • Xu Y.
      • Qin X.
      • Johnson C.H.
      Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
      ). The KaiC crystal structure readily helps rationalize the higher stability of the CI hexamer (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      ) relative to CII that does not form hexamers when expressed as a separate domain with KaiC from both S. elongatus (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ) and T. elongatus (
      • Hayashi F.
      • Iwase R.
      • Uzumaki T.
      • Ishiura M.
      Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.
      ). Similar active site configurations in KaiC and F1-ATPase and the aforementioned lack of large conformational changes between the kinase and “phosphatase” states of KaiC are also in line with the ATP synthase mechanism of dephosphorylation (
      • Egli M.
      • Mori T.
      • Pattanayek R.
      • Xu Y.
      • Qin X.
      • Johnson C.H.
      Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
      ). Lastly, hexameric assembly and kinase, ATP synthase, and ATPase all mapping to the same active sites at CII subunit interfaces are consistent with allosteric control of KaiA-stimulated KaiC phosphorylation (
      • Egli M.
      • Pattanayek R.
      • Sheehan J.H.
      • Xu Y.
      • Mori T.
      • Smith J.A.
      • Johnson C.H.
      Loop-Loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
      ) and with communication and cooperative effects between KaiC protomers (
      • Kitayama Y.
      • Nishiwaki-Ohkawa T.
      • Sugisawa Y.
      • Kondo T.
      KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria.
      ).
      Although the shapes and dimensions of the CI and CII rings appear to be similar at first glance, comparison of separate surface models reveals some important differences. The C-terminal surface of CI is essentially flat (Fig. 1F, center panel), whereas the corresponding side of CII has a dome-like appearance (Fig. 1G, center panel). ATP molecules wedged between subunits are apparent in clefts that open to the upper C-terminal surfaces of both rings (Fig. 1, F and G, right panels). However, these clefts in the CI hexamer are obscured at the waist by the CII hexamer. The electrostatic surface potentials (ESPs) of the bottom (N-terminal) and top (C-terminal surfaces) of the two rings are very different (
      • Pattanayek R.
      • Yadagiri K.K.
      • Ohi M.D.
      • Egli M.
      Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
      ). The former is negatively polarized, and the latter is quite positive. This means that the N-terminal side of full-length KaiC is essentially flat and negatively polarized and the C-terminal side is convex and positively polarized (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ).
      The KaiCII ATPase integrates input signals through the ATP/ADP ratio and fuels the kinase activity, whereas the KaiCI ATPase acts as an input-independent timer and is needed for KaiB-KaiC complex formation (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ,
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      ). Mixing separate CI and CII hexamers abrogates rhythmicity (
      • Hayashi F.
      • Iwase R.
      • Uzumaki T.
      • Ishiura M.
      Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.
      ), and covalent linkage of the CI and CII domains of subunits is therefore needed for proper function. The structural basis for cross-talk between the CI and CII rings remains to be elucidated. It was suggested that rhythmic stacking and unstacking of the two rings drives the oscillator forward (
      • Chang Y.G.
      • Tseng R.
      • Kuo N.W.
      • LiWang A.
      Rhythmic ring-ring stacking drives the circadian oscillator clockwise.
      ), whereby subunit interactions in the CII ring rhythmically tighten and relax (
      • Chang Y.G.
      • Kuo N.W.
      • Tseng R.
      • LiWang A.
      Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria.
      ). It was previously established that phosphorylation across subunit interfaces creates additional salt bridges and H-bonds (
      • Xu Y.
      • Mori T.
      • Pattanayek R.
      • Pattanayek S.
      • Egli M.
      • Johnson C.H.
      Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.
      ) and that the volume of the KaiC particle is smallest in the hyperphosphorylated state (
      • Akiyama S.
      • Nohara A.
      • Ito K.
      • Maéda Y.
      Assembly and disassembly dynamics of the cyanobacterial periodosoome.
      ). Crystal structures of KaiC hexamers offer no evidence for changes in the vertical separation of the CI and CII rings. However, a comparison of the structure of S. elongatus KaiC (
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ) with the recently determined structure of T. elongatus KaiC (
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      ) revealed a difference between the relative rotations of CI and CII rings around the axis along the central channel that amounts to ∼7 degrees. Therefore, it is possible that the KaiC hexamer undergoes a twisting motion over the daily oscillation instead of an up and down movement.

      KaiC Phosphorylation Patterns

      The strict order of phosphorylation of Thr-432 and Ser-431 is a hallmark of the KaiABC clock (
      • Nishiwaki T.
      • Satomi Y.
      • Kitayama Y.
      • Terauchi K.
      • Kiyohara R.
      • Takao T.
      • Kondo T.
      A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.
      ,
      • Rust M.J.
      • Markson J.S.
      • Lane W.S.
      • Fisher D.S.
      • O'Shea E.K.
      Ordered phosphorylation governs oscillation of a three-protein circadian clock.
      ). Researchers have replaced threonine and/or serine with aspartic or glutamic acid (e.g. T432E/S431E) to mimic phospho-threonine and phospho-serine sites, or with alanine to prevent transfer of the phosphate and lock KaiC either in the hypophosphorylated state (T432A/S431A) or in the second stage of dephosphorylation (S431D or S431E) (
      • Qin X.
      • Byrne M.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      Coupling of a core post-translational pacemaker to a slave transcription/translation feedback loop in a circadian system.
      ,
      • Qin X.
      • Byrne M.
      • Mori T.
      • Zou P.
      • Williams D.R.
      • McHaourab H.
      • Johnson C.H.
      Intermolecular associations determine the dynamics of the circadian KaiABC oscillator.
      ,
      • Kim Y.I.
      • Dong G.
      • Carruthers Jr., C.W.
      • Golden S.S.
      • LiWang A.
      The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.
      ,
      • Chang Y.G.
      • Tseng R.
      • Kuo N.W.
      • LiWang A.
      Rhythmic ring-ring stacking drives the circadian oscillator clockwise.
      ,
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ,
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      ,
      • Kitayama Y.
      • Nishiwaki-Ohkawa T.
      • Sugisawa Y.
      • Kondo T.
      KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria.
      ,
      • Xu Y.
      • Mori T.
      • Qin X.
      • Yan H.
      • Egli M.
      • Johnson C.H.
      Intramolecular regulation of phosphorylation status of the circadian clock protein KaiC.
      ,
      • Terauchi K.
      • Kitayama Y.
      • Nishiwaki T.
      • Miwa K.
      • Murayama Y.
      • Oyama T.
      • Kondo T.
      ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.
      ,
      • Murayama Y.
      • Mukaiyama A.
      • Imai K.
      • Onoue Y.
      • Tsunoda A.
      • Nohara A.
      • Ishida T.
      • Maéda Y.
      • Terauchi K.
      • Kondo T.
      • Akiyama S.
      Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution.
      ). Because binding of KaiB to KaiC involves either a particular conformational state and/or electrostatic polarization of the latter, it is important to keep in mind the limitation of such mimicry. Neither shape nor charge (−1) of aspartate or glutamate is a good match for phospho-threonine or -serine (charge −2). Not surprisingly, the ESPs of the hypophosphorylated (e.g. T432A/S431A) and hyperphosphorylated (pTpS) KaiC forms as well as the T432E/S431E mimic of the hyperphosphorylated state show significant differences (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). By comparison, the difference in ESP between the T432A/S431A and the TS hypophosphorylated forms is likely smaller, but functional assays also uncovered differences between the T432A/S431A mutant and unphosphorylated KaiC (
      • Dong G.
      • Yang Q.
      • Wang Q.
      • Kim Y.I.
      • Wood T.L.
      • Osteryoung K.W.
      • van Oudenaarden A.
      • Golden S.S.
      Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus.
      ). Crystal structures of KaiC Thr-432 and/or Ser-431 mutants displayed phosphorylation patterns that are not always consistent with the particular state of phosphorylation that such mutants were intended to mimic (Table 1). Thus, T432E/S431A KaiC (pTS mimic) featured Thr-426 phosphorylation in four subunits, S431D KaiC (TpS mimic) featured three phosphorylated Thr-432 residues (
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      ), and T432E/S431E (pTpS mimic) exhibited a new phosphorylation site at Ser-320 in two of the six subunits (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). The structural data suggest that the mimicry with Asp or Glu residues of particular KaiC phosphorylation states along the daily cycle is not perfect, a caveat one should bear in mind when interpreting the outcome of experiments using the various KaiC single or double mutants discussed above.
      TABLE 1Phosphorylation patterns in WT and mutant KaiC crystal structures
      KaiC proteinNumber of phosphates in the CII halfReference
      Thr-432
      Phosphorylation site.
      Ser-431
      Phosphorylation site.
      Thr-426
      Phosphorylation site.
      Ser-320
      Phosphorylation site.
      Wild type
      S. elongatus; quasi-hyperphosphorylated.
      64
      • Pattanayek R.
      • Wang J.
      • Mori T.
      • Xu Y.
      • Johnson C.H.
      • Egli M.
      Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
      ,
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Xu Y.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
      Wild type
      T. elongatus; hyperphosphorylated.
      66
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      T432A6
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      S431A61
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      S431D3
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      T426N63
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      T432A/T426A6
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      T432E/S431A4
      • Pattanayek R.
      • Mori T.
      • Xu Y.
      • Pattanayek S.
      • Johnson C.H.
      • Egli M.
      Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
      T432E/S431E2
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      E318A5
      • Egli M.
      • Mori T.
      • Pattanayek R.
      • Xu Y.
      • Qin X.
      • Johnson C.H.
      Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
      A422V6
      • Egli M.
      • Pattanayek R.
      • Sheehan J.H.
      • Xu Y.
      • Mori T.
      • Smith J.A.
      • Johnson C.H.
      Loop-Loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
      a Phosphorylation site.
      b S. elongatus; quasi-hyperphosphorylated.
      c T. elongatus; hyperphosphorylated.

      Evidence That Supports Binding of KaiB to KaiCII

      Looking back at the evolution of investigations directed at KaiB function and the KaiB-KaiC binding mode, it is no surprise that the first place to look for KaiB was the CII end. KaiA associates with CII-terminal tails, and because KaiB is thought to antagonize KaiA action, it is certainly possible that it does so by occupying a spot on KaiC in the vicinity of KaiA. Initial SAXS models of complexes featured both KaiA dimer and KaiB tetramer bound near the CII dome (
      • Akiyama S.
      • Nohara A.
      • Ito K.
      • Maéda Y.
      Assembly and disassembly dynamics of the cyanobacterial periodosoome.
      ). Phosphorylation alters the ESP at the CII end but is not “felt” on the opposite side (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ), a change that might be sensed by KaiB and trigger binding to CII (KaiB was reported to favor the phosphorylated form of KaiC (
      • Kageyama H.
      • Nishiwaki T.
      • Nakajima M.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
      )). However, no other evidence besides the assumption that antagonism could involve close association between KaiA and KaiB tied KaiB to KaiCII in the above SAXS study. Rather than surveying the evidence supporting a KaiCII binding mode by KaiB in a chronological order, let us start with the most recent data. An overview of structural and biophysical observations pertaining to the CII binding mode is given in Fig. 2.
      Figure thumbnail gr2
      FIGURE 2Evidence in support of a KaiCII binding mode by KaiB. A, six KaiB monomers forming a third layer on top of the KaiCII ring, consistent with cryo-EM density (16-Å resolution) and a more extensive buried surface at the KaiCII end based on molecular dynamics flexible fitting (MDFF) simulations when compared with a KaiCI binding mode (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ). B, negative stain EM study of KaiC with a C-terminal His6 tail (identical to the construct used for the crystal structure determination; C), bound to KaiB and in the presence of 1.8-nm nickel-nitrilotriacetic acid Nanogold (left panel), bound to KaiB without gold (central panel), and applying a weighted Fourier difference electron density analysis (right panel) (
      • Pattanayek R.
      • Yadagiri K.K.
      • Ohi M.D.
      • Egli M.
      Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
      ). The gold labeling approach supports binding of KaiB to KaiCII because of the co-localization of His tag, KaiB, and the gold cloud on top of the CII ring. C, cryo-EM density of the KaiC6B6 complex (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ). The strongest density (blue regions) maps to the α-helical regions of KaiCI and KaiCII (C), which is supportive of KaiBs bound to the β-rich side of the KaiCII ring. D, SAXS investigation of the KaiBC complex with crystallographic models of KaiC hexamer (magenta, with one subunit highlighted in cyan) and KaiB dimers (green) modeled into the three-dimensional envelope (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). The observation that the protrusions produced by the C-terminal KaiCII tails (C) and by KaiB appear on the same side supports a KaiCII binding mode. E, side view of three KaiC subunits rendered in surface mode with blue patches indicating regions between subunits that are protected from HDX (left panel). HADDOCK-based models of the KaiC6B6 complex with KaiB monomers bound to either the CII (central panel) or the CI ring (right panel) (
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ). The former is favored based on MS-based ion mobility spectrometry-derived collisional cross-section data. F–H, native PAGE assays of the complex formations between full-length KaiC hexamer and KaiB (F), KaiCI hexamer and KaiB (G), and KaiCII and KaiB (H) (KaiCII alone does not form a stable hexamer) (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ). KaiB binds to KaiC and to KaiCII but not to KaiCI (complex bands are marked by an asterisk).
      Cryo-EM at 16 Å resolution on the one hand (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ) and hydrogen-deuterium exchange (HDX) in combination with native MS on the other (
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ) independently provide support for six KaiB monomers interacting with the KaiC hexamer, i.e. a KaiC6B6 stoichiometry (Fig. 2, A and E, respectively). The resolution of the EM density alone is insufficient to unambiguously assign KaiB monomers to the CII side. However, regions of the strongest EM density normally associated with α-helical regions are consistent with a CII binding mode (Fig. 2C). This is because helical portions are concentrated in the N-terminal halves of the CI and CII rings (Fig. 1C). A negative stain EM investigation using KaiC with C-terminal His6 tags and imaging the KaiBC complex with or without 1.8-nm nickel-nitrilotriacetic acid-Nanogold clearly showed gold, His tags, and KaiB congregating on the same side (CII; Fig. 2B) (
      • Pattanayek R.
      • Yadagiri K.K.
      • Ohi M.D.
      • Egli M.
      Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
      ). The HDX data reveal protection in areas that include the subunit interface of one KaiC ring, and collisional cross-sections together with computational simulations favor the CII binding mode (Fig. 1E) (
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ). The cryo-EM results were also complemented by molecular dynamics simulations that indicated a clear advantage of the CII interface in terms of the buried surface when compared with CI (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ).
      The convergence of cryo-EM and HDX/MS in terms of the KaiBC model, the B-C stoichiometry, and the monomeric form of KaiB targeting the C hexamer is remarkable. The monomeric form of KaiB associating with KaiC subunits also eliminates a puzzle arising from a model of the complex formed by two KaiB dimers and a single KaiC hexamer (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ), namely why subunits constituting the KaiB dimer would engage in deviating interactions with underlying KaiC subunits. In the KaiC6B6 complex, all six KaiB monomers are expected to occupy equivalent sites along the KaiCII ring and interact with KaiC in an identical fashion (Fig. 2A). What other evidence exists in support of the CII binding mode? The SAXS envelope computed for the KaiBC complex may seem inadequate to distinguish between KaiB binding at “the top” or at “the bottom” (Fig. 2D) (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). However, if we take into account the SAXS envelope for KaiC alone (Fig. 1E), it becomes evident that KaiBs are unlikely to be associated with the CI ring as the SAXS envelope of KaiBC should otherwise be composed of four layers (KaiB ring, CI ring, CII ring, CII tail protrusion).
      It is important to stress that the conclusion that KaiB binds on the CII side was not reached by essentially trying to distinguish between the CI and CII rings at low resolution. Negative stain EM density does not allow one to assign the KaiC ring contacted by KaiB because of insufficient resolution and pseudo-symmetry of the KaiC hexamer (Fig. 1D). However, gold labeling gave a clear answer, and these experiments were carried out with full-length KaiB and KaiC proteins from S. elongatus (
      • Pattanayek R.
      • Yadagiri K.K.
      • Ohi M.D.
      • Egli M.
      Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
      ) that constitute the in vitro PTO together with KaiA and ATP/Mg2+. That KaiC His6 tags may have introduced an artifact is unlikely because C-terminally His-tagged KaiC was used to reconstitute the PTO in the same fashion as WT KaiC (
      • Kitayama Y.
      • Nishiwaki-Ohkawa T.
      • Sugisawa Y.
      • Kondo T.
      KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria.
      ). The earliest evidence for CII binding actually came from native PAGE experiments that used KaiB with full-length KaiC (Fig. 2F) or separate CI (Fig. 2G) or CII (Fig. 2H) (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ). Although the CII domain does not form a stable hexamer in the absence of the CI ring (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ,
      • Hayashi F.
      • Iwase R.
      • Uzumaki T.
      • Ishiura M.
      Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.
      ), mixing CII and B protein produced a band shift (Fig. 2H). Conversely, no complex formation was observed for CI hexamer alone and B protein (Fig. 2G).
      What are the implications of the KaiCII binding mode for KaiB function? Cryo-EM and HDX/MS in conjunction with modeling both place KaiB monomers at CII subunit interfaces, thereby covering ATP binding clefts (Figs. 1G and 2A) (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ,
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ). Because KaiBC association is closely linked to the phosphorylation of Ser-431 (
      • Nishiwaki T.
      • Satomi Y.
      • Kitayama Y.
      • Terauchi K.
      • Kiyohara R.
      • Takao T.
      • Kondo T.
      A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.
      ,
      • Rust M.J.
      • Markson J.S.
      • Lane W.S.
      • Fisher D.S.
      • O'Shea E.K.
      Ordered phosphorylation governs oscillation of a three-protein circadian clock.
      ), i.e. KaiB binding occurs when KaiC phosphorylation peaks and continues during dephosphorylation, KaiB could possibly flip the switch from kinase to ATP synthase by perturbing active sites at subunit interfaces. Binding by KaiB would initially be triggered by the change in ESP on CII as a result of phosphorylation (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ), but perhaps KaiB does not actively promote KaiC dephosphorylation and instead antagonizes KaiA action only by sequestering KaiA? A recent EM model of the KaiABC ternary complex placed KaiA at the KaiBCII interface, such that KaiAs protrude from the side of the third (KaiB) ring (
      • Pattanayek R.
      • Williams D.R.
      • Rossi G.
      • Weigand S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
      ). In this fashion, each KaiB monomer can sequester a KaiA molecule, thus preventing them from contacting KaiC C-terminal tails and stimulating phosphorylation. It would appear that a CII binding mode by KaiB is consistent with the current (however incomplete) understanding of the function of KaiB. However, a celebratory attitude may be somewhat premature because a further requirement for KaiB binding to KaiC cannot be readily reconciled with the KaiCII binding mode. According to two recent studies, KaiBC complex formation is preceded by a cycle of ATP hydrolysis in the CI half (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ,
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      ).

      Evidence That Supports Binding of KaiB to KaiCI

      Using the proteins from S. elongatus, Rust and co-workers (
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      ) found that the amount of WT KaiB bound to a hexameric KaiC with the T432E/S431E double mutation in CII and catalytic glutamates replaced by glutamine in CI (abolishing the ATPase there) was strongly reduced relative to WT KaiC (to ∼20%). Ishiura and co-workers (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ) observed that the hexameric T. elongatus KaiC T432D/S431D mutant without the ATPase in CI (KaiCCatE1-/DD) and KaiB lacking the C-terminal tail (14 amino acids; KaiB1–94) do not interact (Fig. 3A). The same KaiB mutant does bind to a KaiC mutant hexamer that lacks the ATPase in CII (KaiCCatE2-) and to WT KaiC, but not to the KaiC T432A/S431A mutant hexamer that mimics the hypophosphorylated state (Fig. 3A). Moreover, the KaiB1–94 deletion mutant binds to KaiC monomer and a KaiCI monomeric domain (Fig. 3B), but not to a KaiCII monomeric domain with the T432D/S431D double mutation (Fig. 3C) (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ). Although these observations are intriguing, it is important to remember that WT KaiB does not form a complex with a KaiC monomer (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ). NMR studies of a KaiB mutant lacking the N-terminal tail (seven residues) in addition to the above C-terminal deletion and additional Y8A/Y94A mutations (KaiB*) mixed with a FLAG-tagged monomeric CI domain (CI*) revealed formation of a specific complex (Fig. 3D) (
      • Tseng R.
      • Chang Y.-G.
      • Bravo I.
      • Latham R.
      • Chaudhary A.
      • Kuo N.-W.
      • LiWang A.
      Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
      ). No complex is observed when CI* and KaiA lacking the N-terminal domain (ΔNKaiA) are mixed alone (Fig. 3D). Conversely, shifts in NMR signals are consistent with formation of a ternary CI*-B*-ΔNA complex (Fig. 3D), indicating that the CI*B* complex can sequester KaiA.
      Figure thumbnail gr3
      FIGURE 3Evidence in support of a KaiCI binding mode by KaiB. A, a native PAGE assay indicates that KaiBC complex formation requires KaiCI ATPase activity (T. elongatus Kai proteins (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      )). Clockwise from top left: WT KaiC forms a complex with a KaiB truncation mutant that lacks the 14 C-terminal residues (13% of full-length KaiB; KaiB1–94). No complex is observed with a KaiC mutant that is unable to hydrolyze ATP in CI (KaiCCatE1-) and contains the T432D/S431D double mutation in CII that mimics hyperphosphorylation (DD); conversely, complex formation occurs with a KaiC mutant that lacks kinase and ATPase activity in CII (KaiCCatE2-); the T432A/S431A double mutation in CII that mimics hypophosphorylation (AA) abolishes complex formation. Please also see Ref.
      • Phong C.
      • Markson J.S.
      • Wilhoite C.M.
      • Rust M.J.
      Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
      for the requirement of CI ATPase activity to support KaiBC complex formation (S. elongatus Kai proteins). B, the monomeric KaiCI domain forms a complex with KaiB1–94 (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ) C, on the contrary, complex formation with KaiB1–94 is not observed using the monomeric KaiCII domain that carries the T432D/S431D double mutation (DD) (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ). D, methyl transverse relaxation optimized spectroscopy (TROSY) spectra of U-[15N,2H]Ile-δ1-13C,1H-labeled T. elongatus CI* alone (* indicates N- and C-terminal FLAG tags (column 1); in the presence of KaiB* (= Y8A/Y94A double mutation, 8–94 truncation form) (column 2); in the presence of KaiB* + ΔNKaiA (KaiA lacking the N-terminal domain) (column 3); and in the presence of ΔNKaiA alone (column 4) (
      • Tseng R.
      • Chang Y.-G.
      • Bravo I.
      • Latham R.
      • Chaudhary A.
      • Kuo N.-W.
      • LiWang A.
      Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
      ). These data support binding between KaiCI monomer and KaiB* dimer, whereby the dimer form is supported by combined SDS-PAGE/gel filtration assays (not shown). E, superimposition of eight T. elongatus KaiB monomers (from crystal structures of T. elongatus WT KaiB (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ) and Thr-64 mutant tetramers (
      • Iwase R.
      • Imada K.
      • Hayashi F.
      • Uzumaki T.
      • Morishita M.
      • Onai K.
      • Furukawa Y.
      • Namba K.
      • Ishiura M.
      Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.
      )) with C-terminal tails (residues 94 up to 108) that were resolved to various degrees in electron density maps colored differently for individual monomers. The C-terminal sequences of S. elongatus KaiB (102 amino acids) and T. elongatus KaiB (108 amino acids) are included below the drawing for comparison. Note the different numberings (Tyr-94 in T. elongatus KaiB corresponds to Tyr-93 in S. elongatus KaiB) and highly acidic character of the tails (Glu and Asp are highlighted in red). F, electrostatic surface potentials (ESPs) of T. elongatus KaiB encompassing all 108 residues (left) and with the C-terminal residues 95–108 removed (right; KaiA1–94, see panels A and B). The minimum and maximum values of the electrostatic potential are −5 (red) and +5 kt/e (blue), respectively. G, secondary structure graphic of a monomeric CI domain from the crystal structure of T. elongatus KaiC (
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      ). ATP/Mg2+ and a negatively polarized loop region (now termed B-loop (
      • Tseng R.
      • Chang Y.-G.
      • Bravo I.
      • Latham R.
      • Chaudhary A.
      • Kuo N.-W.
      • LiWang A.
      Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
      )) located at the N-terminal surface of the KaiC hexamer are highlighted in ball-and-stick mode and in magenta, respectively.
      Why use a KaiB deletion mutant in the first place? Removal of the C-terminal tail of KaiB abolished the in vivo rhythm of the KaiABC clock (
      • Iwase R.
      • Imada K.
      • Hayashi F.
      • Uzumaki T.
      • Morishita M.
      • Onai K.
      • Furukawa Y.
      • Namba K.
      • Ishiura M.
      Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.
      ). A subsequent publication reported normal in vitro oscillations based on the KaiB1–94 mutant, but attenuated amplitudes of oscillations and a lack of normal gene expression rhythms in cells expressing KaiB1–94 (
      • Murakami R.
      • Mutoh R.
      • Iwase R.
      • Furukawa Y.
      • Imada K.
      • Onai K.
      • Morishita M.
      • Yasui S.
      • Ishii K.
      • Valencia Swain J.O.
      • Uzumaki T.
      • Namba K.
      • Ishiura M.
      The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.
      ). Both KaiB1–94 and KaiB* appear to favor the dimeric over the tetrameric state (
      • Murakami R.
      • Mutoh R.
      • Iwase R.
      • Furukawa Y.
      • Imada K.
      • Onai K.
      • Morishita M.
      • Yasui S.
      • Ishii K.
      • Valencia Swain J.O.
      • Uzumaki T.
      • Namba K.
      • Ishiura M.
      The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.
      ,
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ,
      • Tseng R.
      • Chang Y.-G.
      • Bravo I.
      • Latham R.
      • Chaudhary A.
      • Kuo N.-W.
      • LiWang A.
      Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
      ). As demonstrated by native MS, the quaternary structure adopted by KaiB, tetramer, dimer, or monomer, is influenced by both protein concentration and temperature, with lower concentrations and reduced temperatures favoring the monomer (
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ). From KaiB crystal structures (no NMR solution structure of KaiB has been reported to date), it is not obvious why removing the C-terminal tail should favor the dimer. The tails adopt a wide range of conformations in the crystal and do not fold back on the core structure in the tetramer (Fig. 3E). However, removal of the tail reduces significantly the number of acidic residues (Fig. 3E) and inverts the ESP on one face of KaiB from strongly negative to weakly positive (Fig. 3F). KaiA and KaiB do not form a complex according to native PAGE and fluorescence assays (
      • Pattanayek R.
      • Williams D.R.
      • Pattanayek S.
      • Mori T.
      • Johnson C.H.
      • Stewart P.L.
      • Egli M.
      Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
      ). However, KaiA (ΔNKaiA) and KaiB* do form a complex as demonstrated by NMR (
      • Tseng R.
      • Chang Y.-G.
      • Bravo I.
      • Latham R.
      • Chaudhary A.
      • Kuo N.-W.
      • LiWang A.
      Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
      ). Clearly, this observation argues against the KaiB C-terminal tail contacting KaiA. Rather, the tail might be involved in the KaiBC interaction such that it is tucked away at the KaiBC interface and then allows sequestration of KaiA. In this context, it is crucial to remember that WT KaiB does not bind KaiC monomer and that for the KaiB C-terminal tail to properly engage, a KaiC hexamer (including subunit interfaces) appears necessary.
      What are the implications of the KaiCI binding mode for KaiB function? The ATPase activity needed to promote KaiB binding there could go along with a conformational change in CI that exposes a new interface or expands an existing one (
      • Mutoh R.
      • Nishimura A.
      • Yasui S.
      • Onai K.
      • Ishiura M.
      The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
      ). The C-terminal tail of KaiB might assist in prying open an already weakened subunit interaction in the CI ring as a result of the hydrolysis of ATP to ADP and thus assist in the formation of a stable interaction between KaiB and CI from individual subunits. It has been suggested that the binding site on CI involves the so-called B-loop (Fig. 3G) (
      • Pattanayek R.
      • Sidiqi S.K.
      • Egli M.
      Crystal structure of the redox-active cofactor DBMIB bound to circadian clock protein KaiA and structural basis for DBMIB's ability to prevent stimulation of KaiC phosphorylation by KaiA.
      ). This loop is highly acidic and contributes to the overall negative ESP at the CI end of the hexamer (Fig. 1F). The loop is more extended when compared with the corresponding CII region that is buried at the waist (
      • Pattanayek R.
      • Xu Y.
      • Lamichhane A.
      • Johnson C.H.
      • Egli M.
      An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
      ). It is somewhat implausible that KaiB with a negatively polarized ESP as a result of the C-terminal tail (Fig. 3F) would interact with a negatively polarized B-loop alone. For one, B-loops are available on the outer surface of the CI ring, and accessing them does not necessitate ATPase activity. Moreover, computational simulations of KaiC6B6 complexes with KaiB monomers located on either the CI or the CII end demonstrated a significantly enhanced buried surface for the latter interaction (
      • Villarreal S.A.
      • Pattanayek R.
      • Williams D.R.
      • Mori T.
      • Qin X.
      • Johnson C.H.
      • Egli M.
      • Stewart P.L.
      CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
      ,
      • Snijder J.
      • Burnley R.J.
      • Wiegard A.
      • Melquiond A.S.J.
      • Bonvin A.M.J.J.
      • Axmann I.M.
      • Heck A.J.R.
      Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
      ) (Fig. 2E). In any case, KaiB binding to CI seems to involve individual C subunits, unlike the proposed interaction at the CII end, where KaiB monomers bridge adjacent KaiC subunits by covering ATP binding clefts (Fig. 2A). Because the CI ring is more stable, KaiB docking there could disrupt the structural platform critical for holding together the entire hexamer, which is more in line with subunit shuffling during the dephosphorylation phase (
      • Kageyama H.
      • Nishiwaki T.
      • Nakajima M.
      • Iwasaki H.
      • Oyama T.
      • Kondo T.
      Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
      ,
      • Ito H.
      • Kageyama H.
      • Mutsuda M.
      • Nakajima M.
      • Oyama T.
      • Kondo T.
      Autonomous synchronization of the circadian KaiC phosphorylation rhythm.
      ,
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ) than a CII binding mode. However, initial experiments to probe the effects of the presence of either KaiA or KaiB on KaiC monomer exchange did not show an enhancement of the process by KaiB (
      • Mori T.
      • Williams D.R.
      • Byrne M.O.
      • Qin X.
      • Egli M.
      • Mchaourab H.S.
      • Stewart P.L.
      • Johnson C.H.
      Elucidating the ticking of an in vitro circadian clockwork.
      ). Both the CI and the CII binding modes by KaiB in principle allow sequestration of KaiA at the KaiBC interface. A key difference lies in the initial trigger of KaiB association with KaiC. The KaiCII interaction is preceded by kinase activity and a concomitant change in the ESP that may be sensed by KaiB; the KaiCI interaction is preceded by ATPase activity and a concomitant conformational change that provides access for KaiB.

      Perspective

      Studies of the cyanobacterial KaiABC circadian clock have led to new insights into the mechanism of circadian time keeping. To a large degree this is due to the discovery that the KaiA, KaiB, and KaiC proteins in the presence of ATP constitute a PTO that can be studied independently from the TTFL. As far as KaiB function and its interactions with KaiC and KaiA are concerned, there are currently more questions than answers. Clearly, sequestration of KaiA by KaiB that is itself bound to KaiC is of central importance for maintaining a stable oscillator. Further, there is strong evidence that ATPase activity in the KaiCI half boosts KaiBC complex formation. KaiB tetramers dissociate, and it is the monomeric form of KaiB that interacts with KaiC. However, no consensus exists in the field as to where KaiB binds on the KaiC hexamer. There are observations that cannot be rationalized with a KaiCI binding mode and others that defy a binding mode that involves only KaiCII. KaiB monomers bound at the CII end are shared between adjacent subunits, but KaiB docking at the CI end involves only individual subunits (monomers).
      A hybrid structural biology approach has been useful to characterize Kai protein-protein interactions at low resolution. A better understanding of KaiB function and its role in KaiA sequestration at the level of KaiC hexamer now requires crystal or high-resolution cryo-EM structures of KaiBC and KaiABC complexes. KaiB interacting with a KaiC monomer or a KaiCI domain opens the door to structure determination of complexes by solution NMR. However, it appears unrealistic to gain a complete understanding of a >400-kDa molecular machine by analyzing 40-kDa fragments in the absence of (i) ATP, (ii) KaiC phosphorylation, and (iii) cooperative effects among KaiC subunits, and with (iv) KaiB mutants that bind KaiC monomers although WT KaiB does not. Recent observations raise the possibility that KaiB can contact KaiC at both the CI and the CII ends. Experiments using tagged KaiC constructs for EM or mutants carrying spin labels for electron paramagnetic resonance could be used to address the hypothesis that KaiB binds both halves. A dual binding mode seems unnecessary just to sequester KaiA. Perhaps KaiB is not just a key component of the PTO but is also involved in the regulation of the clock output pathway. These roles may necessitate different KaiC binding modes and involve either KaiC hexamer or KaiC monomer. Breakthroughs in both the structural and the functional realms are still needed for a comprehensive mechanistic understanding of the cyanobacterial KaiABC PTO.

      Author Profile

      REFERENCES

        • Johnson C.H.
        • Stewart P.L.
        • Egli M.
        The cyanobacterial circadian system: from biophysics to bioevolution.
        Annu. Rev. Biophys. 2011; 40: 143-167
        • Mackey S.R.
        • Golden S.S.
        • Ditty J.L.
        The itty-bitty time machine genetics of the cyanobacterial circadian clock.
        Adv. Genet. 2011; 74: 13-53
        • Egli M.
        • Johnson C.H.
        A circadian clock nanomachine that runs without transcription or translation.
        Curr. Opin. Neurobiol. 2013; 23: 732-740
        • Johnson C.H.
        • Egli M.
        Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.
        Annu. Rev. Biochem. 2014; 83: 221-247
        • Tomita J.
        • Nakajima M.
        • Kondo T.
        • Iwasaki H.
        No transcription-translation feedback in circadian rhythm of KaiC phosphorylation.
        Science. 2005; 307: 251-254
        • Nakajima M.
        • Imai K.
        • Ito H.
        • Nishiwaki T.
        • Murayama Y.
        • Iwasaki H.
        • Oyama T.
        • Kondo T.
        Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.
        Science. 2005; 308: 414-415
        • Murakami R.
        • Mutoh R.
        • Iwase R.
        • Furukawa Y.
        • Imada K.
        • Onai K.
        • Morishita M.
        • Yasui S.
        • Ishii K.
        • Valencia Swain J.O.
        • Uzumaki T.
        • Namba K.
        • Ishiura M.
        The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.
        J. Biol. Chem. 2012; 287: 29506-29515
        • Qin X.
        • Byrne M.
        • Xu Y.
        • Mori T.
        • Johnson C.H.
        Coupling of a core post-translational pacemaker to a slave transcription/translation feedback loop in a circadian system.
        PLoS Biol. 2010; 8: e1000394
        • Hardin P.E.
        • Hall J.C.
        • Rosbash M.
        Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels.
        Nature. 1990; 343: 536-540
        • Aronson B.D.
        • Johnson K.A.
        • Loros J.J.
        • Dunlap J.C.
        Negative feedback defining a circadian clock: autoregulation of the clock gene frequency.
        Science. 1994; 263: 1578-1584
        • Xu Y.
        • Mori T.
        • Pattanayek R.
        • Pattanayek S.
        • Egli M.
        • Johnson C.H.
        Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13933-13938
        • Nishiwaki T.
        • Satomi Y.
        • Nakajima M.
        • Lee C.
        • Kiyohara R.
        • Kageyama H.
        • Kitayama Y.
        • Temamoto M.
        • Yamaguchi A.
        • Hijikata A.
        • Go M.
        • Iwasaki H.
        • Takao T.
        • Kondo T.
        Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13927-13932
        • Nishiwaki T.
        • Satomi Y.
        • Kitayama Y.
        • Terauchi K.
        • Kiyohara R.
        • Takao T.
        • Kondo T.
        A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.
        EMBO J. 2007; 26: 4029-4037
        • Rust M.J.
        • Markson J.S.
        • Lane W.S.
        • Fisher D.S.
        • O'Shea E.K.
        Ordered phosphorylation governs oscillation of a three-protein circadian clock.
        Science. 2007; 318: 809-812
        • Williams S.B.
        • Vakonakis I.
        • Golden S.S.
        • LiWang A.C.
        Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15357-15362
        • Xu Y.
        • Mori T.
        • Johnson C.H.
        Cyanobacterial circadian clockwork: roles of KaiA, KaiB, and the kaiBC promoter in regulating KaiC.
        EMBO J. 2003; 22: 2117-2126
        • Kitayama Y.
        • Iwasaki H.
        • Nishiwaki T.
        • Kondo T.
        KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.
        EMBO J. 2003; 22: 2127-2134
        • Kageyama H.
        • Nishiwaki T.
        • Nakajima M.
        • Iwasaki H.
        • Oyama T.
        • Kondo T.
        Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro.
        Mol. Cell. 2006; 23: 161-171
        • Ito H.
        • Kageyama H.
        • Mutsuda M.
        • Nakajima M.
        • Oyama T.
        • Kondo T.
        Autonomous synchronization of the circadian KaiC phosphorylation rhythm.
        Nat. Struct. Mol. Biol. 2007; 14: 1084-1088
        • Mori T.
        • Williams D.R.
        • Byrne M.O.
        • Qin X.
        • Egli M.
        • Mchaourab H.S.
        • Stewart P.L.
        • Johnson C.H.
        Elucidating the ticking of an in vitro circadian clockwork.
        PLoS Biol. 2007; 5: e93
        • van Zon J.S.
        • Lubensky D.K.
        • Altena P.R.
        • ten Wolde P.R.
        An allosteric model of circadian KaiC phosphorylation.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7420-7425
        • Clodong S.
        • Dühring U.
        • Kronk L.
        • Wilde A.
        • Axmann I.
        • Herzel H.
        • Kollmann M.
        Functioning and robustness of a bacterial circadian clock.
        Mol. Syst. Biol. 2007; 3: 90
        • Qin X.
        • Byrne M.
        • Mori T.
        • Zou P.
        • Williams D.R.
        • McHaourab H.
        • Johnson C.H.
        Intermolecular associations determine the dynamics of the circadian KaiABC oscillator.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14805-14810
        • Brettschneider C.
        • Rose R.J.
        • Hertel S.
        • Axmann I.M.
        • Heck A.J.
        • Kollmann M.
        A sequestration feedback determines dynamics and temperature entrainment of the KaiABC circadian clock.
        Mol. Syst. Biol. 2010; 6: 389
        • Ye S.
        • Vakonakis I.
        • Ioerger T.R.
        • LiWang A.C.
        • Sacchettini J.C.
        Crystal structure of circadian clock protein KaiA from Synechococcus elongatus.
        J. Biol. Chem. 2004; 279: 20511-20518
        • Garces R.G.
        • Wu N.
        • Gillon W.
        • Pai E.F.
        Anabaena circadian clock proteins KaiA and KaiB reveal potential common binding site to their partner KaiC.
        EMBO J. 2004; 23: 1688-1698
        • Hitomi K.
        • Oyama T.
        • Han S.
        • Arvai A.S.
        • Getzoff E.D.
        Tetrameric architecture of the circadian clock protein KaiB: a novel interface for intermolecular interactions and its impact on the circadian rhythm.
        J. Biol. Chem. 2005; 280: 19127-19135
        • Iwase R.
        • Imada K.
        • Hayashi F.
        • Uzumaki T.
        • Morishita M.
        • Onai K.
        • Furukawa Y.
        • Namba K.
        • Ishiura M.
        Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.
        J. Biol. Chem. 2005; 280: 43141-43149
        • Pattanayek R.
        • Williams D.R.
        • Pattanayek S.
        • Mori T.
        • Johnson C.H.
        • Stewart P.L.
        • Egli M.
        Structural model of the circadian clock KaiB-KaiC complex and mechanism for modulation of KaiC phosphorylation.
        EMBO J. 2008; 27: 1767-1778
        • Villarreal S.A.
        • Pattanayek R.
        • Williams D.R.
        • Mori T.
        • Qin X.
        • Johnson C.H.
        • Egli M.
        • Stewart P.L.
        CryoEM and molecular dynamics of the circadian KaiB-KaiC complex indicates KaiB monomers interact with KaiC and block ATP binding clefts.
        J. Mol. Biol. 2013; 425: 3311-3324
        • Pattanayek R.
        • Wang J.
        • Mori T.
        • Xu Y.
        • Johnson C.H.
        • Egli M.
        Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.
        Mol. Cell. 2004; 15: 375-388
        • Pattanayek R.
        • Xu Y.
        • Lamichhane A.
        • Johnson C.H.
        • Egli M.
        An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.
        Acta Crystallogr. D Biol. Crystallogr. 2014; 70: 1375-1390
        • Johnson C.H.
        • Egli M.
        • Stewart P.L.
        Structural insights into a circadian oscillator.
        Science. 2008; 322: 697-701
        • Pattanayek R.
        • Williams D.R.
        • Rossi G.
        • Weigand S.
        • Mori T.
        • Johnson C.H.
        • Stewart P.L.
        • Egli M.
        Combined SAXS/EM based models of the S. elongatus post-translational circadian oscillator and its interactions with the output His-kinase SasA.
        PLoS ONE. 2011; 6: e23697
        • Kim Y.I.
        • Dong G.
        • Carruthers Jr., C.W.
        • Golden S.S.
        • LiWang A.
        The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12825-12830
        • Vakonakis I.
        • LiWang A.C.
        Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: implications for KaiC regulation.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10925-10930
        • Pattanayek R.
        • Williams D.R.
        • Pattanayek S.
        • Xu Y.
        • Mori T.
        • Johnson C.H.
        • Stewart P.L.
        • Egli M.
        Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods.
        EMBO J. 2006; 25: 2017-2028
        • Egli M.
        • Pattanayek R.
        • Sheehan J.H.
        • Xu Y.
        • Mori T.
        • Smith J.A.
        • Johnson C.H.
        Loop-Loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.
        Biochemistry. 2013; 52: 1208-1220
        • Hayashi F.
        • Ito H.
        • Fujita M.
        • Iwase R.
        • Uzumaki T.
        • Ishiura M.
        Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC.
        Biochem. Biophys. Res. Comm. 2004; 316: 195-202
        • Pattanayek R.
        • Mori T.
        • Xu Y.
        • Pattanayek S.
        • Johnson C.H.
        • Egli M.
        Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
        PLoS One. 2009; 4: e7529
        • Chang Y.G.
        • Tseng R.
        • Kuo N.W.
        • LiWang A.
        Rhythmic ring-ring stacking drives the circadian oscillator clockwise.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 16847-16851
        • Mutoh R.
        • Nishimura A.
        • Yasui S.
        • Onai K.
        • Ishiura M.
        The ATP-mediated regulation of KaiB-KaiC interaction in the cyanobacterial circadian clock.
        PLoS ONE. 2013; 8: e80200
        • Akiyama S.
        • Nohara A.
        • Ito K.
        • Maéda Y.
        Assembly and disassembly dynamics of the cyanobacterial periodosoome.
        Mol. Cell. 2008; 29: 703-716
        • Snijder J.
        • Burnley R.J.
        • Wiegard A.
        • Melquiond A.S.J.
        • Bonvin A.M.J.J.
        • Axmann I.M.
        • Heck A.J.R.
        Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 1379-1384
        • Chang Y.G.
        • Kuo N.W.
        • Tseng R.
        • LiWang A.
        Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 14431-14436
        • Phong C.
        • Markson J.S.
        • Wilhoite C.M.
        • Rust M.J.
        Robust and tunable circadian rhythms from differentially sensitive catalytic domains.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 1124-1129
        • Egli M.
        • Mori T.
        • Pattanayek R.
        • Xu Y.
        • Qin X.
        • Johnson C.H.
        Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism.
        Biochemistry. 2012; 51: 1547-1558
        • Nishiwaki T.
        • Kondo T.
        Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate.
        J. Biol. Chem. 2012; 287: 18030-18035
        • Nishiwaki-Ohkawa T.
        • Kitayama Y.
        • Ochiai E.
        • Kondo T.
        Exchange of ADP with ATP in the CII ATPase domain promotes auto-phosphorylation of cyanobacterial clock protein KaiC.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 4455-4460
        • Leipe D.D.
        • Aravind L.
        • Grishin N.V.
        • Koonin E.V.
        The bacterial replicative helicase DnaB evolved from a RecA duplication.
        Genome Res. 2000; 10: 5-16
        • Mori T.
        • Saveliev S.V.
        • Xu Y.
        • Stafford W.F.
        • Cox M.M.
        • Inman R.B.
        • Johnson C.H.
        Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 17203-17208
        • Hayashi F.
        • Suzuki H.
        • Iwase R.
        • Uzumaki T.
        • Miyake A.
        • Shen J.-R.
        • Imada K.
        • Furukawa Y.
        • Yonekura K.
        • Namba K.
        • Ishiura M.
        ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.
        Genes Cells. 2003; 8: 287-296
        • Hayashi F.
        • Iwase R.
        • Uzumaki T.
        • Ishiura M.
        Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.
        Biochem. Biophys. Res. Commun. 2006; 348: 864-872
        • Kitayama Y.
        • Nishiwaki-Ohkawa T.
        • Sugisawa Y.
        • Kondo T.
        KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria.
        Nat. Commun. 2013; 4: 2897
        • Pattanayek R.
        • Yadagiri K.K.
        • Ohi M.D.
        • Egli M.
        Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator.
        Cell Cycle. 2013; 12: 810-817
        • Xu Y.
        • Mori T.
        • Qin X.
        • Yan H.
        • Egli M.
        • Johnson C.H.
        Intramolecular regulation of phosphorylation status of the circadian clock protein KaiC.
        PLoS ONE. 2009; 4: e7509
        • Terauchi K.
        • Kitayama Y.
        • Nishiwaki T.
        • Miwa K.
        • Murayama Y.
        • Oyama T.
        • Kondo T.
        ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381
        • Murayama Y.
        • Mukaiyama A.
        • Imai K.
        • Onoue Y.
        • Tsunoda A.
        • Nohara A.
        • Ishida T.
        • Maéda Y.
        • Terauchi K.
        • Kondo T.
        • Akiyama S.
        Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution.
        EMBO J. 2011; 30: 68-78
        • Dong G.
        • Yang Q.
        • Wang Q.
        • Kim Y.I.
        • Wood T.L.
        • Osteryoung K.W.
        • van Oudenaarden A.
        • Golden S.S.
        Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus.
        Cell. 2010; 140: 529-539
        • Tseng R.
        • Chang Y.-G.
        • Bravo I.
        • Latham R.
        • Chaudhary A.
        • Kuo N.-W.
        • LiWang A.
        Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria.
        J. Mol. Biol. 2014; 426: 389-402
        • Pattanayek R.
        • Sidiqi S.K.
        • Egli M.
        Crystal structure of the redox-active cofactor DBMIB bound to circadian clock protein KaiA and structural basis for DBMIB's ability to prevent stimulation of KaiC phosphorylation by KaiA.
        Biochemistry. 2012; 51: 8050-8052