Life Science : Medical Biology

Visualization of circadian tickingof cyanobacterial clock protein KaiC in real time

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mutant having only a C1 domain forms a C1 ring andexhibits approximately 70% of the ATPase activityof full-length KaiC. KaiC has two phosphorylationsites, i.e., Ser431 and Thr432, in the C2 domain,and both residues are phosphorylated and thendephosphorylated in a programmed sequence during
the phosphorylation cycle as follows: KaiC S/pT→KaiCpS/pT →KaiCpS/T →KaiCS/T (where ‘S’ represents
Ser431, ‘pS’ represents phosphorylated Ser431, ‘T’represents Thr432, and ‘pT’ represents phosphorylated
Thr432). The phospho-mimicking mutant of KaiCS/T
exhibits a higher ATPase activity than the phosphomimicking mutant of KaiCpS/pT, indicating a functional
coupling between ATPase and the phosphorylation
state through the KaiC hexamer. Hence, thedetermination of the structural change of KaiC
interlocked with ATPase and the phosphorylationstate is of great importance.
Taking advantage of the fact that the ATPase
activity is closely coupled with the phosphorylation
state, we investigated the structure of KaiC in solution
along the reaction sequence of the phosphorylation
cycle. Auto-dephosphorylation reaction was induced
by a temperature jump from ‘on ice’ to 30°C, and thestructural transition was monitored using time-resolved
small-angle X-ray scattering (SAXS) at beamline
BL45XU [5]. Concomitantly with the shift in thephosphorylation state, we observed a gradual increasein the apparent radius of gyration (Rg) estimated fromthe measured SAXS curves. As a result of the detailed
Guinier analysis, a substantial increase in Rg by 5%
was observed in the KaiCpS/pT →KaiCpS/T transition.
The 5% increase in Rg indicates the occurrence of a

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Circadian clocks are endogenous timing systems
for various organisms to adapt to dairy alterations
in external environments. The cyanobacterium
Synechococcus elongatus PCC7942 is one of thesimplest organisms known to possess a circadianoscillator consisting of the three clock proteins
KaiA, KaiB, and KaiC [1]. KaiC is rhythmicallyphosphorylated and then dephosphorylated in vivo
without a transcriptional-translational negative
feedback mechanism, which has been proposed tounderlie eukaryotic circadian clocks. In addition,
Nakajima et al. have shown that the phosphorylation
cycle of KaiC is reconstituted simply by co-incubating
the three Kai proteins in the presence of ATP [2].
KaiC possesses autokinase/autophosphataseactivities. KaiA stimulates the auto-phosphorylationof KaiC, whereas KaiB promotes the autodephosphorylation of KaiC by attenuating theeffect of KaiA. During the KaiC phosphorylation
cycle, the three Kai proteins are assembled into
hetero-multimeric complexes and then disassembled
in a rhythmic manner [3]. The frequency of thephosphorylation cycle of KaiC is closely correlatedwith the rate of ATP hydrolysis exhibited by KaiC
only [4]. The ATPase activity of KaiC, which isextremely low and is kept constant in a range ofphysiological temperatures, plays a central role ingenerating the temperature-compensated circadianperiod of the Kai oscillator.
KaiC is a dumbbell-shaped molecule composedof tandemly duplicated N-terminal (C1) and C-terminal(C2) domains. Six protomers are assembled into ahexamer to attain a double-doughnut shape. A KaiC

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Projection of C2 Ring

Expansion of C2 Ring

Contraction of C2 Ring

Fig. 1. Expansion and contraction motions of C2 ring of KaiC interlocked with ATPase activity. The protomerprotomer arrangement of the known X-ray structure of KaiC was refined against the SAXS data under the assumptionof P6 symmetry. The ATPase activity of each phosphorylation state is plotted relative to that of KaiC-WT at 30°C.

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large conformational change in the KaiC hexamer.
The structural transition of the KaiC hexamer was
further monitored by time-resolved fluorescence
spectroscopy. KaiC intrinsically has three tryptophan(Trp) residues, which are useful for monitoring local
structural changes. One Trp residue is locatedin the C1 ring, whereas the other two are located in
the C2 ring. We observed a gradual change in thefluorescence intensity during auto-dephosphorylation,and then identified that the observed change wasmainly attributed to the change in the local
environments around the two Trp residues in theC2 ring.
To visualize the structural change, we built lowresolution SAXS models of the KaiC hexamer by
the rigid-body refinement of a known X-ray crystal
structure (Fig. 1). The global shape remains almost
unchanged in the transition from KaiCS/pT to KaiCpS/pT,
whereas the radius of the C2 ring is dramaticallyenlarged in the subsequent transition from KaiCpS/pT
to KaiCpS/T. The expanded C2 ring is partly contracted
in the transition from KaiCpS/T to KaiCS/T, and further
contracted in the subsequent transition from KaiCS/T
to KaiCS/pT. Our model implies that KaiC ticks throughexpanding and contracting motions of the C2 ring.
The Trp fluorescence coming from KaiC displays
a circadian oscillation in the presence of both KaiA(a)and KaiB (Fig. 2(a)), providing direct evidence ofthe dynamic structural ticking of KaiC in solution. Theobserved oscillation in the fluorescence intensity
can be well explained by a combination of thefluorescence intensity of the each phosphorylation
state and its abundance, supporting the expanding
and contracting motions of the C2 ring during
circadian oscillation.
The dynamic structural change of the KaiC
hexamer revealed in this study provides notable
insights into the cyanobacterial circadian clock.
The ATPase activity of KaiC is less sensitive to theC2 ring expansion in the transition from KaiCpS/pT toKaiCpS/T, and the partial contraction of the C2 ringin the transition from KaiC pS/T to KaiCS/T results ina significant activation of ATPase (Fig. 1). Theseobservations suggest that ATPase activity is not as
simple as an on/off (contraction/expansion) switchingprocess. Our results may reflect the auto-inhibitory
mechanism proposed in a previous study [4].
Additionally, the timing of the accumulation of KaiCpS/T
with the expanded C2 ring is coincident with that of
the accumulation of the ternary complex with KaiA
and KaiB [3]. Considering that KaiA and KaiB bindto the C2 ring of KaiC, the conformational ticking ofKaiC serves as a timing cue for assembly/disassemblywith KaiA and KaiB (Fig. 2(b)).

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Fig. 2. Circadian ticking of KaiC. (a) Trp fluorescence intensity in the presence of KaiA,KaiB, and KaiC. (b) Schematic presentation of Kai protein oscillator. The dynamic
structural change of the C2 ring of KaiC serves as a timing cue for recruiting KaiA and KaiB.

References[1] M. Ishiura et al.: Science 281(1998) 1519.
[2] M. Nakajima et al.: Science 308 (2005) 414.
[3] S. Akiyama et al.: Mol. Cell 29 (2008) 703.
[4] K. Terauchi et al.: PNAS 104 (2007) 16377.
[5] Y. Murayama, A. Mukaiyama, K. Imai, Y. Onoue,
A. Tsunoda, A. Nohara, T. Ishida, Y. Maéda, K. Terauchi,T. Kondo and S. Akiyama: EMBO J. 30 (2011) 68.

Atsushi Mukaiyama a,b, Takao Kondo a,b andShuji Akiyama a,b,c,d,e,*a Division of Biological Science, Nagoya Universityb CREST, Japan Science and Technology Agency
c SPring-8/RIKEN
d PRESTO, Japan Science and Technology Agency
e Institute for Molecular Science*E-mail: akiyamas@ims.ac.jp

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