Life ScienceResearch Frontiers 2015

Absolute slowness encoded in the circadian clock protein KaiC
Intracellular events are rhythmically regulatedby an oscillator with a 24 h period (circadian clock)to adapt to daily changes in the environment onEarth. The slowness of the clock oscillator is one of
the characteristics distinguishing it from well-known
faster chemical oscillators (with periods of seconds
or minutes) [1], raising a fundamental question about
the origins of slow but ordered rhythms. We thus
searched for determinants of the circadian periodusing a protein-clock system: the cyanobacterial
circadian clock can be reconstituted in a test tube bymixing three clock proteins, KaiA, KaiB and KaiC, withadenosine triphosphate (ATP) [2].
We found that the activity of ATP hydrolysis(ATPase) in KaiC is a determinant of the oscillatory
frequency (reciprocal of the period). As the ATPase
activity of KaiC itself is increased by a series ofsingle amino acid replacements in KaiC (Fig. 1), thefrequency of the in vitro protein-clock system (Fig.
1(a)) as well as that of the in vivo system (Fig. 1(b))
increased in a well-correlated manner. We thus
conducted X-ray crystallographic studies of KaiC [3] atbeamline BL44XU to search for the structural originsof the slow ATPase orchestrating the frequency of theintracellular events in cyanobacteria.

Frequency
in vitro (d–1)

1.2
1.0

S157P
Q153A
WT
T42S
S157C
S48T

(a)

0.8
0.6
0.4
0.2
0.0

Frequency
in vivo (d–1)

1.2
1.0

(b)

0.8
0.6
0.4
0.2
0.0
05

10152025

ATPase Activity of KaiC (d–1 KaiC –1)
Fig. 1. ATPase activity of KaiC governing thefrequencies of both protein-clock rhythms ina test tube (a) and intracellular rhythms (b).
These figures are reproduced from Ref. 3.

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We identified two structural factors slowing theATP hydrolysis reaction to the circadian time scale(Fig. 2(a)). The first is an unusual positioning of a lytic
water molecule surrounding a γ-phosphate of ATP
in the crystal structure with the P212121 space group(right inset in Fig. 2(a)). The lytic water molecule
is sequestered from an ideal position to attack aphosphorus atom of the γ-phosphate by hydrogenbonding to the carbonyl oxygen atom of F199 and to
the nitrogen atom (Nη) of R226 (Fig. 2(b)). In anothercrystal structure with the P3 121 space group, thelytic water molecule is positioned farther from theγ-phosphate of ATP than that in the P212121 crystal
structure (right inset in Fig. 2(a)). The impact of thesetwo different states on ATPase activity was studied byintroducing KaiC mutations perturbing their relative
stability (S157P, Q153A, and S157C in Fig. 1). On
the basis of the experimental evidence, we conclude
that the water positions in both states are basicallyunfavorable to the ATP hydrolysis (right inset in Fig.
2(a)) as they deviate considerably from the ideal
position, similarly to the behavior of other hyperactive
enzymes (right inset in Fig. 2(a)), and that theposition in the P3121 crystal structure is much more
unfavorable than that in the P212121 crystal structure.
The second is peptide isomerization, one of theslowest reactions in protein chemistry. The coupling ofATP hydrolysis to a cis-trans isomerization is clearlyreflected in the crystal structure of a KaiC hexamer
harboring ATP-bound and adenosine diphosphate(ADP)-bound subunits asymmetrically. In the prehydrolysis state, the peptide bond between D145 andS146 mainly adopted the cis-conformation, whereas,in the post-hydrolysis state, the peptide completely
adopted the trans-conformation (left inset in Fig. 2(a)).
Our molecular dynamics simulations indicate that thepre-hydrolysis state should overcome a barrier of 14
to 16 kcal·mol–1 for cis-trans isomerization itself and abarrier of 11 to 17 kcal·mol–1 for ATP hydrolysis [3].
The two structural origins uncovered in thepresent study indicate an unexpectedly high energy
barrier upon ATP hydrolysis concomitant withpeptide isomerization (Fig. 3). Assuming a frequency
factor of 1012 s–1, the barrier can be estimated to beapproximately 22 kcal·mol–1 using the rate constant of
0.5 h–1 (12 ATP d–1) at 30°C. Our results show that thecircadian time scale is designed in the atomic structureof KaiC to regulate the efficiency of ATP hydrolysis.
An analogous mechanism may be found in otherorganisms, because all of the key factors such asResearch Frontiers 2015

(a)

(b)
F199
R226Positions of lytic water moleculesin various ATPases

Peptide isomerization
between D145 and S146
cis trans

Oxygen atom oflytic water molecule

KaiC (P3121)~142°~3.9 Å

~156°

Kinesin
F1Myosin

Ideal position 3.0 Å

γ phosphate
O
180°
P O

Ideal position=~4.3 ÅKaiC (P212121)

O–

O

ATPγS

Sphere model of a phosphorus
atom of the γ-phosphate group–Fig. 2. Structural origins of slowness in a KaiC hexamer. (a) Unusual positioning of alytic water molecule relative to ATP (right inset) and coupling of the peptide isomerization
with the ATP hydrolysis reaction (left inset). (b) Steric hindrance surrounding aγ-phosphate of ATP. The black filled circle represents the ideal position for a lytic watermolecule to attack a phosphorus atom of the γ-phosphate of ATP. Van der Waals radii ofthe oxygen atom of the lytic water molecule, a carbonyl oxygen atom of F199, Nη of R226,
and a phosphorus atom of the γ-phosphate group of ATP γS are drawn using sphere models.

Free Energy

the water molecule, ATP and peptide isomerization
are ubiquitously shared and available for achievingabsolute slowness. In mammals, the acceptedmodel of the circadian clock is the transcriptional
and translational oscillation (TTO); the clock proteins
negatively regulate their own expression, thereby
producing rhythmic transcription and translation.
However, the slowness of the TTO model is often

Reaction via cis
Reaction via transquestioned because the transcriptional andtranslational events can proceed within minutes [4].
Recently, a circadian clock independent of the TTO inmammals was reported [5]. In the future, a structural
analysis focusing on the slowness and the correlation
with the period of a chemical reaction (the ATPhydrolysis reaction in this study) might reveal thedesign principle of the circadian clock.

cis-trans
isomerization
14 – 16 kcal/mol
ATP hydrolysis
11 – 17 kcal/mol

Reaction Coordinate
Fig. 3. Circadian time scale encoded in the atomic structure ofKaiC. The pre-hydrolysis state with the cis-conformation (red line)
overcomes a higher energy barrier (exceeding 22 kcal·mol−1) during
the reaction than that with the trans-conformation (blue line).

Jun Abe a, Atsushi Mukaiyama a,b and Shuji Akiyama a,b,*ab

Research Center of Integrative Molecular Systems (CIMoS),
Institute for Molecular Science
Dept. of Functional Molecular Science, SOKENDAI,The Graduate University for Advanced Studies*E-mail: akiyamas@ims.ac.jp

References[1] K.I. Agladze et al.: Nature 308, (1984) 834.
[2] M. Nakajima et al.: Science 308 (2005) 414.
[3] J. Abe, T.B. Hiyama, A. Mukaiyama, S. Son, T. Mori,S. Saito, M. Osako, J. Wolanin, E. Yamashita, T. Kondo andS. Akiyama: Science 349 (2015) 312.
[4] B. Schwanhäusser et al.: Nature 473 (2011) 337.
[5] J.S. O’Neill and A.B. Reddy: Nature 469 (2011) 498.

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