Life ScienceResearch Frontiers 2022

Master allostery in clock protein KaiC
orchestrates circadian rhythm
C1 or C2 results in arrhythmicity, but the mechanismsof the rhythm emergence via C1-C2 coupling andthe transmission of temporal information to KaiA andKaiB are still unresolved. To address this issue, we
conducted comprehensive structural analyses of KaiC.
To gain insights into the structural basis of
the rhythmicity, we crystallized KaiC in different
phosphorylation states and obtained electron density
maps at 2.2 – 3.1 Å resolutions through diffractionexperiments at SPring-8 BL44XU [1][2][3]. Our
structure library consisting of 13 KaiC coordinates
covered the entire C2-phospho cycle via KaiC-ST,KaiC-SpT, KaiC-pSpT, and KaiC-pST. Similarly, for
C1, we identified ATP, nucleophilic water molecules,
and adenosine diphosphate (ADP) in the active sites
and confirmed that pre- and post-hydrolysis statesin C1-ATPase cycle were captured.
The KaiC structure library captured three
conformational changes in C1 and C2. First, a secondary
structural transition associated with the C2-phosphocycle was identified (C2-PSw, phosphorylation switch)(Fig. 2(a)). The upstream region of the phosphosites adopted helical and coiled conformations due
to differences in the volumes and charges aroundS431/pS431 [1]. This dynamic structural change
was also confirmed in the solution phase during thein vitro oscillation [3]. The C2-phospho cycle throughthe biochemical 4-state was nearly a conformationalswitch between the two structural states.
The second was a whole domain positional
rearrangement in which the neighboring C1 shifts
systematically at the protomer-protomer interfaces(C1-domain slide) (Fig. 2(b)). This was related tothe oscillation of the ATP hydrolysis activity, through
ADP-ATP exchange process and the regulation of lytic
water positions (Fig. 2(c)) [2].

(a)

(b)

CATP
ADP

KaiC

P

ATP
ADP

ATP
ADP

ATP
ADP
P

P

P

Gene Expression
Clock Protein System
in Cyanobacterial Cell KaiA, KaiB, & KaiC
(in vitro) (24 hours)(in vivo) (24 hours)

C1KaiC

ATP
ADP
P

P

P

Assembly & Disassembly(in vitro) (24 hours)

C2ATP
S431
T432

Overall Structure of KaiC

4030
20100Relative
AbundanceAkai

B
kaiKaiB

KaiA

Earth Rotation KaiB KaiA
(24 hours)

ATPase Activity(/day)Allostery, a biological (or ubiquitous) event in whicha chemical process occurring at one site of a protein
or enzyme affects the catalytic activity of anothersite, is the basis of non-linear cellular responses. The
circadian rhythm, a major example of such regulatory
phenomena, is a biological timekeeping systemthat enables organisms to adapt to environmental
fluctuations caused by Earth’s rotation. Efforts havebeen dedicated to understanding the mechanismsby which activities and structures of molecules,complexes, and cells autonomously oscillate with a
24-hour period. However, the precise relationshipbetween protein allostery and rhythmicity remains tobe elucidated.
The core oscillator of circadian rhythms incyanobacteria consists of three clock proteins, KaiA,KaiB, and KaiC (Fig. 1(a)). The three Kai proteins
repeatedly associate and disassociate in vitro in thepresence of adenosine triphosphate (ATP). KaiC, acomponent with the highest molecular mass among thethree, orchestrates the assembly states of KaiA andKaiB through autonomous activation and deactivationof its own catalytic activities.
The cyanobacterial circadian rhythm is generated
by two reaction cycles in KaiC (Fig. 1(b)). The KaiC
hexamer hydrolyzes ATP in its N-terminal domain(C1) to determine the period length of the rhythm(C1-ATPase cycle). In its C-terminal domain (C2),phosphorylation and dephosphorylation of S431 andT432 configure the cycle with a circadian period (C2phospho cycle) (ST→SpT→pSpT→pST: S for S431,T for T432, p for phosphorylated states). Although
the active sites of C1 and C2 are 4 nm distant, theyfunction cooperatively through mutual allosteric
regulations. The importance of this cooperativity isevident from the fact that the loss of function of either

10.5002472

Oscillation of Reaction Activities

Fig. 1. Clock protein KaiC mastering the cyanobacterial circadian rhythm. (a) Cross-scale
relationships between the oscillation in vitro, the rhythm in vivo, and Earth’s rotation.
(b) The overall architecture of KaiC and the oscillations of states in C1 and C2. KaiC-ST, KaiCSpT, KaiC-pSpT, and KaiC-pST, are distinguished by green, red, blue, and orange, respectively.

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Time (h)Research Frontiers 2022

Third, the above structural alternations in C1 and C2
were mediated by the hydrogen bond rearrangementsin their interface (Fig. 2(d)). The neutral Q394 was thekey residue for the functional and structural C1-C2
communication, which is accomplished by switching its
hydrogen bonding partner between the basic R217 andthe acidic E214 (ERQ triad) [1].
KaiC integrates elementary reactions of C1-ATPase
cycle and C2-phospho cycle through these secondary,tertiary, and quaternary structural arrangements togenerate the circadian rhythm. This observation wasexperimentally verified through the examination ofa mutant with the single phosphorylation at S432
(KaiC-SV). Despite the mutation of T432 into valine,
KaiC-SV still retained a rhythmicity based on C2PSw. Furthermore, its prolonged period could beallosterically shortened by activating C1-ATPase(Fig. 2(e)) [1].
The C1-C2 allostery is indeed a core of the clockthat transmits the time information throughout the cell.

(a)

The disassembly of KaiA-KaiB-KaiC complexes
formed during the cycle is essentially suppressed withan extremely low dissociation rate constant [4]. Thecue for the disassembly is provided by the activationof ATP hydrolysis accompanied by C1-domain
slide [2]. Moreover, the circadian rhythm is known
as a temperature-insensitive phenomenon, in which
the period length is almost constant in the range ofphysiological temperature, and this ability referred
to as temperature compensation also relies on C1-C2
allostery [5].
For understanding biological phenomena suchas the circadian rhythm that span a wide range ofspatio-temporal scales (from fast atomic dynamics toslow complex disassembly dynamics), it is important
to observe the structural changes of biomolecules
underlying the cellular events. Slight structural changes
in the active sites can be particularly significant in thesesystems, so it is still necessary to solve the crystalstructures as demonstrated in this study.

(b)
S431

S431

ERQ

pT432

T432

pS431

C2PSwST

pST

SV

pST

pSpT

pSV

C2-Phosphorylation Cycle
in Wildtype-KaiC

KaiC-pSV

pS431(e)C1Domain
Slide0.6

ADPATP
R217

E214
Q394

Q394
0.60.30.30H2O(d)KaiC-pSV

T432(c)024 48 72 96

Time (h)Reduced Cycle with 2-statesin KaiC-SV (T432V)

0024 48 72 96

Time (h)Accelerated Cycle
in KaiCD30A,S157P-SV

Fig. 2. Master allostery integrating C1-ATPase cycle and C2-phospho cycle in KaiC. (a) Helical and coiled
structures found at the upstream region of S431. (b) Structural coupling between C1-domain slide andC2-phosphorylation switch. The direction of C1-domain slide is indicated by an arrow. (c) ATP, nucleophilicwater molecules, and ADP found in C1-ATPase cycle. The positional rearrangements of the waters arerepresented by red (high activity), green (middle activity), and blue (low activity) spheres. (d) Hydrogenbond switch among E214, R217, and Q394. (e) Scheme of the experiments verifying the identified allostery.
The period length of KaiC-SV oscillation was 47 hours in the presence of KaiA and KaiB. The mutations,
D30A and S157P introduced into C1, resulted in the reduction of the period length down to 29 hours.

Yoshihiko Furuike a,b,*, Atsushi Mukaiyama a,b,c
and Shuji Akiyama a,b
Research Center of Integrative Molecular Systems (CIMoS),
Institute for Molecular Scienceb
Department of Functional Molecular Science, SOKENDAI,The Graduate University for Advanced Studiesc
Department of Bioscience and Biotechnology,
Fukui Prefectural Universitya*Email: furuike@ims.ac.jp

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