Article

Temperature-Sensitive Substrate and ProductBinding Underlie Temperature-CompensatedPhosphorylation in the Clock
Graphical Abstract

Authors
Yuta Shinohara, Yohei M. Koyama,
Maki Ukai-Tadenuma, ...,
Takashi Umehara, Kazuki Tainaka,
Hiroki R. Ueda

Correspondence
uedah-tky@umin.ac.jp

In Brief
The circadian clock is able tocompensate for fluctuations intemperature, and Shinohara et al. reveal
two underlying mechanisms: ‘‘lower
substrate affinity to CKId-ATP complex’’and ‘‘higher product affinity to CKId-ADP
complex.’’ They also identify a key CKIspecific domain that is necessary fortemperature compensation.

HighlightsdTemperature-sensitive affinity can counteract the speedup ofphosphorylationd

CKId agonist ATA is identified by inhibitor screening of
dephosphorylation activityd

K224D mutation significantly changed the temperaturedependency of circadian rhythmdTemperature compensation is conferred by inserting CKIspecific domain around K224

Shinohara et al., 2017, Molecular Cell 67, 783–798
September 7, 2017 ª 2017 Elsevier Inc.
http://dx.doi.org/10.1016/j.molcel.2017.08.009

Molecular Cell

ArticleTemperature-Sensitive Substrate and ProductBinding Underlie Temperature-CompensatedPhosphorylation in the ClockYuta Shinohara,1 Yohei M. Koyama,1,2,3 Maki Ukai-Tadenuma,1 Takatsugu Hirokawa,4,5 Masaki Kikuchi,6
Rikuhiro G. Yamada,1 Hideki Ukai,1 Hiroshi Fujishima,1 Takashi Umehara,6,7 Kazuki Tainaka,8,9 and Hiroki R. Ueda1,8,10,*
1Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
2Laboratory for Computational Molecular Design, RIKEN Quantitative Biology Center, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan
3Research and Development Project of Special Purpose Supercomputers for Drug Design, RIKEN Quantitative Biology Center,
6-2-4 Furuedai, Suita, Osaka 565-0874, Japan
4Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST),
2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
5Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305-8575, Japan
6Epigenetics Drug Discovery Unit, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku,
Yokohama 230-0045, Japan
7PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
8Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
9System Pathology for Neurological Disorders, Brain Research Institute, Niigata University, 1 Asahimachi, Chuo-ku, Niigata 951-8585, Japan
10Lead Contact*Correspondence: uedah-tky@umin.ac.jp
http://dx.doi.org/10.1016/j.molcel.2017.08.009

SUMMARY

Temperature compensation is a striking feature of
the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKId-dependent multi-site phosphorylationin mammals. We identify two mechanisms fortemperature-insensitive phosphorylation at higher
temperature: lower substrate affinity to CKId-ATP
complex and higher product affinity to CKId-ADP
complex. Inhibitor screening of ADP-dependent
phosphatase activity of CKId identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase
activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKId that impaired product binding andtemperature-compensated primed phosphorylation.
Importantly, K224D mutation shortens behavioral
circadian rhythms and changes the temperaturedependency of SCN’s circadian period. Interestingly, temperature-compensated phosphorylation
was evolutionary conserved in yeast. Molecular
dynamics simulation and X-ray crystallographydemonstrate that an evolutionally conserved
CKI-specific domain around K224 can provide
a structural basis for temperature-sensitive substrate and product binding. Surprisingly, thisdomain can confer temperature compensation ona temperature-sensitive TTBK1. These findings
suggest the temperature-sensitive substrate- andproduct-binding mechanisms underlie temperaturecompensation.

INTRODUCTION
The circadian clock is a molecular mechanism underlying
endogenous, self-sustained oscillations with a period of 24 hr
manifest in diverse physiological and metabolic processes. Its
striking and defining feature is its ability to maintain constantperiod length over a wide range of temperatures (known as‘‘temperature compensation’’) (Hastings and Sweeney, 1957;
Hogenesch and Ueda, 2011; Millius and Ueda, 2017; Pittendrigh,1954). This characteristic is evolutionarily conserved in a wide
range of organisms from photosynthetic bacteria (Kondo et al.,1993) to warm-blooded mammals (Izumo et al., 2003; Tsuchiya
et al., 2003). Although mammals are warm blooded, it is known
that temperature gradients exist between the body’s skin andcore (Lenhardt and Sessler, 2006), which indicates that the temperature compensation is also important for mammals to keep
the circadian rhythm among tissues. Since the rate of most enzymatic reactions doubles or triples with every 10 C increase intemperature (i.e., Q10 = 2–3) (Segel, 1975), the temperaturecompensation of the circadian period (Q10 = 0.8–1.4) (Dunlap
et al., 2004) has attracted the attention of a broad spectrum of
researchers from different disciplines (Hogenesch and Ueda,
2011; Millius and Ueda, 2017). However, despite many genetic
and molecular studies, the detailed biochemical mechanisms
underlying temperature compensation remain poorly understood (Hogenesch and Ueda, 2011; Millius and Ueda, 2017).
The simplest explanation for temperature compensation of
the circadian clock is that the key period-determining reactions

Molecular Cell 67, 783–798, September 7, 2017 ª 2017 Elsevier Inc. 783

are insensitive to temperature. Indeed, Pittendrigh proposed
the existence of a temperature-insensitive component in theclock system in 1954 (Pittendrigh, 1954), and in 1968, he andhis colleagues demonstrated that both the wave form and theperiod of a circadian oscillation are invariant with temperature(Zimmerman et al., 1968). However, the idea of temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature sensitive(Segel, 1975). Exceptions first came from the observationsof the cyanobacterial clock, where temperature-insensitive
biochemical reactions have been observed in vitro (Nakajima
et al., 2005; Tomita et al., 2005). Although the clock proteinsin cyanobacterial clock seem distinct from other clock systems,
the temperature-insensitive biochemical reactions were also
discovered in mammals, where we observed that CKId/
ε-dependent phosphorylation, a period-determining process
in the mammalian circadian clock, had temperature compensation in vitro and in cellulo (Isojima et al., 2009). Here, we show
two mechanisms underlie temperature compensation: ‘‘lower
substrate affinity to CKId-ATP complex at higher temperature’’and ‘‘higher product affinity to CKId-ADP complex at higher
temperature.’’RESULTS
Temperature-Sensitive Binding of a Substrate to an
Enzyme Underlies the Temperature Compensation of
CKId-Dependent Single-Serine Phosphorylation
In a previous study, we demonstrated that CKId-dependent
phosphorylation was temperature compensated in vitro (Isojima
et al., 2009). Interestingly, temperature dependency of the phosphorylation by the catalytic domain of wild-type CKId (DCKId;
2–317 residues of CKId to delete C-terminal regulatory
domain) is highly affected by the peptide sequence of theirsubstrate. For example, although the phosphorylation of casein
by DCKId is highly temperature sensitive, it is temperaturecompensated for bTrCP- (RKKKPHSGSSGYGSLGSNGSHEHLMSQTSSSDSN) and FASPS- (RKKKTEVSAHLSSLTLPGKAESVVSLTSQ) peptides that include typical CKI-substrate motif
S-X-X-S (Gallego and Virshup, 2007). However, these peptides
contain more than twelve and nine possible phosphorylationsites, respectively, whose complexity confounds a detailed
biochemical understanding of temperature-compensated phosphorylation. Therefore, in this study, we introduced derivatives ofa simple synthetic peptide (CKItide; KRRRALpSVASLPGL),
which is used as a substrate for CKI. In the following analysis,
we introduced a CKItide derivative (*CKItide; KRRRALSVASLPGL), which includes only a single S-X-X-S motif. In orderto precisely quantify the amount of substrate and phosphorylated peptides, we used a capillary electrophoresis-based
mobility shift assay system (Figure S1A). The initial velocityof DCKId-dependent phosphorylation from a two-serine
substrate *CKItide to double-phosphorylated product CKItide(2pS) (KRRRALpSVApSLPGL) at low-concentration substrate(20 mM) seemed highly temperature compensated over a wide
range of temperatures from 20 C to 45 C (Figure S1B). Initial velocities were also temperature compensated for a wide range ofsubstrate concentrations (Figures 1A and S1C), although the

784 Molecular Cell 67, 783–798, September 7, 2017

substrate inhibition observed for 640 mM at 25 C indicated thedeviation from the Michaelis-Menten model.
We further simplified the two-serine substrate because it
seemed still too complex to determine the detailed kinetic parameters for each process. We thus prepared a series of single-serine substrates, *CKItide(S10X) (KRRRALSVAXLPGL), bymutating the second serine (at position 10) of *CKItide to anamino acid X. As a negative control, we utilized DTTBK1 (a catalytic domain of wild-type Tau-tubulin kinase 1, TTBK1) because
TTBK family is one of the closest neighbors to CKI family
in mammalian kinases (Manning et al., 2002). Interestingly, thephosphorylation reactions of all single-serine substrates by
DCKId were also temperature compensated, whereas those by
DTTBK1 were temperature sensitive (Figure 1B). These results
suggest that a single-serine substrate is sufficient for temperature-compensated, DCKId-dependent phosphorylation.
To determine each kinetic parameter (Figure 1C) of phosphorylation activity of DCKId, which showed ATPase activity (Figure S1D), we used a single-serine substrate *CKItide(S10A)(KRRRALSVAALPGL) and evaluated how its phosphorylation isdependent on the concentration of ATP (Figures 1D and S1E).
Interestingly, the catalytic efficiency (kcatTS/KmTS) was temperature compensated whereas the catalytic constant (kcatTS) andthe Michaelis constant (KmTS) were highly sensitive to temperature (Figure 1D). Hence, the apparent temperature compensation of kcatTS/KmTS would be attributed to the counterbalance
between the temperature-dependent increase in kcatTS andtemperature-dependent decrease in the affinity between DCKId
and a single-serine substrate (1/KmTS).
To validate the role of enzyme-substrate binding in the temperature compensation of DCKId-dependent phosphorylation,
we measured the phosphorylation activity of a series of alanine
mutants of *CKItide by DCKId (Figure S1F). Since the phosphorylation of R4A, S7A, and V8A mutant peptides was remarkably
suppressed, we considered that these residues would participate in the enzyme-substrate binding. The previous study
demonstrated that the phosphorylation activity of CKI was
increased by introducing a negatively charged residue such as
aspartate and glutamate to the 3 position from the residue tophosphorylate in the substrate (Flotow and Roach, 1991). We
focused on the arginine at the position 4 (R4) because it was
located at the 3 position from the serine at position 7 of the single-serine substrate *CKItide(S10A). We synthesized a new
substrate, *CKItide(R4D,S10A), by replacing the arginine at theposition 4 (R4) with the aspartate (D). Although kcat/Km in thephosphorylation of the single-serine substrate *CKItide(S10A)
was temperature compensated (Figure 1E), that of *CKItide(R4D,S10A) was sensitive to temperature with temperature-independent Km (Figure 1F). We next focused on the serine at
the position 7 (S7) because it was located at the 3 position
from the second serine (S10). We synthesized a new substrate,*CKItide(S7D), by replacing S7 with the aspartate (D). We foundthat kcat/Km in the phosphorylation of *CKItide(S7D) was sensitive to temperature with temperature-independent Km (Figure S1G). Taken together, temperature-compensated phosphorylation of the single-serine substrates could be achievedby the temperature-sensitive interaction between enzyme andsubstrate.

0.0
0

200
400
600
Substrate (μM)
n = 3 (± SEM)C

E
kaD[D]

kdD

ED

kD

kaDP[P] kdDP
EDP

0

0

kcatTS ([T])

k [T]kdT aTET

KmTS ([T])ΔTTBK1
ΔCKIδ
1
2
3
4
Relative Activity at 25˚Cn = 3 (± SD)k= KcatTS
mTS[T]
KT + [T]KmTS ([T]) = KmTS

ETS

0.002

KT + [T]

0

100

1000

5
35˚C
25˚C

0

200 300
ATP (μM)

0

100

70

0.010

0.005

KTS + [T]

40
30
20

25˚C 35˚C

Q10 = 1.17 ± 0.11

0

60
40

0

25˚C 35˚C

Q10 = 1.27 ± 0.42

35˚C
25˚C
0

2000

80

100

8000

1500
1000

0

25˚C 35˚C

Q10 = 5.82 ± 1.51

200 300
ATP (μM)

10000

500

20

10
0

6000
4000
2000
0

25˚C 35˚C

Q10 = 3.93 ± 1.45

25˚C 35˚C

Q10 = 4.95 ± 1.36

F

0

400
800
1200
Substrate (μM)
n = 3 (± SEM)

2

0

0

300
200
100
0

25˚C 35˚C
Q10 = 1.07 ± 0.04

25˚C 35˚C
Q10 = 1.49 ± 0.03

25˚C 35˚C
Q10 = 1.40 ± 0.06

n = 3 (± SEM)n = 3 (± SEM)n = 3 (± SEM)

4

0.015

25

3
2
1

35˚C
25˚C

0
0

200
400
600
Substrate (μM)
n = 3 (± SEM)

2500

n.s.

2000

20
*

0.010

15
10

Km (μM)

400

0.020
-1

*

kcat (min-1)

4

0.005

500

***

*CKItide(R4D, S10A)KRRDALSVAALPGL
6 S0 → S1
5

kcat/Km (min μM )

35˚C
25˚C

0.010

6

600
Initial velocity (μM/min)

1

0.015

n.s.
kcat (min-1)

2

kcat/Km (min-1 μM-1)

3

0

8

0.020

Km (μM)*CKItide(S10A)KRRRALSVAALPGL
4 S0 → S1

-1

E

50

500
0

100 200 300
ATP (μM)

60

E : Enzyme, T : ATP, D : ADP, S : Substrate, P : ProductInitial velocity (μM/min)

10

35˚C
25˚C

0.000

KmTS ([T]) (μM)

0.004

0.015

[T]
kcatTS ([T]) = kcatTS
KTS + [T]kdTS kaTS[S]
kDP

2

0.006

S0 → S1
1500

KmTS (μM)

35˚C
25˚C

15 S0 → S1

KTS (μM)

1.0

Q10 = 1.0

4

S0 → S1
0.008

kcatTS (min-1)

1.5

6

KT (μM)

2.0*CKItide(S10A)
1 serine substrate KRRRALSVAALPGL

kcatTS ([T]) / KmTS ([T])(min-1 μM-1)

Relative Activity at 35˚C

Initial velocity (μM/min)

8

2.5 S0 → S2

0.5

D*CKItide(S10X)KRRRALSVAXLPGL
X = variable a.a.
Q10 = 2.0

kcatTS ([T]) (min-1)

B*CKItide (2 serine substrate)
KRRRALSVASLPGL

kcatTS-1-1
KmTS (min μM )

A

n.s.

1500
1000

0.005

5

500

0

0

25˚C 35˚C
Q10 = 1.55 ± 0.12

25˚C 35˚C
Q10 = 1.80 ± 0.75

025˚C 35˚C
Q10 = 1.16 ± 0.41

n = 3 (± SEM)n = 3 (± SEM)n = 3 (± SEM)

Figure 1. Temperature-Sensitive Binding of a Substrate to an Enzyme Underlies the Temperature Compensation of CKId-Dependent SingleSerine Phosphorylation(A) Substrate saturation curves of DCKId-dependent phosphorylation on a two-serine substrate *CKItide at 25 C and 35 C.
(B) Temperature dependency of DCKId- or DTTBK1-dependent phosphorylation for a series of single-serine substrates, *CKItide(S10X). Activities at 25 C for*CKItide were set as 1.0 in the relative activity. Data are mean ± SD (n = 3).
(C) A computational model of single-serine phosphorylation (left) and its kinetic parameters (right).
(D) Temperature and ATP dependency of kinetic parameters (upper), and ATP-saturation kinetic parameters in (C) (lower) for DCKId-dependent phosphorylation
on single-serine substrate *CKItide(S10A). Data are parameter ± SE estimated from the nonlinear least-square fitting (n = 1).
(E and F) Substrate saturation curves (left) and kinetic parameters (right) of DCKId-dependent phosphorylation on single-serine substrate *CKItide(S10A) (E) and*CKItide(R4D, S10A) (F) with 640 mM ATP at 25 C and 35 C.
For (A), (E), and (F), data are mean ± SEM (n = 3); *p < 0.05, ***p < 0.001, and n.s. (not significant); two-tailed Welch’s t test. Solid line and shade represent mean ±SEM of curves of Michaelis-Menten model with estimated parameters.
See also Figure S1.

Temperature-Sensitive and ADP-Dependent Binding ofa Product to an Enzyme Underlies the Temperature
Compensation of CKId-Dependent Multi-site
Phosphorylation
Multi-site phosphorylation after priming phosphorylation seems
critical in mammalian circadian clock because we recently foundthat the multi-site phosphorylation in a clock protein, CRY1,
worked as a molecular timer by additively contributing to theperiod determination of mammalian circadian clocks (Odeet al., 2017). Therefore, we investigated CKItide, which contains
a phosphorylated serine. We found that the production rate of
CKItide(2pS) decelerated only after 10 min (Figure S2A), which
was in contrast to the almost constant production rate of
CKItide(2pS) from *CKItide (Figure S1C). Although the observed
non-linear behavior made it difficult to determine the accurate
kinetic parameters, the phosphorylation of CKItide was also

temperature compensated, especially for higher substrate concentrations described by kcat (Figure 2A).
Initial velocities of CKItide phosphorylation both at 35 C and25 C were significantly decelerated by the addition of ADP or a
mixture of ADP and CKItide(2pS) (Figures 2B, S2B, and S2C), suggesting the phosphorylation of CKItide could be inhibited by ADP
and CKItide(2pS). Isothermal titration calorimetry (ITC) revealed
that DCKId and ADP form a stable binary complex with temperature-insensitive affinity (Figure S2D). Interestingly, the ADPdependent binding of CKItide(2pS) to DCKId was strengthened
for higher temperature (Figure 2C). In addition, the binding was
exothermic, and enthalpically and entropically favored (Figure 2C).
Since the binding of a product to an enzyme is the first step toward a reverse reaction, we hypothesized that the high affinity of
CKItide(2pS) to DCKId may result in the reverse reaction(dephosphorylation) of CKItide(2pS) by DCKId. To verify this

Molecular Cell 67, 783–798, September 7, 2017 785

B

50
100
150
Substrate (μM)
n = 3 (± SEM)

10

60

400

n.s.

40

200

20

0
25˚C 35˚C
Q10 = 1.42 ± 0.14

025˚C 35˚C
Q10 = 1.33 ± 0.13

025˚C 35˚C
Q10 = 0.94 ± 0.05

n = 3 (± SEM)n = 3 (± SEM)n = 3 (± SEM)

5

0.2

50

60

0

10

50

μcal/sec

−0.02
−0.04
−0.06

-0.20

25˚C
0.0

0.5

1.0
1.5
2.0
Molar Ratio
Kd = 11.9 μM
N = 1.29 sites
ΔH = -234.7 cal/mol ΔS = 21.7 cal/mol/deg

0.0

15
10
5

2

2p

0.2
0.1
25˚C

0.0
0

e(

S1

0A

S)

)

500

e(

10
50
100
ADP (μM)n = 3 (± SEM)

tid

5

KI

0

0

C

0

0.3

tid

4

20

Dephosphorylation [E]0 = 0.2 μM
S2 → S1
0.4

C
KI

6

Initial velocity (μM/min)

*

8

40
20
(1)

(2)

160
320
ADP + S2 (μM)
n = 5 (± SEM)

Reaction time 30 min
(2)

80
60
40
20

(1)

3.0
2.0
1.0

*

3.0
2.0
1.0
0.0

0
30
Reaction Time (min)n = 3 (± SEM)

G
Dephosphorylationn = 3 (± SEM)

25

10

60

0
30
Reaction Time (min)n = 3 (± SEM)

1.0
1.5
2.0
2.5
Molar Ratio
N = 0.91 sites
Kd = 7.3 μM
ΔH = -256.0 cal/mol ΔS = 22.7 cal/mol/deg

Conversion (%)

Conversion (%)

12 S2 → S1

80

0.0

0.5

F

E

100

n.s.

35˚C

100
200
300
S2 (μM)n = 3 (± SEM)

[E]0 = 0.05 μM
Initial velocity (μM/min)

-0.20

0.00

Signal intensity ofMass spectrometry

kcal mol-1 of injectant

ΔCKIδ + ADP + CKItide(2pS)-0.15

Reaction time 0 min

80

0
45.5
46.0
46.5
45.5
46.0
46.5
Retention Time (min)Retention Time (min)88(x 10 ) (1)ALSVApSLPGL(x 10 ) (2)ALpSVASLPGL
4.0
4.0

−0.08
ΔCKIδ + ADP + CKItide(2pS)

-0.10

160
320
ADP (μM)n = 5 (± SEM)

0

-0.02
-0.05

80

100

60

0.00

-0.01

0
n=3
(± SEM)

0.20
0.15
0.10
0.05
0.00
0

50

100
S2 (μM)n = 3 (± SEM)

35˚C
150

1000

0.20

0.25 S2 → S1
kcat/Km (min-1 μM-1)

10Time (min)
20
30
40

Relative AbundanceTime (min)
20
30
40

0.00
μcal/sec

0.4

D0kcal mol-1 of injectant

0.6

0.0

C

**
n.s.

800

0.15
**
0.10
0.05
0.00

25˚C 35˚C
Q10 = 9.01 ± 1.08
n = 3 (± SEM)

Km (μM)

0600

Relative Abundance

35˚C
25˚C

0.0

*
15

1.0 [S1] = 20 μM
0
35˚C
0.8

Signal intensity ofMass spectrometry

1.0

Initial velocity (μM/min)

1.5

80

*

Km (μM)

2.0

0.5

800

20

2.5 S1 → S2

kcat/Km (min-1 μM-1)

Initial velocity (μM/min)

KRRRALpSVASLPGL

kcat (min-1)

A

n.s.

600
400
200
0
25˚C 35˚C
Q10 = 0.13 ± 0.05
n = 3 (± SEM)

Figure 2. Temperature-Sensitive and ADP-Dependent Binding of a Product to an Enzyme Underlies the Temperature Compensation of CKIdDependent Multi-site Phosphorylation(A) Substrate saturation curves (left) and kinetic parameters (right) of phosphorylation on a single-phosphoserine substrate CKItide at 25 C and 35 C.
(B) Initial velocities of CKItide phosphorylation at 35 C with initial ADP or mixture of ADP and CKItide(2pS).
(C) ITC assay for the binding of CKItide(2pS) to DCKId in the presence of ADP at 25 C (left) and 35 C (right).
(D) Mass spectrometry for dephosphorylated peptides produced by DCKId from CKItide(2pS) in the presence of ADP.
(E) ADP concentration dependency of dephosphorylation activity by DCKId on CKItide(2pS).
(F) Substrate specificity of ADP-dependent dephosphorylation activity by DCKId.
(G) Substrate saturation curves (left) and kinetic parameters (right) of DCKId-dependent dephosphorylation on a CKItide(2pS) at 25 C and 35 C.
For (A), (B), and (D)–(G), data are mean ± SEM (n = 5 for B with ADP or ADP + S2 and n = 3 otherwise); *p < 0.05, **p < 0.01, and n.s. (not significant); two-tailed
Welch’s t test. For (A) and (G), solid line and shade represent mean ± SEM of curves of Michaelis-Menten model with estimated parameters. See also Figure S2.

hypothesis, we incubated double-phosphorylated CKItide(2pS)
in the presence of 1 mM ADP and then measured the stateof the peptide by mass spectrometry. We noted that a singlephosphorylated pS-X-X-S product was specifically yielded(Figure 2D). In addition, the dephosphorylation was significantly facilitated by an ADP concentration (Figure 2E), much
more favorable in CKItide(2pS) compared to CKItide(S10A)(KRRRALpSVAALPGL) (Figure 2F), and markedly enhanced by

786 Molecular Cell 67, 783–798, September 7, 2017

the increase of temperature (Figure S2E). According to kcat/Km,
the observed dephosphorylation was highly temperature sensitive (Figure 2G). In particular, Km values indicate that an affinity
of CKItide(2pS) to DCKId-ADP at 35 C is higher than that at
25 C, which is consistent with the ITC assay (Figure 2C). Therefore, temperature-compensated phosphorylation of CKItide
might be explained by the balance between thermal accelerationof phosphorylation and thermal retardation of DCKId turnover

B

EDS1
EDS2

kaDS2[S2]

kDS1
kTS1
kDS2

ETS0
ETS1

kdTS1

kaTS1[S1]E : Enzyme, T : ATP, D : ADP,
Si : i-sites phosphorylated peptide

Phosphorylation (S0 → S2)[S0]0 = 20, 40, 80, 160, 320 μM
WT
2.5
2.5

2.0
1.5

1.0

1.0

0.5

1/2-fold at 35˚C 0.5

0.8
0.5
5

10 15 20
Time (min)

25

0

5

10 15 20
Time (min)

25

kTS1

kdDS2

kaDS2

kDS2

kdTS1

kaTS1

kdDS1

2.0
1.4 1.5

1.0

kdTS0

2.5

1.5

Phosphorylation (S0 → S1)
Q10 (WT) = 1.24 ± 0.07

1/2-fold at 35˚C

kDS1

2.5

2.0

2-fold at 35˚C

2.0

1.0

1.0

0

5

10 15 20
Time (min)1/2-fold at 35˚C

0.5

25

2-fold at 35˚C

1.5

1.5

2-fold at 35˚C

kdDS1

2.5

0

5

10 15 20
Time (min)1/2-fold at 35˚C

0.5

25

0

5

10 15 20
Time (min)251400 Q10 (WT) = 1.21 ± 0.29

15 Q10 (WT) = 1.50 ± 0.29

kcat (min-1)

0.03
0.02
0.01

Km (μM)

1200

10

5

1000
800
600
400

kaTS0

kdTS0

kDS1

kdDS1

kdDS1

kaTS1

kdTS1

kD

k dT

WT

kdD

1/2-fold at 35˚C

kaDS1

WT at 35˚C

kaT

kdDS1

0

kdD

WT at 25˚C

kDS1

kdD

kaT

WT

0

kDS1

kaTS0

kdTS0

kD

kdD

kdT

0.00

WT

200

kaT

kcat/Km (μM-1 min-1)

0.04

kaTS0
2-fold at 35˚C

2.0

0

C

kDS1

kdTS0 kaTS0[S0]

kaDS1

0

kdTS0

20
kdD

kaDS1[S1] kdDS1

kdDS2

ET

kD

kaTS0

kdT

kaD

kdD

ED

60
40

kD

kaT[T]

Q10 of [S2] for S0 → S2

kaD[D]

80

WT at 35˚C
1/2-fold at 35˚C
2-fold at 35˚C

kdT

E

100

WT

S0 → S2

120

kaT

RMSRE (%) of [S2] for S0 → S2A

2-fold at 35˚CD
150 Q10 (WT) = 0.71 ± 0.31

2500 Q10 (WT) = 1.21 ± 0.39

50

2000

WT at 35˚C

kTS1

kdDS2

kDS2

kD

kdT

0

WT

kdDS2

kTS1

kDS2

kdTS1

kaTS1

kdDS1

50

kaT

Km (μM)WT at 25˚C

kaDS1

kdDS2

kTS1

kDS2

kdTS1

kaTS1

kD

kdD

0

kdT

0

WT

500
kdD

1000

10

kD

20

100

1500

kaT

30

WT

kcat (min-1)

40

kaT

kcat/Km (μM-1 min-1)

Phosphorylation (S1 → S2)
60 Q10 (WT) = 1.70 ± 0.19

1/2-fold at 35˚C 2-fold at 35˚C

Figure 3. A Computational Model Recapitulated the Importance of Substrate and Product Binding Mechanisms in Temperature-Compensated, CKId-Dependent Multi-site Phosphorylation(A) A computational model for temperature-compensated, CKId-dependent multi-site phosphorylation.
(B) Perturbation analysis of the product concentration ([S2]) from S0 to S2 phosphorylation by changing each rate parameter value 2-fold or 1/2-fold at 35 C. The
upper panel shows root-mean-square relative errors (RMSREs) from experimental product concentration by the perturbation. Lower panels show the ratio (Q10) of
perturbed mean time course of [S2] at 35 C to unperturbed one at 25 C. The shaded region (pink) indicates that the reaction is temperature compensated (0.8 %Q10 % 1.4) in this region.
(C and D) Perturbation analysis of kinetic parameters in a priming phosphorylation from S0 to S1 (C) and a second phosphorylation from S1 to S2 (D) by changing
each rate parameter value 2-fold or 1/2-fold at 35 C.
For the upper panel in (B), and (C) and (D), data are mean ± SD (sampled from 108 step MCMC simulation with 10,000 step interval).
See also Figures S3 and S4.

due to the stable product binding, which might also trigger thedephosphorylation of the product.
A Computational Model Recapitulated the Importance ofSubstrate and Product Binding Mechanisms in
Temperature-Compensated, CKId-Dependent Multisite Phosphorylation
To clarify which reaction process contributes to temperaturecompensation of complicated two-step phosphorylation of*CKItide or non-linear phosphorylation of CKItide, we attempted
to construct a comprehensive computational model based on
experimental data (Figures 3A and S3A–S3I; STAR Methods).
*CKItide and its phosphorylated forms were simply described
as S0, S1, and S2, respectively, corresponding to the numberof phosphate groups in the peptide.
In order to understand the molecular mechanism underlying
the observed temperature compensation in DCKId-dependent
phosphorylation of *CKItide (Figure 1A), we first focused on the

Molecular Cell 67, 783–798, September 7, 2017 787

multi-site phosphorylation from S0 to S2 at 35 C. The perturbation of kaTS0, kdTS0, kDS1, and kdDS1, all of which related to S0 to
S1 reaction, affected the concentration of S2 (Figure 3B), suggesting the rate-limiting step in this multi-site phosphorylation
would be priming phosphorylation from S0 to S1. Consistent
with the initial crude analysis of experimental data (Figure 1E),
kcat/Km for the priming phosphorylation (Figure 3C) was temperature compensated, whereas kcat/Km for other reactions such as
ATPase activity (Figure S4A) or dephosphorylation from S2 to S1(Figure S4B) was temperature sensitive. The perturbation of association (kaTS0) and dissociation (kdTS0) rate between substrate
S0 and ATP-bound enzyme (ET) strongly affected Q10 of kcat/Km(Figure 3C). These results imply that temperature-sensitive substrate-binding mechanism (weaker at higher temperature) could
effectively counteract temperature-sensitive enzyme activity(faster at higher temperature).
In order to understand the molecular mechanism underlying
the observed temperature compensation in DCKId-dependent
phosphorylation of CKItide (Figure 2A), we next focused on
the phosphorylation from S1 to S2 at 35 C. Compared with
S0 to S2 (Figure 3B), the perturbation of various rate parameters
affected the concentration of S2 for S1 to S2 (Figure S4C). We
noted that kcat for S1 to S2 (Figure 3D) was temperaturecompensated, which is consistent with the initial crude analysisof experimental data (Figure 2A). Interestingly, the perturbationof dissociation rate (kdDS2) between product S2 and ADP-boundenzyme (ED) strongly affected Q10 of kcat (Figure 3D). Furthermore, by slowing down kdDS2 (i.e., strengthen the affinity between S2 and ED), kcat at 35 C decreased and lowered Q10.
In addition to the kinetic parameter, Q10 of mean time courseof S2 concentration also decreased for higher substrate concentrations by slowing down kdDS2 (dashed lines in panel
kdDS2 in Figure S4C). These results imply that the temperature-sensitive product-binding mechanism (stronger at higher
temperature) could effectively counteract with the temperature-sensitive enzyme activity (faster at higher temperature).
The detailed analysis revealed that the temperature-sensitive
substrate-binding mechanism would be important for a priming
phosphorylation, whereas the temperature-sensitive productbinding mechanism would be critical for the following
phosphorylation.
Identification of a Chemical, ATA, that Inhibits ADPDependent Product Binding of CKId by Comprehensive
Chemical Screening
If we could find specific chemicals competing with putative
substrate- and product-binding processes, we could use
such chemical inhibitors to identify putative substrate- andproduct-binding sites and further investigate the underlying
molecular mechanisms. Previously, we have discovered
several CKId inhibitors from LOPAC1280 library, which would
potentially antagonize the substrate-binding step (Isojima
et al., 2009). However, temperature compensation of the
mammalian circadian clock was still preserved even underhigh concentrations of such potent inhibitors. Therefore, we
focused on chemicals competing with a stable complex formation between DCKId-ADP and product. Such chemicals werehypothesized to inhibit the dephosphorylation, and therefore

788 Molecular Cell 67, 783–798, September 7, 2017

would work as a CKId agonist by acceleration of DCKId turnover in phosphorylation. Based on this hypothesis, we firstscreened 1,259 chemical compounds in LOPAC1280 libraryfor chemical inhibitors of dephosphorylation activity (Figure 4A). The chemical screening reliably identified 13 candidates, including known CKId kinase inhibitors such as
SB 202190 and SP600125 (Bain et al., 2007) (Figures S5A
and 4B). We evaluated the inhibitory specificity of dephosphorylation against phosphorylation among these chemicals andcharacterized aurintricarboxylic acid (ATA) as a dephosphorylation-specific inhibitor (Figures 4C and 4D). As we expected,
ATA strongly inhibited dephosphorylation activity and inversely
enhanced phosphorylation activity of DCKId in a dose-dependent manner (Figures 4E and 4F). Importantly, we found that
ATA significantly changed Q10 of DCKId-dependent phosphorylation (Figure 4F), supporting the significant contribution of astable product-binding process to temperature-compensatedphosphorylation predicted by the computational model.
Identification of Putative ATA-Binding Site of CKId by In
Silico Docking Simulation
According to ITC assay, ATA could bind to DCKId with a stoichiometry of approximately 2:1 (Figure 4G). High concentration of
ATA could inhibit ATPase activity of DCKId in addition to an inhibition of dephosphorylation activity, implying an ATA molecule
could bind to an ATP-binding site of DCKId (Figure S5B). To
predict another ATA-binding site of DCKId, we searched putative druggable sites on DCKId-ATP complex, which wereroughly divided into five binding regions (A to E) (Figure 4H).
Among these putative ATA-binding sites, C5 site displayed
the smallest binding-free energy (Figure 4I). The same bindingsite was also predicted by an ATA analog (NS-05199909), which
also enhanced phosphorylation activity (Figures S5C–S5E). In
these docking simulations, the carboxyl group of ATA andNS-05199909 was estimated to make hydrogen bonds to
the guanidinium group of Arg178, the amino group of Lys130
and Lys224, and the amide backbone of Gly215 (Figures 4I
and S5E).
Comprehensive Genetic Screening Identified K224D
and K224E Mutants of CKId that Impaired ADPDependent Product Binding and Temperature
Compensation of Multi-site Phosphorylation
Previously reported crystal structures of mammalian CKId haveidentified four binding sites of phosphate analogs (Long et al.,2012; Longenecker et al., 1996) (Figure 5A). Combined with theprediction of ATA-binding site, R178 and K224 residues were
found to be the most prominent candidates for mediating
enzyme-phosphopeptide binding (Figure 5B).
R178C mutant of CKIε, which is well known as tau mutant(Lowrey et al., 2000), shortens the circadian period of mammalian circadian clocks by destabilizing PER protein (Meng et al.,2008). Although tau mutant in a Syrian hamster affected temperature compensation in the circadian period of peripheral
clocks (Tosini and Menaker, 1998), the mouse tau mutant(Meng et al., 2008) had an intact temperature-compensatedcircadian period in vivo and temperature-compensated phosphorylation of bTrCP-peptide in vitro (Isojima et al., 2009)

B
Chemical screening workflow
CKItide(2pS) + ADP in 384-well plates
Chemical library
1,259 chemicals
ΔCKIδ
Incubation at 35˚C

Reaction stop

Mobility shift assay

C
Normalized
PP activity

Name

Function

Aurintricarboxylic acid (ATA)

Inhibitor of TopoII

0.290

Farnesylthiosalicylic acid

Antagonist of Ras

0.356

PD 169316

Inhibitor of p38 MAPK

0.358

SP600125

Inhibitor of c-JNK
Antagonist of P2Y

0.412

Reactive Blue 2
SB 202190

Inhibitor of p38 MAPK

Morin

0.513

Me-3,4-dephostatin

Inhibitor of Antioxidant
Inhibitor of PP1B/SHPTP-1(±)-8-Hydroxy-DPAT hydrobromide

Agonist of 5-HT1A

0.542

N-arachidonylglycine

Inhibitor of FAAH

0.580

GW5074

Inhibitor of Raf1 kinase

0.601

6-Hydroxy-DL-DOPA

Neurotoxin

0.683

MRS 2211 sodium salt hydrate

Antagonist of A1

0.699

0.5

SP600125
SB 202190

0

0
0.5
1.0
1.5
2.0
Normalized Dephosphorylation activity
n = 3 (± SD)

G

1.2

S2 → S1

35˚C

1.0
0.8
0.6
0.4
0.2
0

0

5 10 15 20 25 30
ATA concentration (μM)
n = 3 (± SEM)

H
0.05

0

Time (min)
10 20
30

40

A

-0.15

3.0

OH
Aurintricarboxylic acid (ATA)*CKItide 20 μM
S0 → S2

2.5 S0 → S2

2.0
1.5
1.0
0.5
0

35˚C
25˚C
0

1.5

ΔCKIδ
1.85 ± 0.09

2.0
1.27 ± 0.03

1.0
0.5
0.0

5 10 15 20 25 30
ATA concentration (μM)
n = 3 (± SEM)

DMSO

C1(0.9)ATA (25 μM)

Lys130 ATA(C5)C2(-19.2)
C-0.20
kcal mol-1 of injectant

HO

2.5

ATP
A(-19.0)

ATP

-0.10

C3(-16.5)

ΔCKIδ + ATA

-0.80

D

B

-1.00

E

-1.20
-1.40

OH

50

-0.05

-0.60

HO

0.529

I

0.00
μcal/sec

Phosphorylation (ATA / DMSO)

1.0

O

F
Dephosphorylation (ATA / DMSO)Normalized Phosphorylation activity
n = 3 (± SD)

1.5

ATA

O

1.000

E
2.0

O
OH

0.425
0.464

DMSO

D

O

Q10(Initial velocity)

A

35˚C

B(-15.7)
D(-11.4)
C5(-28.9)

C4(-19.8)
E(-25.3)

Arg178

Lys224Gly2150.0

0.5
1.0 1.5
2.0
Molar Ratio
Kd = 14.2 μM
N = 1.91 sites
ΔH = -1405 cal/mol
ΔS = 17.6 cal/mol/deg

Figure 4. Identification of ATA that Inhibits ADP-Dependent Product Binding of CKId by Comprehensive Chemical Screening and Putative
ATA-Binding Site of CKId by In Silico Docking Simulation(A) The workflow of the chemical inhibitor screening against DCKId-dependent dephosphorylation activity.
(B) The first chemical screening identified 13 candidates (over 3 SD).
(C) Chemical structure of ATA.
(D) The second chemical screening to evaluate the specificity of chemical inhibitors to dephosphorylation activity against phosphorylation activity, whichcharacterized ATA as a dephosphorylation-specific inhibitor. Data are mean ± SD (n = 3).
(E) DCKId-dependent dephosphorylation on a double-phosphorylated CKItide(2pS) was inhibited by ATA in a dose-dependent manner. Data are mean ± SEM(n = 3).
(F) Temperature and ATA concentration dependency of DCKId-dependent phosphorylation on *CKItide. Data are mean ± SEM (n = 3).
(G) ITC assay for the binding of ATA to DCKId at 35 C.
(H) Putative druggable sites on DCKId-ATP complex identified by SiteMap program, which were roughly divided into five binding regions (A to E).
(I) Putative ATA binding sites identified by in silico docking simulation. The binding-free energy (kcal/mol) was indicated in parentheses (left). The detailed
structure of putative ATA-binding site C5 with the lowest binding-free energy was indicated in the right panel. See also Figure S5.

despite a significant acceleration of circadian period (Menget al., 2008) and phosphorylation reaction (Isojima et al.,2009). Thus, there remains a discrepancy between the relationship of phosphorylation-dependent period acceleration by thetau mutation to temperature compensation in the mammalian
circadian clock.
We examined a series of CKId mutants of K224 as wellas R178 for their effect on temperature dependency of

Molecular Cell 67, 783–798, September 7, 2017 789

A

B
site 2

site 1
K130
R98

R178

H46

K221

WO42-

WO42-

H126
K224
K217

H185

R127

R227

K232

K155

K154
K171
R168

site 3

K122

R160

site 4

R270
R192

H162
H164

WO42-

R274

K263

site 1

H278

SO42-

R259

C

H120
K122
R157
R160
site 3 H162
H164
R192
R259
K263

H120

R157

site 3site 4

R115

15

H46
H126
R127
site 2 K154
K155
R168
K171

K231

site 2

Distance (Å)
5
10

0
R98
K130
R178
H185
K217
site 1 K221
K224
R227
K231
K232

R115
site 4 R270
R274
H278
WO42-

SO42- (3UYS)

D
10
E

8

Per2

Luc

S
M

2
0

Transfection

N

F G
Y

2

3

4

Slope = 1.0

N

M
P

K

2
I

K224 mutants
1

W

S

4

Y

0

A

6

Slope = 1.0

D
A

W

4

Slope = 2.0

8

6

Csnk1d mutant

10

Slope = 2.0

Plasmid DNA

0

5

0

1

R178 mutants2n = 3 (± SD)

3

4

5

n = 3 (± SD)

E
12

10

Slope = 2.0

site 1
site 2
D, E mutants

10

Slope = 2.0

site 3site 4
D, E mutants

8

8
6

Slope= 1.0

6

2

2
0

Slope = 1.0

4

4

0

1

2

3

4

5

0

6

0

1

2

n = 3 (± SD)

F

3

4

5

40

50

n = 3 (± SD)

H
K224D mutant [E]0300kcat (min-1)

0.3
0.2

50

0.0

0
0

100
200
300
Substrate (μM)
n = 4 (± SEM)

Q10 = 1.98 ± 0.27
n = 4 (± SEM)

150

100
200
300
Substrate (μM)
n = 3 (± SEM)

0kcal mol-1 of injectant

kcat (min-1)

200

50
0

50

0

60

10

20

30

-0.02

-0.02
0.80

0.00
*

100

0.00

40

-0.04

300
250

0.05

30

-0.06

K224E mutant [E]0 = 1 nM

0.10

20

0.00

G
0.15

10

0.00

100

0.1

Time (min)Time (min)0150kcal mol-1 of injectant

0.4

*

200

μcal/sec

250

0.6
0.5

μcal/sec

0.7

-0.20
-0.40
-0.60
-0.80

Q10 = 2.06 ± 0.24
n = 3 (± SEM)

0.40
0.00
-0.40
-0.80

0.0

0.5

1.0
Molar Ratio

1.5

2.0

0.0

0.5

1.0

1.5

2.0

2.5

Molar Ratio(legend on next page)790 Molecular Cell 67, 783–798, September 7, 2017

CKId-dependent phosphorylation measured by the stability of
the PER2::LUC protein in HEK293T cells, which do not harbor
functional circadian oscillators (Isojima et al., 2009) (Figure 5C).
A number of K224 and R178 mutants significantly promoted thedegradation of PER2::LUC protein in a temperature-dependent
manner (Figure 5D); however, these mutations did not altertemperature dependency of the stability of the LUC protein
alone (Figures S6A and S6B). Interestingly, the charge-deleted
R178 mutants significantly altered temperature dependency of
CKId-dependent phosphorylation measured by the stabilityof the PER2::LUC protein (Figure 5D). The charge-inversed
K224 mutants (K224D/E) also remarkably changed temperaturedependency (Figure 5D). On the other hand, temperature dependency was not altered by D/E mutations of all the othercandidate residues in four sites (Figures 5B, 5E, S6A, andS6C), highlighting K224 as a unique residue. As a negative control, we selected H185, which was also a basic residue locatedin site 1, but did not directly interact with phosphate analogs(Figure 5B) or ATA (Figure 4I). Then, we confirmed that all
H185 mutants did not significantly promote the degradationof PER2::LUC or LUC proteins in a temperature-dependent
manner (Figures S6D and S6E).
Since the charge inversion of K224 markedly facilitated
CKId-dependent phosphorylation at higher temperature whenmeasured by the stability of the PER2::LUC protein (Figure 5D),
we hypothesized that K224D/E mutants might fail to counteract
the activated phosphorylation activity at higher temperature dueto the unstable enzyme-phosphopeptide complex formation. To
verify this hypothesis, we investigated the phosphorylation activity of K224D/E mutants against CKItide. As expected, both mutants exhibited remarkably accelerated initial velocities at higher
temperature, and also demonstrated temperature dependency
for kcat (Figures 5F and 5G) and kcat/Km (Figures S6F and S6G)
in spite of temperature-insensitive Km (Figures S6F and S6G).
In the ITC assay, we could not detect any signals for the bindingof CKItide(2pS) to both mutants with ADP (Figure 5H). These
results suggested that K224D/E mutants of CKId impair ADPdependent product binding, probably by inducing phosphopeptide release via electrostatic repulsion. These results also
confirmed that ADP-dependent product binding was critical in
the temperature compensation of CKId-dependent multi-site
phosphorylation.

K224D Mutation Remarkably Shortens Behavioral
Circadian Rhythm in Mammalian Circadian Clocks
Since K224D/E mutants of CKId exhibited more than four times
greater degradation activity of PER2 protein at higher temperature compared to wild-type CKId (Figure 5D), circadian periodsof these mutant mice would be expected to be shortened as
was observed in that of CKIε (tau) mutant mice (Meng et al.,2008). To investigate in vivo significance of K224 mutants, we
generated embryonic stem cell-derived mice (ESC mice) of
these mutants (Figure S7A; STAR Methods) (Ode et al., 2017).
Although K224E knockin mice failed to be created, wild-type
CKId and K224D knockin mice (named WT-KI and K224D-KI
mice, respectively) were successfully generated. We thenanalyzed the behavioral circadian rhythms of WT-KI andK224D-KI mice by monitoring their locomotor activity in thelight-dark (LD) cycle, constant light (LL), or constant darkness(DD) conditions (Figures 6A, S7B, and S7C). Interestingly,
K224D-KI mice showed markedly shorter circadian period(19.6 ± 1.2 hr) than WT-KI mice (23.7 ± 0.1 hr) in DD condition
while no significant circadian period difference was observedin LD condition (Figures 6A and S7D). The strikingly shortened
circadian period was comparable to that of CKIε(tau) mutant
mice (20.0 ± 0.1 hr) (Meng et al., 2008). In addition, alteredbehavioral circadian rhythms of K224D-KI mice in DD condition
were adjusted by second LD cycles, and circadian period lengthof K224D-KI mice in LL condition was again shorter than that of
WT-KI mice (Figure S7E).
K224D Mutation Significantly Changed the Temperature
Dependency of Circadian Rhythm of SCN in Mammalian
Circadian Clocks
It is difficult to evaluate how K224D mutation of CKId contributes
to temperature dependency of circadian rhythms in organisms
because of the homeostasis of body temperature in warmblooded mammals. Thus, we instead investigated temperaturedependency of circadian rhythms in suprachiasmatic nucleus(SCN). To record endogenous circadian rhythms of SCN slices,
WT-KI or K224D-KI mice were mated with PER2::Luciferase(PER2::LUC) protein-fused reporter mice by in vitro fertilization(Yoo et al., 2004). Time course of luciferase activity in eachlong-term cultured organotypic SCN slice was monitoredby photomultiplier tubes (PMTs) under different temperature

Figure 5. Comprehensive Genetic Screening Identified K224D and K224E Mutants of CKId that Impaired ADP-Dependent Product Binding
and Temperature Compensation of Multi-site Phosphorylation(A) Overview (left) and close-up views (right) of four binding sites of phosphate analogs such as tungstate and sulfate ions.
(B) Minimum distance between phosphate analogs and basic amino acids (H, R, and K). Data are mean ± SD, whose sample n is the number of chains for specified
site and PDB ID.
(C) Workflow of the dual-luciferase assay to examine a series of CKId mutants for their effect on temperature dependency of phosphorylation measured by thestability of the PER2::LUC protein in HEK293T cells.
(D) K224 and R178 mutations significantly promoted the degradation of PER2::LUC protein in a temperature-dependent manner.
(E) The aspartate or glutamate mutation of all other candidate residues in four sites did not promote the degradation of PER2::LUC protein in a temperaturedependent manner.
For (D) and (E), data are mean ± SD (n = 3).
(F and G) Temperature and substrate concentration dependency of the phosphorylation activity on a single-phosphorylated CKItide by (F) K224D and (G) K224E
mutants. Data are mean ± SEM (F, n = 4; G, n = 3); *p < 0.05; two-tailed Welch’s t test. Solid line and shade represent mean ± SEM of curves of Michaelis-Menten
model with estimated parameters.
(H) ITC assay for the binding of CKItide(2pS) to K224D (left) and K224E (right) mutants in the presence of ADP.
See also Figure S6.

Molecular Cell 67, 783–798, September 7, 2017 791

A
ROSA26::P(CAG)-CKIδ(WT)Time of day

0

48

ROSA26::P(CAG)-CKIδ(K224D)Time of day

0

48 (hr)0Time of day

48

0

Time of day

30

48(hr)

20

20

30

30

40

40

50

50

60

60

LD

DD

LD
LL

10

10

20

20

30

30

40

40

50

50

60

60

LD

DD

Period (hr)

10

Days

Days

28
10

26

***
***

24
22
20

LD

18

LL

WT K224D WT K224Dn = 9 n = 14 n = 9 n = 14
LD
DD(± SD)(± SD)B
ROSA26 (+/+), PER2::LUC

ROSA26 (+/WT), PER2::LUC
23.7 ± 0.3 hr

0

5
0

20

40

23.3 ± 0.6 hr

8
6
4
2

20

40

10
8
6
4
2
0

0
0

20 40

60 80 100 120 140
Time (hr)1020

40 60 80 100 120 140
Time (hr)

Slope
-0.03
-0.05

22
20

0

18

-0.17
0

20

40

60 80 100 120 140
Time (hr)1.2

19.1 ± 0.5 hr

10
8
6
4
2
0

0

ROSA26 (+/+) (± SD)
ROSA26 (+/WT) (± SD)
ROSA26 (+/K224D) (± SD)

24560 80 100 120 140
Time (hr)23.4 ± 0.3 hr

12Bioluminescencex 104 counts/min

100

60 80 100 120 140
Time (hr)Bioluminescencex 104 counts/min

037˚CBioluminescencex 104 counts/min

20.3 ± 0.5 hr
15Period (hr)

5

10

26

Estimated Q10

1015Bioluminescencex 104 counts/min

15

Bioluminescencex 104 counts/min

30˚C
Bioluminescencex 104 counts/min

23.6 ± 0.5 hr

ROSA26 (+/K224D), PER2::LUC

0

20

40

60 80 100 120 140
Time (hr)2830

32 34 36 38
Temerature (˚C)
***

40

***

1.1
1.0
0.9

0.8
ROSA26 (+/+) (+/WT) (+/K224D)n = 32 n = 12
n=8(± SD) (± SD) (± SD)

Figure 6. K224D Mutation of CKId Remarkably Shortened Behavioral Circadian Rhythm and Significantly Changed the Temperature Dependency of Circadian Rhythm of SCN in Mammalian Circadian Clocks(A) Locomotor activity of wild-type CKId knockin mice (WT-KI mice; left) and K224D mutant knockin mice (K224D-KI mice; middle) in the light-dark (LD) cycle,
constant light (LL), or constant darkness (DD) conditions. Behavioral period in LD and DD conditions (right). Data are mean ± SD (n = 9 for WT and n = 14 forK224D); ***p < 0.001; two-tailed Welch’s t test.
(B) Temperature dependency of circadian rhythms of Per2Luc SCN of the control mice without knockin (first column), WT-KI mice (second column), and K224D-KI
mice (third column); the circadian period (top right); and the estimated Q10 (bottom right). The shaded band represents 1 SD from the mean. Data are mean ± SD
(n = 32 for control mice without knockin, n = 12 for WT-KI mice, and n = 8 for K224D-KI mice); ***p < 0.001; two-tailed Welch’s t test.
See also Figure S7 and Movie S1.

conditions over 6 days (Figure 6B; Movie S1). The circadianperiod and the robustness to temperature perturbation of
WT-KI slices were quite similar to those of slices without theknockin (Figures 6B and S7F). On the other hand, K224D-KI
slices displayed remarkably shorter circadian periods comparedwith WT-KI slices, closely matching with behavioral circadianrhythms in organisms. Furthermore, circadian period of
K224D-KI slices was significantly shortened by temperatureelevation; therefore, the estimated Q10 value was also significantly increased (Figure 6B). These results highlighted theimportance of K224 residue in circadian period and its temperature dependency of mammalian circadian clock in the SCN.
Temperature Compensation of Multi-site
Phosphorylation Is Evolutionary Conserved in CKI family
A budding yeast demonstrated temperature-compensated ultradian metabolic cycles (Mellor, 2016). Importantly,

792 Molecular Cell 67, 783–798, September 7, 2017

CKI has also been reported in the period determinationof ultradian metabolic cycles (Causton et al., 2015). We
note that amino acid residues including K224 were highly
conserved in the CKI family of budding and fission yeasts(Figures 7A and S8A). Interestingly, CKI homolog 1 (Figure S8B)-dependent phosphorylation of *CKItide is temperature compensated (Figures 7B and S8C). In addition, ATA
also increased the initial velocity of this multi-site phosphorylation in a dose-dependent manner (Figure S8D). Moreover,
ITC assay revealed CKI homolog 1 exhibited a remarkableaffinity to CKItide(2pS) with ADP (Figure 7C). Structuralalignment of newly solved budding yeast CKI homolog 1(Table 1) with mammalian CKId displayed strong structuralconservation (Figures S8E and S8F). Based on these results,
we concluded that temperature-compensated phosphorylation was evolutionarily conserved in budding yeast CKI
homolog 1.

A

224

B

C
CKI homolog 1 + ADP + CKItide(2pS)

0.10

0
0

30

400
200

0

-2.00

10
0

100
200
300
Substrate (μM)
n = 3 (± SEM)

D

n.s.

600

40
20

0.05

35˚C
25˚C

n.s.

50

0.00

μcal/sec2n.s.

Km (μM)

4

800

60
0.15

kcat (min-1)

6

70kcal mol-1 of injectant

0.20

8

kcat/Km (min-1 μM-1)

Initial velocity (μM/min)

CKI homolog 1 (S.cerevisiae)Time (min)20 30 40

025˚C 35˚C
Q10 = 1.38 ± 0.68
n = 3 (± SEM)

25˚C 35˚C
Q10 = 1.85 ± 0.82
n = 3 (± SEM)

0.0

50

-0.4
-0.6

35˚C

-0.8

0

25˚C 35˚C
Q10 = 0.78 ± 0.12
n = 3 (± SEM)

10

0.0
-0.2

0.5

1.0
1.5
2.0
Molar Ratio
Kd = 0.5 μM
N = 0.373 sites
ΔH = -2178 cal/mol ΔS = 21.8 cal/mol/deg

β7

310 B

CKIδ residue index

135

140

CKIδ WO4 / SO4 site22222233333333322 222 2222211111
21 22 2223
1
1111111111 11 111 111 11
LMGL--G--KKGNLVYIID-FGLAKKYRDARTHQHIPYREN--KNLTGTARYASINTHLGI-EQSRRDDLESLGYVLMYFNLGSLPWQGLKAATKRQKYERISEKKMSTCKIδ 1CKJ : B WO4

β8

β9

αD

150

160

170

αE

180

190

αF

200

210

220

230

235

LMGL--G--KKGNLVYIID-FGLAKKYRDARTHQHIPYREN--KNLTGTARYASINTHLGI-EQSRRDDLESLGYVLMYFNLGSLPWQGL+++++++KYERISEKKMST
LMGL--G--KKGNLVYIID-FGLAKKYRDA++++HIPYREN--KNLTGTARYASINTHLGI-EQSRRDDLESLGYVLMYFNLGSLPWQGLKAATKRQKYERISEKKMSTCKIδ 1CKI : B
CKIδ 3UYS : C SO4CKIε 4HOK : I SO4
LMGL--G--KKGNLVYIID-FGLAKKYRDARTHQHIPYREN--KNLTGTARYASINTHLGI-EQSRRDDLESLGYVLMYFNLGSLPWQGLKAATKRQKYERISEKKMSTCK1 1CSN : A ATP, MG, SO4 LIGRpnS--KNANMIYVVD-FGMVKFYRDPVTKQHIPYREK--KNLSGTARYMSINTHLGR-EQSRRDDLEALGHVFMYFLRGSLPWQGLKAATNKQKYERIGEKKQSTCKIγ3 4G17 : A inhibitor
TTBK1 4BTJ : A ADP, PO4
TTBK1 4BTJ : B ATP, PO4
VRK2 2V62 : B EDO
AurA 4DEE : A ADP, MG
CDK2 1HCK : A ATP, MG

LIGR--PgnKTQQVIHIID-FGLAKEYIDPETKKHIPYREH--KSLTGTARYMSINTHLGK-EQSRRDDLEALGHMFMYFLRGSLPWQGLKADTLKERYQKIGDTKRATAMGR--L-pSTYRKCYMLD-FGLARQYTN-TTGDVRPPRNV--AGFRGTVRYASVNAHKNR-EMGRHDDLWSLFYMLVEFAVGQLPWRKI------kDKEQVGMIKEKY
AMGR--L-pSTYRKCYMLD-FGLARQYTN-TTGDVRPPRNV--AGFRGTVRYASVNAHKNR-EMGRHDDLWSLFYMLVEFAVGQLPWRKIK------DKEQVGMIKEKY
LLGY--K---NPDQVYLAD-YGLSYRYCPN--GNHKQYQENprKGHNGTIEFTSLDAHKGV-ALSRRSDVEILGYCMLRWLCGKLPWEQN-----lKDPVAVQTAKTNLLLGS-------AGELKIAD-FGWSVHAP---------sSRR--DTLCGTLDYLPPEMIEGR-MHDEKVDLWSLGVLCYEFLVGKPPFEAN-------TYQETYKRISRV
LIN-------TEGAIKLADfGLAR----------afgvpvrtythevvTLWYRAPEILLGCkYYSTAVDIWSLGCIFAEMVTRRALFPGD-------SEIDQLFRIFRT

Secondary structure elements:

310 helix

α helix

turn

bend

β strandE

β bridgeno secondary structure assigned

F
ΔCKIδ-ATP
8

2

4
2

0
0

50 100 150 200 250 300
Amino acid0

50 100 150 200 250 300
Amino acid

4

I228A
Q214A

Inserted sequence

K232A
Slope = 1.0

2

214

224

CKIδ LPWQGLKAATKRQKYERISEKKMSTTBK1 LPWRKI------KDKEQVGMIKEK

E197A

240

244

4

TTBK1 WT

2

I228

K224
Q214
R240
αF helix

D244
Blue TTBK1Green CKIδ5

10 15 20 25
Time (min)n = 5 (± SEM)

2.0
1.5

*
1.86 ± 0.16
1.27 ± 0.11

1.0
0.5

30

TTBK1 WT TTBK1 rMT
n = 5 (± SEM)

3.0

[S1]0 = 320 μM
35˚C
25˚C

40

TTBK1 rMT

30
20
10
0

2.5

0.0
0

50

R204

Q214

K232

20 30 40 50 >60
B-factor (Å2)

3.0

J

R178

E197

TTBK1 rMT

6

3

R178
αE helix

50 100 150 200 250 300<10Amino acid[S0]0 = 20 μM

35˚C
8 25˚C0
1
2
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

00

TTBK1(rMT) LPWQGLKAATKRQKKEQVGMIKEK
0

40

10
Produced Product (μM)

R178A

60

I

P(CMV)-Per2-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

Produced Product (μM)

Relative Activity at 37˚C (WT = 1)

6

80

0

RMSF
25˚C > 35˚C
25˚C < 35˚C

H

G

100

20

0

RMSF
25˚C > 35˚C
25˚C < 35˚C

Chain A
Chain B

Q10 (Initial velocity)

4

6

ΔCKIδ-ADP

Q10 (Initial velocity)

6

120

35˚C
25˚C

B-factor(Å2)

35˚C
25˚C
RMSF (Å)

RMSF (Å)

8

ΔCKIδ-ADP

TTBK1 WT
05

10 15 20 25
Time (min)n = 3 (± SEM)

30

2.5

***
2.52 ± 0.08

2.0
1.5

1.49 ± 0.05

1.0
0.5
0.0

TTBK1 WT TTBK1 rMT
n = 3 (± SEM)

(legend on next page)

Molecular Cell 67, 783–798, September 7, 2017 793

A CKI-Specific Domain around K224 Can Provide an
Evolutionally Conserved Structural Basis of
Temperature Sensitivity of Substrate and ProductBinding
Multiple sequence alignment revealed that TTBK1 and TTBK2
lacked the CKI-specific domain around the residue K224 (Figure 7A). The multiple structural alignment further confirmedthat TTBK1 was also deficient in K217 to Q223 regions of CKId
(Figure 7D, green shaded region), although it is difficult to judge
the exact conservation for K224 of CKId because of the existence of K243 or K245 in TTBK1. Since TTBK1 doesn’t preserve
the temperature compensation, we speculated that the CKIspecific domain around K224 and K224 itself, which is criticalfor second-serine phosphorylation (Figure 5), might provide a
structural basis of the temperature sensitivity in the substrate
and product binding, and hence temperature-compensatedphosphorylation.
To gain structural insights into the temperature sensitivity
inherent in CKId, we performed molecular dynamics (MD) simulations, which predicted that K217-Q223 around aF helix was
highly fluctuated (Figures 7E, S9A, and S9B; Movies S2 andS3). In addition, B factors (Figure 7F) of the newly solved
DCKId-ADP complex (Table 1; Figure S9C) and previously determined apo-DCKId structure (Longenecker et al., 1996) (Figure S8F) showed higher fluctuations in K217-Q223. Furthermore,
B factors of apo-CKI homolog 1 structure indicate higher fluctuations in the CKI-specific domain (K279-Q285) (Figure S8F), indicating that higher fluctuations in the CKI-specific domain were
evolutionarily conserved from yeast to mammals. These results
suggested that a CKI-specific domain (K217-Q223) aroundK224 displayed large fluctuations, which could provide an evolutionally conserved structural basis of temperature sensitivity
in substrate and product binding, and hence temperaturecompensated multi-site phosphorylation.
A CKI-Specific Domain around K224 Can Be a Structural
Basis of Temperature Compensation of CKIdDependent Multi-site Phosphorylation
To identify critical interactions for the large fluctuations, we performed potential energy principal component analysis (PEPCA)(Koyama et al., 2008, 2011) (Figures S9D–S9I). For DCKId-ADP
complex at 35 C and DCKId-ATP complex at 25 C, the first prin-cipal components identified the large conformational change
of aF helix including K224 away from the loop including R178
(Figure S9B), and the first eigenvector components highlighted
critical interactions, which significantly changed their minimum
distance (Figures S9D–S9G). As predicted, the alanine mutations
on aF helix (i.e., I228 and K232) or on its spatially adjacent regions (i.e., E197 and Q214) significantly altered temperature dependency of CKId-dependent phosphorylation measured by thestability of the PER2::LUC protein (Figures 7G and S10A). Taken
together, these results were consistent with the hypothesis thata CKI-specific domain around K224 can be a structural basisof temperature compensation of CKId-dependent multi-site
phosphorylation.
A CKI-Specific Domain around K224 Can Confer
Temperature Compensation on TTBK1, Which Shows
Temperature Sensitivity in the First- and Second-Serine
Phosphorylation
In order to verify whether the CKI-specific domain aroundK224 residue was sufficient for temperature compensation,
we next attempted to reconstitute temperature compensationin vitro by introducing the CKI-specific domain into TTBK1,
which shows temperature-sensitive phosphorylation for a
two-serine and for a variety of single-serine substrates (Figure 1B). To our surprise, with rationally designed TTBK1 mutant(rMT, reconstituted mutant type; Figures 7H and S10B;
STAR Methods), the phosphorylation of a two-serine substrate*CKItide (Figure 7I) and a single-serine substrate *CKItide(S10A)(Figure S10D) for a lower substrate concentration (20 mM) wastemperature compensated, whereas the phosphorylation by
wild-type TTBK1 was temperature sensitive. Furthermore, fora higher substrate concentration (320 mM), when we utilized
two-serine substrate *CKItide (Figure S10C), single-serine
substrate *CKItide(S10A) (Figure S10D), and single-phosphorylated CKItide (Figure 7J), TTBK1 rMT significantly reduced
the temperature sensitivity of the phosphorylation comparedto that of wild-type TTBK1. These results, together with theresults shown in Figure 5, thus demonstrated that the Q214K224 domain could work as a regulatory module of temperature compensation in the CKI family, probably mediatedthrough temperature-sensitive substrate- and/or product-binding mechanisms.

Figure 7. A Conserved CKI-Specific Domain around K224 Can Provide a Structural Basis for Temperature-Sensitive Substrate and ProductBinding, and Confer Temperature Compensation to a Temperature-Sensitive Kinase(A) Multiple sequence alignment among CKI and TTBK family.
(B) Temperature dependency of CKI homolog 1-dependent phosphorylation on a two-serine substrate.
(C) ITC assay for the binding of CKItide(2pS) to CKI homolog 1 in the presence of ADP.
(D) Multiple structural alignments among CKI, TTBK1, VRK2, AurA, and CDK2.
(E) RMSF for each Ca atom of MD simulation of DCKId-ATP and DCKId-ADP complexes.
(F) B factor for each Ca atom of X-ray crystal structure analysis of DCKId-ADP complex.
(G) Dual-luciferase assay to examine a series of CKId mutants for their effect on temperature dependency phosphorylation measured by the stability of thePER2::LUC protein in HEK293T cells. Data are mean ± SD (n = 3).
(H) TTBK1 mutant (rMT) was rationally designed by replacing five amino acid residues (R240-D244) of TTBK1 with eleven amino acid residues (Q214-K224) of
CKId (pink), which contained the CKI-specific domain (green) (upper). The structural alignment of TTBK1 (blue) and CKId (green) (lower).
(I) Temperature dependency of phosphorylation on a two-serine substrate by TTBK1 (WT) and TTBK1 mutant (rMT).
(J) Temperature dependency of phosphorylation on a single-phosphorylated CKItide by TTBK1 (WT) and TTBK1 mutant (rMT).
For (B), (I), and (J), data are mean ± SEM (B, n = 3; I, n = 5; J, n = 3); *p < 0.05, ***p < 0.001, and n.s. (not significant); two-tailed Welch’s t test. See also Figures S8–
S10, Table 1, and Movies S2 and S3.

794 Molecular Cell 67, 783–798, September 7, 2017

Table 1. Data Collection and Refinement Statistics
DatasetCKId

CKI Homolog 1

BL26B2,SPring-8

BL26B2,SPring-8

Data Collection and Processing
X-ray source
Temperature (K)

100

100

Space group

P21

P21
45.50

Cell dimensions: a (Å)

50.14

Cell dimensions: b (Å)

130.3597.92

Cell dimensions: c (Å)

53.18

84.74

Cell dimensions: b ( )115.45

93.89

Wavelength (Å)

1.000

1.000

Resolution range (Å)

50.0–2.00(2.03–2.00)a

50.0–1.80(1.83–1.80)

No. of unique reflections

41687

67599

Multiplicity

3.8 (3.7)

3.7 (3.2)

Completeness (%)

100.0 (99.9)

98.4 (86.6)

Rsym (%)b8.2 (59.6)

4.8 (57.5)

I /s(I)

15.3 (2.2)

27.0 (2.0)

Resolution range (Å)

50–2.00(2.04–2.00)

50–1.80(1.85–1.80)

No. of reflections, working set

41,62965,536

Model Refinement

No. of reflections, test set

2,047

1,951

Rwork/Rfree (%)c18.72/24.16

15.97/20.85

RMSD for bond length (Å)

0.014

0.008

RMSD for bond angles ( )

1.667

1.226

No. of non-H atoms: protein

4671

4759

No. of non-H atoms: watermolecules

311

315

No. of non-H atoms: ligand

54–

Mean B factors (Å2): protein

36.55

43.14

Mean B factors (Å2): watermolecule

39.76

46.62

Mean B factors (Å2): ligand

25.64

–

Residues in the Ramachandranplot: most favored (%)

97.4

97.1

Residues in the Ramachandranplot: allowed (%)

2.6

2.9

Residues in the Ramachandranplot: outliers (%)

00

PDB entry

PDB: 5X17

PDB: 5X18

RMSD, root-mean-square deviation.
a
Values in parentheses are for the outermost resolution shell.
P P
P P
b
Rsym = ( h ijIhi –hIhij/ h ijIhij), where h indicates unique reflection
indices, and i indicates symmetry equivalent indices.
P
P
c
Rwork = jFobs–Fcalcj/ Fobs for all reflections; Rfree was calculated byusing randomly selected reflections.

(2) ‘‘higher product affinity to CKId-ADP complex at higher temperature,’’ underlie the temperature compensation. PER proteinsfunction in a large complex (PER complex) over 1 MDa, including
CRY and other proteins (Kim et al., 2015). Furthermore, PER2 wasshown to be phosphorylated on many sites by CKId (Schlosser
et al., 2005). Here, we briefly discuss how two mechanisms cancontribute to the temperature compensation of the multi-site
phosphorylation of PER proteins in PER complex. If the priming
phosphorylation of PER is the rate-limiting step in PER complex,
we expect that mechanism (1) will be important to keep the temperature compensation (Figures 3B and 3C). On the other hand, if
the multi-site phosphorylation of PER induces the tight binding
between CKI and PER complex, then the rate-limiting step becomes the dissociation of CKI from PER complex. In this case,
we expect that mechanism (2) can contribute to the temperaturecompensation. In reality, we showed that K224D mutation
increased the temperature dependency of kcat (Figure 5F) andthe period in the SCN slice experiment (Figure 6B). Since K224
is an important residue to recognize a phosphate (analog) insite 1 (Figures 5A, 5B, and 5H), this implies mechanism (2) ismore important for the temperature compensation in PER complex. In another respect, it was proposed that the phosphorylation of two sites in PER could contribute differently to the temperature compensation (Zhou et al., 2015). One CKI target unprimed
site is bTrCP binding site and the other primed site by unknown
kinase is FASPS mutant site (Isojima et al., 2009). Since two regions are different in the priming conditions, mechanisms (1)and (2) may be able to contribute to the two sites in a different
way to realize the temperature compensation in PER complex.
The observed temperature dependency in substrate binding
and product release is intrinsically encoded in CKId structure,
which is supported by temperature-sensitive fluctuations of a
CKI-specific domain around K224 revealed by MD simulations(Figures 7E and S9A), and the large fluctuations observed by
X-ray crystallography (Figures 7F and S8F). Furthermore, it is
supported by the impaired temperature dependency of CKIddependent phosphorylation measured by PER2::LUC degradation in a series of mutations on a CKI-specific domain or on its
spatially adjacent regions (Figures 5 and 7). Finally, we successfully conferred temperature-compensated phosphorylation ontemperature-sensitive TTBK1 by replacing the original R240D244 residues of TTBK1 with Q214-K224 residues of CKId.
Interestingly, TTBK1 (rMT) significantly reduced temperaturesensitivity in phosphorylation of two-serine substrate *CKItide(Figures 7I and S10C), single-serine substrate *CKItide(S10A)(Figure S10D), and a single-phosphorylated peptide CKItide(Figure 7J). Therefore, incorporated residues in TTBK1 (rMT)
would work as a regulatory module of temperature-dependent
substrate- and/or product-binding mechanisms essential for
temperature compensation in the CKI family.
STAR+METHODSDISCUSSION
In the current study, we showed two mechanisms, (1) ‘‘lower substrate affinity to CKId-ATP complex at higher temperature’’ andDetailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING

Molecular Cell 67, 783–798, September 7, 2017 795

d EXPERIMENTAL MODEL AND SUBJECT DETAILSB Transfection and Luciferase Assay
B Mice Productiond METHOD DETAILSB Protein Expression for Kinase Assay
B Mobility Shift Assay
B Substrate Screening
B Initial Velocity Analysis
B ATP-dependent Kinase Assay
B ATPase Activity Measurements
B Product Inhibition Assay for Kinase Activity
B ITC Assay
B Dephosphorylation Assay
B Mass Spectrometry for Dephosphorylation Assay
B Bayes Estimation of Rate Parameters
B Rate Equation for the ATPase Activity
B Rate Equation for the Phosphorylation Activity
B Chemical Screening
B Docking Simulation of ATA and Its Derivative
B Basic Residues around Phosphate Analogs
B Plasmid Construction

valuable comments; and H. Niwa (RIKEN CLST) and M. Yamamoto and G.
Ueno (RIKEN SPring-8 Center) for assistance with X-ray data collection. We
thank A. Millius and K. Wilkins for critical reading and valuable comments on
the manuscript. We thank Y. Okada for valuable discussions. We thank M.
Taiji, M. Takeichi, S. Hayashi, and F. Matsuzaki for encouraging the current
study. We thank the RIKEN Integrated Cluster of Clusters (RICC). This work
was supported by a grant from AMED-CREST (AMED/MEXT, H.R.U.), CREST(JST/MEXT, H.R.U.), Brain/MINDS (AMED/MEXT, H.R.U.), and Basic Scienceand Platform Technology Program for Innovative Biological Medicine (AMED/
MEXT, H.R.U.); a Grant-in-Aid for Scientific Research (S) (JSPS KAKENHI
Grant Number 25221004, H.R.U.); a Grant-in-Aid for Scientific Research on
Innovative Areas (JSPS KAKENHI Grant Number 23115006, H.R.U.); a
Grant-in-Aid for challenging Exploratory Research (JSPS KAKENHI Grant
Number 16K15124, K.T.); the strategic programs for R&D (President’s Discretionary Fund) of RIKEN (to H.R.U.); an intramural Grant-in-Aid from the RIKEN
Quantitative Biology Center (to H.R.U.); Grants-in-Aid for Japan Society for the
Promotion of Science (JSPS) Fellows 25-5989 (to Y.S.); and MEXT as ‘‘Priority
Issue on Post-K computer’’ (Building Innovative Drug Discovery Infrastructure
Through Functional Control of Biomolecular Systems) (hp160213) (to T.H.).
Received: May 12, 2017
Revised: July 4, 2017
Accepted: August 16, 2017
Published: September 7, 2017

B Cultivation of Mouse ES Cells
B Targeting into Rosa26 Locus
B Locomotor Analysis of CKId Mutant MiceB Luminescence Monitoring of SCN SliceB Multiple Sequence Alignment
B Protein Preparation for X-ray Crystallography
B Crystallization and Structure Determination
B Multiple Structural Alignment
B Molecular Dynamics Simulations
B Alanine Scanning of CKId around K224
B Design of a Temperature-compensated Kinase
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION
Supplemental Information includes ten figures, one table, and three movies
and can be found with this article online at http://dx.doi.org/10.1016/j.
molcel.2017.08.009.
AUTHOR CONTRIBUTIONS
H.R.U. and Y.S. designed the study. Y.S. performed and analyzed most of thebiochemical experiments. Y.M.K. constructed the computational model andperformed Bayes estimation and MD simulation. Y.S. and H.F. performed
chemical screening. T.H. performed chemical-enzyme docking simulation.
Y.S., M.U.-T., and H.U. made the constructs. M.U.-T. performed dual-luciferase assay. M.U.-T. and H.U. generated CKI-KI mouse. R.G.Y. performed
SCN slice experiment. Y.S. analyzed behavioral data. M.K. and T.U. performed
X-ray crystal structure analysis. Y.S. and Y.M.K. designed synthetic temperature-compensated enzyme. H.R.U., K.T., Y.S., and Y.M.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.
ACKNOWLEDGMENTS
We thank past and present lab members at RIKEN QBiC, and University of Tokyo, in particular M. Nakajima, M. Ishida, R. Narumi, C. Imai, J. Yoshida-Garcon, S. Morino, A. Kishimoto, J. Hara, S. Fujino, A. Nishiyama, K. Yamanaka, K.
Sumiyama, and Y. Wada, for their kind help in preparing the materials and supporting experiments; LARGE, RIKEN CLST for housing the mice; K.L. Ode for796 Molecular Cell 67, 783–798, September 7, 2017

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STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

E. coli Rosetta2 (DE3) (for X-ray
crystallography)

Merck Millipore

Cat#71400-4

E. coli Rosetta2 (DE3) (for TTBK1 design)

Merck

Cat#71397

Library of Pharmacologically Active
Compounds (LOPAC 1280)

Sigma-Aldrich

Cat#LO3300-1KT

cOmplete, Mini, EDTA-free Protease
Inhibitor Cocktail

Sigma-Aldrich

Cat#11836170001

PreScission Protease

GE Healthcare

Cat#27084301

Aurintricarboxylic acid

Sigma-Aldrich

Cat#A1895-5G

Farnesylthiosalicylic acid

Sigma-Aldrich

Cat#F8175-50MG

PD 169316

Sigma-Aldrich

Cat#P9248-5MG

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins

SP600125

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Cat#S5567-10MG

Reactive Blue 2

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Cat#R115-10MG

SB 202190

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Cat#S7067-5MG

Morin hydrate

Sigma-Aldrich

Cat#M4008-2G

Methyl-3,4-dephostatin

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Cat#M9440-1MG(R)-(+)-8-Hydroxy-DPAT hydrobromide

Sigma-Aldrich

Cat#H140-5MG

GW5074Sigma-Aldrich

Cat#G6416-5MG

6-Hydroxy-DL-DOPA

Sigma-Aldrich

Cat#H2380-5MG

Mighty Cloning Reagent Set (Blunt End)

Takara Bio

Cat#6027

Lambda Protein Phosphatase

Bio-Rad

Cat#P0753L

HisGST-CKId (kinase domain)

This paper

N/A

HisGST-CKI homolog 1 (kinase domain)

This paper

N/A

His-TEV protease (S219V mutant)

Kapust et al., 2001

Addgene Plasmid #8827

l phosphatase

Cosmo Bio

Cat#SC-200312A

FuGene6

Roche

Cat#11814443001

Dual-Luciferase Reporter Assay System

Promega

Cat#E1910

Raw images of SDS-PAGE analysis

This paper; Mendeley Data

http://dx.doi.org/10.17632/8v6y744r3t.1

CKId structure complexed with ADPThis paper

PDB: 5X17

CKI homolog 1 structureThis paper

PDB: 5X18

ATCC

CRL-3216

Mouse: WT-KI: B6N-Gt(ROSA)26Sor <em(CAG-Csnk1dWT/-)>

This paper

N/A

Mouse: K224D-KI: B6N-Gt(ROSA)26Sor <em(CAG-Csnk1dK224D/-)>

This paper

N/A

Mouse: ROSA26 (+/+), PER2::LUC:
B6.129S6(B6N)F1-mPER2::LUC+/
Gt(ROSA)26Sor < em(CAG-Csnk1d/)>This paper

N/A

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Deposited Data

Experimental Models: Cell Lines
Human: HEK293T
Experimental Models: Organisms/Strains(Continued on next page)

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Continued
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Mouse: ROSA26 (+/WT), PER2::LUC:
B6.129S6(B6N)F1-mPER2::LUC+/
Gt(ROSA)26Sor < em(CAG-Csnk1dWT/-)>

This paper

N/A

Mouse: ROSA26 (+/K224D), PER2::LUC:
B6.129S6(B6N)F1-mPER2::LUC+/
Gt(ROSA)26Sor < em(CAG-Csnk1dK224D/-)>

This paper

N/A

Mouse: ROSA26 (+/+), PER2::LUC(+/+):
B6.129S6-mPER2::LUC

Yoo et al., 2004

RRID: IMSR_JAX:006852

Oligonucleotides
Primers for CKId mutagenesis, see Table S1

This paper

N/A

Forward primer for the region upstreamof the 50 homologous arm: 50 - TGCTGGCC
TACTGCTGCCTCGATCTTAC-30

Susaki et al., 2014

N/A

Reverse primer for the region downstreamof the 50 homologous arm: 50 - AGGACAACGCCCACACACCAGGTTAGC 30

Susaki et al., 2014

N/A

Forward primer annealing to the region upstreamof the 30 homologous arm: 50 -CGTGGTGGAGCCG
TTCTGTGAGACA-30

Susaki et al., 2014

N/A

Reverse primer annealing to the region downstreamof the 30 homologous arm: 50 -GGTGAAATG
CTTGACTCCTAGACTT-30

Susaki et al., 2014

N/A

Reverse primer annealing to between 50
homologous arm and the CAG promoter:
50 -GGCTTTTAGTAAGCGAATTCGGTACC-30

Susaki et al., 2014

N/A

Forward primer annealing to the poly-A regionof the puromycin resistance gene: 50 -TCCATCAGAAGCTGGTCGATC-30

Susaki et al., 2014

N/A

Recombinant DNA
pGEX6P-1 vector (2-317DCKId)

Isojima et al., 2009

N/A

pMU2-Luc

Isojima et al., 2009

N/A

pMU2-Per2::Luc

Isojima et al., 2009

N/A

phRL-CMV

Promega

Cat#E2261

pMU2-CKId vector

Narumi et al., 2016

N/A

pHisGST CKId

This paper

N/A

pHisGST CKI homolog 1

This paper

N/A

His-TEV protease (S219V mutant): pRK793

Kapust et al., 2001

Addgene Plasmid #8827

CKI homolog 1

Thermo Fisher Scientific

N/A

TTBK1

Thermo Fisher Scientific

N/A

Microsoft

N/A

Software and Algorithms
Excel 2013
Origin 7.0

OriginLab

N/AR

R Core Teamhttps://www.r-project.org

deSolve package

Soetaert et al., 2010https://cran.r-project.org/web/packages/
deSolve/index.html

FME package

Soetaert and Petzoldt, 2010https://cran.r-project.org/web/packages/
FME/index.html

Glide

Friesner et al., 2004;
Halgren et al., 2004

https://www.schrodinger.com/

SiteMap

Halgren, 2007, 2009https://www.schrodinger.com/

MaestroSchrödinger, LLC

https://www.schrodinger.com/(Continued on next page)e2 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

Continued
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Protein Preparation Wizard

Sastry et al., 2013https://www.schrodinger.com/

ClockLab

Actimetrics

http://actimetrics.com/products/clocklab/

MetaMorph

Molecular device

https://www.moleculardevices.com/

Fiji/ImageJ

Schindelin et al., 2012https://fiji.sc/

Analysis of rhythmicity period lengthand oscillation phase of the SCN

Ukai-Tadenuma et al., 2011

N/A

Mathematica 9

Wolframhttps://www.wolfram.com/mathematica/

HKL2000

Otwinowski and Minor, 1997

http://www.hkl-xray.com

MOLREP

Vagin and Teplyakov, 2010

http://www.ccp4.ac.uk/html/molrep.html

Coot

Emsley et al., 2010https://www2.mrc-lmb.cam.ac.uk/
personal/pemsley/coot/

REFMAC

Murshudov et al., 1997

http://www.ccp4.ac.uk/html/refmac5.html

PHENIX

Adams et al., 2010

http://www.phenix-online.org/

PyMOL

Schrödinger, LLC

http://www.pymol.org/

Clustal Omega

Sievers et al., 2011

http://www.uniprot.org/align/

ESPript

Robert and Gouet, 2014

http://espript.ibcp.fr/ESPript/ESPript/

Dali server

Holm and Rosenström, 2010

http://ekhidna.biocenter.helsinki.fi/
dali_server/

Amber 10

Case et al., 2008

http://ambermd.org/

PTRAJ

Roe and Cheatham, 2013

http://ambermd.org/

CPPTRAJ

Roe and Cheatham, 2013

http://ambermd.org/

VMD

Humphrey et al., 1996

http://www.ks.uiuc.edu/Research/vmd/

CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hiroki R.
Ueda (uedah-tky@umin.ac.jp).
EXPERIMENTAL MODEL AND SUBJECT DETAILSTransfection and Luciferase Assay
HEK293T cells (American Type Culture Collection) were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS(JRH Biosciences) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Thermo Fisher Scientific) at 37 C with 5% CO2.
One day prior to transfection, cells were plated onto 35-mm dishes at a density of 4 3 105 cells per well. The following day, HEK293T
cells were co-transfected using FuGene6 (Roche) with 0.5 mg of a Luciferase reporter plasmid (pMU2-Per2::Luc for Figures 5D, 5E,
7G, and S6D, or pMU2-Luc for Figures S6B, S6C, S6E, and S10A; Isojima et al., 2009) in the presence of 0 or 1 mg each of wild-type ormutated pMU2-CKId (Narumi et al., 2016; K224 mutants and R178 mutants for Figures 5D and S6B, H185 mutants for Figures S6D
and S6E, D/E mutants for Figures 5E and S6C, aF mutants for Figures 7G and S10A, see ‘‘Plasmid Construction’’ section and Table
S1 for mutants construction; Narumi et al., 2016), according to the manufacturer’s instructions (n = 3), and cultured at 37 C. Empty
vector was used to make up the total amount of DNA to 2 mg per well. Additionally, 50 ng of a phRL-CMV plasmid (Renilla luciferase(RLuc) reporter vector, Promega) was added into each transfection as an internal control for transfection efficiency. On the next day,cells in each well were harvested separately, and plated into two wells of 35-mm dishes. The following day, pre-warmed medium(27 C and 37 C) was added into each well, and then cells were cultured at 27 C and 37 C, respectively. After 24 hr, cells were harvested and assayed with Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized by Rluc activity.
Data are representative of two independent experiments.
Mice Production
The ROSA26::P(CAG)-CKId knockin mouse strains were established in our laboratory (see ‘‘Targeting into Rosa26 Locus’’ section).
mPer2Luc mice were gifted from Joseph Takahashi (Yoo et al., 2004). CKId knockin 3 mPer2Luc mice were produced by in vitro fertilization. All experimental procedures and housing conditions were approved by Institutional Animal Care and Use Committee of
RIKEN Kobe Branch, and all of the animals were cared for and treated humanely in accordance with the Institutional Guidelines
for Experiments Using Animals.

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e3

METHOD DETAILS
Protein Expression for Kinase Assay
Purification condition of CKId was described by reference to previous our paper (Isojima et al., 2009). After N-terminal GST tag wascleaved by PreScission Protease (GE Healthcare) overnight at 4 C, CKId was dephosphorylated by l phosphatase 3-4 hr at 25 C. The
dephosphorylated CKId was purified by cation exchange chromatography with 50 mM Tris-HCl buffer (pH 7.5) containing 600 mM
NaCl and 1 mM DTT.
Mobility Shift Assay
Enzyme reaction was analyzed by mobility shift assay (LabChip EZ Reader; PerkinElmer). Substrate peptide and produced peptide
were separated by applying a voltage in microchannel (Figure S1A). Reactant product was flowed with separation buffer(PerkinElmer). These peptides are labeled by the fluorescein amidite (FAM) fluorophore at N-terminal residue.
Substrate Screening
Substrate peptides based on the CKItide (KRRRALpSVASLPGL) were synthesized on a peptide synthesizer (Syro Wave; Biotage)using Fmoc solid-phase chemistry. The synthesized peptide was purified using HPLC system and the product was confirmed byusing MALDI-TOF Mass. DCKId protein (1 mM) or DTTBK1 protein (Carna Biosciences; GST-fusion human TTBK1, catalytic domain(1-343 amino acids)) were added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 1 mM ATP, 1 mM DTT, 0.1 mg/mL
BSA and 20 mM synthetic peptide) at 35 C and 25 C. Reaction was stopped by stop solution (20 mM Tris-HCl pH7.5, 100 mM NaCl,
5 mM EDTA) at 30 min. These solutions were moved to 384-well plates and it was analyzed by mobility shift assay.
We prepared a series of single-serine substrates, *CKItide(S10X) (KRRRALSVAXLPGL), by mutating the second serine (at position10) of *CKItide to an amino acid X, and investigated their temperature dependency in DCKId-dependent phosphorylation. We alsoutilized a temperature-sensitive DTTBK1 (a catalytic domain of wild-type Tau-tubulin kinase 1, TTBK1) as a negative control. Activities at 25 C for the two-serine *CKItide peptide were set as 1.0 in the relative activity (Figure 1B).
Initial Velocity Analysis
Initial velocities of product concentrations were computed by linear least square fittings conducted by Microsoft Excel 2013 or
R 2.15.3 (https://www.r-project.org/). Substrate-saturation curve of initial velocities V were fitted by Michaelis-Menten equationV=kcat ½E0 ½S;Km + ½S

where [E]0, [S], kcat, and Km represent enzyme concentration, substrate concentration, catalytic constant, and Michaelis constant,respectively. Nonlinear least square fittings were performed with R. Error of a ratio value y/x such as Q10 was computed by the error
propagation formulasﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
y  ε 2 ε 2
 
x
y
;
εy=x z +xxywhere x and y represent values such as means, εx and εy represent corresponding errors such as SEM.
In Figure 1D, ATP dependency of kinetic parameters are shown. Theoretical standard error (SE) of kcatTS([T])/KmTS([T]) and kcatTS([T])
from n = 1 data shown in error bars in upper panels of Figure 1D were obtained from the result of nonlinear least square fittings. Theoretical SE of KmTS([T]) was computed by the error propagation formula of the ratio of y = kcatTS([T]) to x = kcatTS([T])/KmTS([T]) with theoretical covariance εx,y obtained by fitting assﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
y  ε 2 ε 2
εx;y
 
x
y
εy=x z 
+
2 :
x
x
y
xy
Kinetic parameters kcatTS([T])/KmTS([T]) and kcatTS([T]) were fitted according to equations in Figure 1C. Theoretical SE of kcatTS/KmTS,
KT, kcatTS, and KTS obtained from the result of nonlinear least square fittings are shown in error bars in bottom panels of Figure 1D.
Theoretical SE of KmTS was computed by the error propagation formula of the ratio of kcatTS to kcatTS/KmTS without theoreticalcovariance.
If not mentioned, we used product concentrations at 0, 5, 10, 15, 20, and 25 min to estimate initial velocities. For Figures 2A andS2A, 7B and S8C, S10C, and S10D (right), initial velocities were estimated with product concentrations at 0, 5, 10, and 15 min. For
Figure 2A, due to the substrate inhibition observed at 320 mM substrate (Figure S2A), we used initial velocities estimated at 20, 40, 80,
and 160 mM substrate for Michaelis-Menten analysis. Analysis including 320 mM substrate was conducted in Figure 3 (Figures S3C
and S3D).

e4 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

ATP-dependent Kinase Assay
DCKId protein (1 mM) was added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, and 20,
40, 80, 160, 320 mM *CKItide(S10A) (KRRRALSVAALPGL), respectively) at 35 C and 25 C. ATP concentration was changed the 20,40, 80, 160, 320 mM respectively. Reaction was stopped by stop solution (20 mM Tris-HCl pH7.5, 100 mM NaCl, 5 mM EDTA) at 0, 5,
10, 15, 20, 25 min. These solutions were moved to 384-well plates and were analyzed by mobility shift assay. Initial velocities were
estimated with product concentrations at 0, 5, 10, and 15 min (Figure S1E). See also ‘‘Initial Velocity Analysis’’ section.
ATPase Activity Measurements
ATPase activity of DCKId was measured using an ACQUITY UPLC system (Waters Corporation, USA). The measurement condition
was described by reference to ATPase activity of Kai protein (Murayama et al., 2011). DCKId protein (1 mM) was added to the reaction
buffer (25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, and 10, 25, 50, 100 mM ATP) at 35 C and 25 C. Reaction
was auto-sampling by UPLC at 0, 5, 10, 15, 20, 25 min. ADP was separated from ATP on a BEH C18 column (2.1 3 50 mm, 1.7 mm)(Waters Corporation, USA) at a flow rate of 0.8 ml/min with mobile phase of 50 mM ammonium formate, 10 mM tetrabutylammoniumhydrogen sulfate (pH 8.5) and 17% (v/v) acetonitrile. The ADP concentrations were calculated from their peak areas. See also ‘‘Initial
Velocity Analysis’’ section.
Product Inhibition Assay for Kinase Activity
CKItide(2pS) (KRRRALpSVApSLPG) was synthesized by peptide synthesizer, see ‘‘Substrate Screening’’ section. DCKId protein (1 mM)
was added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 640 mM ATP, 1 mM DTT, 0.1 mg/mL BSA and 20 mM CKItide(KRRRALpSVASLPGL)) at 35 C and 25 C. Synthesized CKItide(2pS) peptide and ADP (80, 160, 320 mM) was added reaction mixture.
Reaction was stopped by stop solution (20 mM Tris-HCl pH7.5, 100 mM NaCl, 5 mM EDTA) at 0, 5, 10, 15, 20, 25 min. These solutions
were moved to 384-well plates and analyzed by mobility shift assay. See also ‘‘Initial Velocity Analysis’’ section.
ITC Assay
For ITC assay, MicroCal iTC200 was used. With Origin 7.0 software provided by MicroCal, data were fitted with a one-site binding
model and the baselines were corrected automatically with minor manual adjustment. DCKId (WT, K224D, and K224E) purified as
described above, were both dialyzed against a buffer consisting of 25 mM Bis-Tris pH 7.5, 50 mM NaCl and 10 mM MgCl. Protein
concentrations were determined by UV spectra. CKItide(2pS) (500 mM at 25 C and 750 mM at 35 C) was loaded into the syringe and
50 mM DCKId into the sampling cell in presence of 1 mM ADP. Titrations were performed at 25 C and 35 C using an initial injection of
0.5 ml, followed by 2 mL injections. CKItide(2pS) was titrated with amount of DCKId protein until saturation was reached.
Dephosphorylation Assay
DCKId protein (0.2 mM at 25 C and 0.05 mM at 35 C) was added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 640 mM
ADP, 1 mM DTT, 0.1 mg/mL BSA and CKItide(2pS) (KRRRALpSVApSLPGL)) at 35 C and 25 C. The reaction was sampled at a seriesof time points at 0, 5, 10, 15, 20, and 25 min. These solutions were added to the stop solution at each time point. The solutions weremoved to 384-well plates and were analyzed by mobility shift assay. See also ‘‘Initial Velocity Analysis’’ section.
Mass Spectrometry for Dephosphorylation Assay
We confirmed contamination in purified protein and the dephosphorylation activity of DCKId using LC-MS analysis. The dephosphorylation assay was performed conditions described in ‘‘Dephosphorylation Assay’’ section and time series sampling at 0 min and
30 min. The trypsin digested sample was prepared to a phase transfer surfactant protocol (Ode et al., 2017). The samples in a buffer(12 mM sodium deoxycholate, 12 mM sodium N-lauroylsarcosinate, 50 mM NH4HCO3) were digested with 1 mg trypsin using microwave (Rapid Enzyme Digestion System, AMR, Japan) for 20 min. After the digestion, an equal volume of ethyl acetate was added tothe sample, which was acidified with 0.5% TFA and well mixed to transfer the detergents into organic phase. The sample was centrifuged at 10,000 x g for 10 min at room temperature, and an aqueous phase containing peptides was collected and dried with
SpeedVac (Thermo Fisher Scientific). The dried peptides were solubilized in 100 mL of 2% acetonitrile and 0.1% TFA and thencleaned up by self-prepared C18 tips (Rappsilber et al., 2007). The LC-MS analyses were performed by data-dependent MS/MS
with IT-FT mass spectrometer (Orbitrap velos mass spectrometer, Thermo Fisher Scientific) equipped with an HPLC system containing a nano HPLC equipment (Advance UHPLC, Bruker Daltonics) and an HTC-PAL autosampler (CTC Analysis) with a trap column(0.3 3 5 mm, L-column, ODS, Chemicals Evaluation and Research Institute, Japan). An analytical sample solubilized in 2% acetonitrile and 0.1% TFA was loaded to the LC-MS system to be separated by a gradient using mobile phases A (0.5% acetic acid) and B(0.5% acetic acid and 80% acetonitrile) at a flow rate 300 nL/min. The eluted peptides were electrosprayed (2.4 kV) and introduced
into the MS equipment (positive ion mode, data-dependent MS/MS). The obtained raw data was subjected to database search with
MASCOT (Matrix Science) algorithm running on Proteome Discoverer (Thermo Fisher Scientific).
Bayes Estimation of Rate Parameters
Figure 3A is a comprehensive model to explain experimental data observed in the current work. Since the model includes manyrate parameters (16 parameters), it is possible to fit experimental data by several combination of parameters (Figures S3A–S3D).

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e5

Therefore, we cannot determine a parameter set uniquely (point estimation). Instead of determining a parameter set, we generate
several rate parameter set to fit experimental data (Bayes estimation). For Bayes estimation of rate parameters, the statisticalcomputing platform R 3.1.1 (https://www.r-project.org/) was used. Since the model equations cannot be solved analytically, we
solved equations numerically. To determine the initial parameter set to fit experimental data, we applied global fittings. Then, we
sampled rate parameters by Markov chain Monte Carlo (MCMC) simulations with the initial best fit parameters. Finally, we performed
perturbation analyses of sampled rate parameters to identifying key parameters for temperature compensated reactions. These procedures are described in detail as follows:
Ordinary Differential Equations (ODE) of the Model
ODEs of the model shown in Figure 3A are described as follows:d½T
= kdT ½ET  kaT ½E½T;dt(Equation 1)

d½D= kdD ½ED  kaD ½E½D;dt(Equation 2)

d½S0
= kdTS0 ½ETS0  kaTS0 ½ET½S0;dt(Equation 3)

d½S1
= kdDS1 ½EDS1  kaDS1 ½ED½S1 + kdTS1 ½ETS1  kaTS1 ½ET½S1;dt(Equation 4)

d½S2
= kdDS2 ½EDS2  kaDS2 ½ED½S2;dt(Equation 5)

d½E
= kdT ½ET  kaT ½E½T + kdD ½ED  kaD ½E½D;dt(Equation 6)

d½ET
= kaT ½E½T  kdT ½ET  kD ½ET + kdTS0 ½ETS0  kaTS0 ½ET½S0 + kdTS1 ½ETS1  kaTS1 ½ET½S1;dt(Equation 7)

d½ED
= kD ½ET + kaD ½E½D  kdD ½ED + kdDS1 ½EDS1  kaDS1 ½ED½S1 + kdDS2 ½EDS2  kaDS2 ½ED½S2;dt(Equation 8)

d½ETS0
= kaTS0 ½ET½S0  kdTS0 ½ETS0  kDS1 ½ETS0;dt(Equation 9)

d½EDS1
= kDS1 ½ETS0 + kaDS1 ½ED½S1  kdDS1 ½EDS1;dt(Equation 10)

d½ETS1
= kaTS1 ½ET½S1  kdTS1 ½ETS1 + kTS1 ½EDS2  kDS2 ½ETS1;dt(Equation 11)

d½EDS2
= kDS2 ½ETS1  kTS1 ½EDS2 + kaDS2 ½ED½S2  kdDS2 ½EDS2:dt(Equation 12)

ODEs were numerically solved with deSolve package (Soetaert et al., 2010).

e6 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

Experimental Data to Construct a Computational Model
We used the following experimental data to construct a computational model:(1) ATPase assay: T to D (Figure S1D; n = 3)
d [E]0 = 1 mM, [T]0 = 10, 25, 50, 100 mM(2) Phosphorylation assay: S0 to S2 (Figures 1A and S1C; n = 3)
d [E]0 = 1 mM, [T]0 = 640 mM, [S0]0 = 20, 40, 80, 160, 320 mM
d Data for [S0]0 = 640 mM were excluded in the current analysis(3) Phosphorylation assay: S1 to S2 (Figures 2A and S2A; n = 3)
d [E]0 = 0.005 mM, [T]0 = 640 mM, [S1]0 = 20, 40, 80, 160, 320 mM(4) Dephosphorylation assay: S2 to S1 (Figure 2G; n = 3)
d [E]0 = 0.2 mM, [D]0 = 640 mM, [S2]0 = 20, 40, 80, 160, 320 mM for 25 C
d [E]0 = 0.05 mM, [D]0 = 640 mM, [S2]0 = 20, 40, 80, 160 mM for 35 C(5) Product inhibition assay: S1 to S2 (Figures 2B, S2B, and S2C; n = 5)(a) with ADP
d [E]0 = 0.005 mM, [T]0 = 640 mM, [S1]0 = 20 mM, [D]0 = 80, 160, 320 mM
d n = 3 data were used for the current analysis(b) with ADP + S2
d [E]0 = 0.005 mM, [T]0 = 640 mM, [S1]0 = 20 mM, [D]0 = [S2]0 = 80, 160, 320 mM
d n = 3 data were used for the current analysis
Global Fitting to Experimental Data
To find a rate parameter set to represent experimental data, product concentrations of ATPase, phosphorylation, dephosphorylation,and product inhibition assays were fitted simultaneously with identical parameter set (global fitting). To circumvent to search in unphysical values, association rate parameters were searched between 1 M-1s-1 and 1010 M-1s-1. Reaction or dissociation rate constants were searched between 105 s-1 and 106 s-1. Numerical optimizations with box constrains were performed by ‘‘optim’’ function
with ‘‘L-BFGS-B’’ option in R. To search wide range of parameters, rate parameterskhðkaT ; kdT ; kD ; kaD ; kdD ; kaTS0 ; kdTS0 ; kDS1 ; kaDS1 ; kdDS1 ; kaTS1 ; kdTS1 ; kDS2 ; kTS1 ; kaDS2 ; kdDS2 Þ(Equation 13)

were converted to
xhlog10 k:(Equation 14)

The score function for the global fitting is a squared sum of relative error of experimental product concentrations with respect tosimulated ones:
ninit X
nt 
na X
n X
X
½Pi expðtm ; j; lÞ  ½Pi simðtm ; j; k = 10x Þ 2:(Equation 15)
ScoreðxÞ =½Pi simðtm ; j; k = 10x Þ
i=1 j=1 l=1 m=1[Pi]exp(tm;j,l) represents an experimental product concentration of i-th assay at time tm and l-th measurement. The index j expresses
initial concentrations of substrate or product in the i-th assay. For the production inhibition assay, the product concentration wascomputed by subtracting initial product concentration from the total product concentration. [Pi]sim(tm;j,k) represents a simulated
product concentration of i-th assay at time tm with rate parameters k and initial concentrations indexed by j. Initial parameters x
were generated from uniform distribution with box constraints. The best fit parameter set x0 was determined by global fittings generated from 1000 random parameter set.
Markov Chain Monte Carlo (MCMC) Simulation to Sample Rate Parameters
For Bayes estimation, parameters x were sampled from the posterior distribution p(xjdata). Based on Bayes’ theorem, the posterior
distribution can be represented as p(xjdata) f p(datajx) p(x), where p(datajx) and p(x) represent the likelihood function and the priordistribution, respectively. We used a normal distribution for the likelihood function p(datajx) f exp(-Score(x)/2s2). The model variance s2 was determined from the score function Score(x0) of the best fit parameter x0 by dividing the total number of all experimental
data points. We used non-informative prior (p(x) = const) with box constraints identical to the global fitting.
With the likelihood function and the prior, parameters that follow the posterior distribution can be generated numerically by the
MCMC simulation. We used FME package (Soetaert and Petzoldt, 2010) for MCMC simulations. For the MCMC simulation, trial parameters were generated from a multivariate normal distribution. To determine the SD of the normal distribution, we conducted10,000 step MCMC simulations by changing the SD value. Then, we determined the SD from the simulation whose acceptance ratio
was around 40%. With this criterion, SDs were determined to 0.008 at 25 C and 0.007 at 35 C. The best fit parameter set x0 was used
as an initial parameter set. MCMC simulations were performed for 108 steps (Figures S3A and S3B). As a result, we obtained wellfitted plots in all experimental data (Figures S3C and S3D), which provided detailed information on each reaction process in multi-site

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e7phosphorylation (Figures S3E and S3F). For analysis, 10,000 rate parameter sets were sampled from 108 steps MCMC simulation
with 10,000 step interval.
Perturbation Analyses of Rate Parameters Sampled by the MCMC Simulation
To understand the contribution of each rate parameter to ATPase, phosphorylation, and dephosphorylation activities, we performed
perturbation analyses by observing the response by changing rate parameters at 35 C. First, we quantify the impact of each rate
parameter to overall time course of product concentrations ([S2]) for S0 to S2 reaction (Figure 3B). As an example, we consider
the perturbation analysis of the association rate parameter kaTS0 as follows: (i) Pick up a rate parameter set from the MCMC simulation. (ii) kaTS0 is multiplied by 2 or 0.5. Other 15 rate parameters are not changed. (iii) ODE for S0 to S2 reaction is solved under these16 rate parameters. (iv) Compute the root mean square relative error (RMSRE) between experimental and simulated product timecourse [S2](t). By repeating (i)-(iv) for sampled parameter set by the MCMC simulation, we obtain (10,000 in the current analysis)values of RMSRE. From these values, we can compute the mean and the SD of RMSRE. The bar graph in Figure 3B was computedin this way. The large deviation from experimental data suggests that the perturbed rate parameter is important process for S0 to S2reaction. The contribution of the parameter to the reaction can be investigated in detail by observing perturbed time courses of product concentrations. The ratio (Q10) of perturbed mean product concentrations at 35 C to unperturbed one at 25 C is shown in
Figure 3B.
We also conducted the perturbation analysis of rate parameters by observing the response of kinetic parameters such as kcat/Km,
kcat, and Km, which characterize the reaction quantitatively. In the following sections, we derive kinetic parameters by rate parameters. Procedures (i) and (ii) are identical to the previous ones. (iii) Compute kinetic parameters by using 16 parameters. By repeating(i)-(iii), we can compute the mean and the SD of the perturbed kinetic parameter. In Figure 3C, unperturbed kcat/Km, kcat, and Km at
25 C and 35 C, and perturbed one at 35 C are shown.
Rate Equation for the ATPase Activity
As shown in Figure S1D, DCKId has ATPase activity. To understand the contribution of ATPase activity to the phosphorylation process, we derive the rate equation. Since DCKId also has dephosphorylation activity (Figures 2D–2G), the ADP binding process was
explicitly introduced. ODEs of the ATPase with product (ADP) binding are given as
d½T= kdT ½ET  kaT ½E½T;dt(Equation 16)

d½D= kdD ½ED  kaD ½E½D;dt(Equation 17)

d½E
= kdT ½ET  kaT ½E½T + kdD ½ED  kaD ½E½D;dt(Equation 18)

d½ET
= kaT ½E½T  kdT ½ET  kD ½ET;dt(Equation 19)

d½ED
= kD ½ET + kaD ½E½D  kdD ½ED:dt(Equation 20)

Under quasi steady state approximation (QSSA), d[E]/dt = d[ET]/dt = d[ED]/dt = 0, Equation 17 is expressed as
d½D
kcatT ½E0 ½T=;
dt KmT 1 + ½D + ½T

(Equation 21)

kdD kDaD=
kdD ;kdD + kD 1 + aD

(Equation 22)

kD
;
kdD

(Equation 23)

KdD

where we defined
kcatT haD he8 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

KmT hðkdT + kD ÞkdD;kaT ðkdD + kD Þ

(Equation 24)

kdD
:
kaD

(Equation 25)

KdD hThus, the rate Equation 21 follows Michaelis-Menten kinetics with ADP independent kcat denoted by kcatT and ADP dependent Km
denoted by KmT (1 + [D]/KdD). The inhibition that does not change kcat but kcat/Km is called competitive inhibition. Therefore, the product (ADP) behaves as a competitive inhibitor and the competitive inhibition constant is identical to the dissociation constant of the
ADP (KdD) in the current model.
Rate Equation for the Phosphorylation Activity
Derivation of the Rate Equation
To understand phosphorylation process S0 to S1 and S1 to S2 quantitatively, we derive rate equations and represent kinetic parameters by rate parameters. To proceed from S1 to S2, the binding process between ET and S1 is required. Therefore, we included theprocess in the model S0 / S1 (Figure S3G). In a similar way, the binding process between ED and S1 was included in the model
S1 / S2 (Figure S3H). Instead of deriving each rate equation for S0 / S1 and S1 / S2, we use the model S / P that includes
both models as shown in Figure S3I. To derive the rate equation, we applied the King-Altman method (Cook and Cleland, 2007).
The King-Altman method is a schematic method to derive the rate equation by systematically deriving concentrations of all enzyme
complexes under QSSA. The ODE of the product concentration is given as
d½P
= kdDP ½EDP  kaDP ½ED½P + kdTP ½ETP  kaTP ½ET½P:dt(Equation 26)

After expressing all enzyme complex concentrations by [T], [D], [S], [P], and [E]0 with the King-Altman method, Equation 26 can be
expressed as
d½P
d½S nð½T; ½D; ½S; ½PÞ
=
=
½E ;
dt
dt dð½T; ½D; ½S; ½PÞ 0(Equation 27)

nð½T; ½D; ½S; ½PÞhnTS ½T½S  nTP ½T½P  nDP ½D½P;(Equation 28)

dð½T; ½D; ½S; ½PÞhd1 + dT ½T + dD ½D + dS ½S + dP ½P
+ dTS ½T½S + dTP ½T½P + dDS ½D½S + dDP ½D½P
+ dTSS ½T½S½S + dTSP ½T½S½P + dTPP ½T½P½P
+ dDSS ½D½S½S + dDSP ½D½S½P + dDPP ½D½P½P:(Equation 29)

We note that the equality d[P]/dt = - d[S]/dt holds in Equation 27 under QSSA. Coefficients in the numerator and the denominatorare given asnTS hkaT kdD kaTS kdTP kDP kdDP kdDS ;(Equation 30)

nTP hkaT kD kdTS kdTP kTS kaDP kdDS ;(Equation 31)

nDP hðkdT + kD ÞkaD kdTS kdTP kTS kaDP kdDS ;(Equation 32)

d1 hðkdT + kD ÞkdD kdTP ðkdTS kTS + kDP kdDP + kdTS kdDP ÞkdDS ;(Equation 33)

dT hkaT ðkD + kdD ÞkdTP ðkdTS kTS + kDP kdDP + kdTS kdDP ÞkdDS ;(Equation 34)

dD hðkdT + kD ÞkaD kdTP ðkdTS kTS + kDP kdDP + kdTS kdDP ÞkdDS ;(Equation 35)

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e9

dS hkdD kaTS kdTP kDP kdDP kdDS ;(Equation 36)

dP hkdT kdTS kdTP kTS kaDP kdDS ;(Equation 37)

dTS hkaT kdTP ðkaTS ðkDP kdDP + kdD ðkTS + kdDP + kDP ÞÞkdDS + kD ðkdTS kTS + kDP kdDP + kdTS kdDP ÞkaDS Þ;(Equation 38)

dTP hkaT ðkdTP kaDP ðkdTS kTS + kD ðkTS + kDP + kdTS ÞÞ + kdD kaTP ðkdTS kTS + kDP kdDP + kdTS kdDP ÞÞkdDS ;(Equation 39)

dDS hkaD kdTP ðkaTS kDP kdDP kdDS + ðkdT + kD ÞðkdTS kTS + kDP kdDP + kdTS kdDP ÞkaDS Þ;(Equation 40)

dDP hkaD kdTP ððkdT + kD + kdTS ÞkTS + ðkdT + kD ÞðkdTS + kDP ÞÞkaDP kdDS ;(Equation 41)

dTSS hkaT kaTS kdTP kDP kdDP kaDS ;(Equation 42)

dTSP hkaT kaTS kdTP ðkDP + kTS ÞkaDP kdDS ;(Equation 43)

dTPP hkaT kdTS kaTP kTS kaDP kdDS ;(Equation 44)

dDSS hkaD kaTS kdTP kDP kdDP kaDS ;(Equation 45)

dDSP hkaD kaTS kdTP ðkDP + kTS ÞkaDP kdDS ;(Equation 46)

dDPP hkaD kdTS kaTP kTS kaDP kdDS :(Equation 47)

In the model S0 / S1 (Figure S3G), the dephosphorylation rate is zero. By putting kTS = 0 into Equations 44 and 47, we have thatdTPP = dDPP = 0. In the model S1 / S2 (Figure S3H), the binding process between ET and S2 is not included. By putting kaTP = 0
into Equations 44 and 47, we have that dTPP = dDPP = 0. Thus, in the both models S0 / S1 and S1 / S2, equalities
dTPP = dDPP = 0(Equation 48)

hold. In the following derivations, we assume Equation 48 hold.
Rate Equation for the Phosphorylation without Initial ADP and ProductWithout initial ADP and product ([D] = [P] = 0), Equation 27 is expressed as
d½P kcatTS ð½TÞ=
dt KmTS ð½TÞ 1 +½E0 ½S;
1 + KiTS½Sð½TÞ(Equation 49)

kcatTS ð½TÞnTS ½T
kcatTS ½Th=;KmTS ð½TÞ d1 + dT ½T KmTS KT + ½T

(Equation 50)

kcatTS nTS
h
;
KmTS
dT

(Equation 51)½S
KmTS ð½TÞwhere we defined

e10 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

d1
;
dT

KT h

kcatTS ð½TÞhnTS ½T½T
= kcatTS;
dS + dTS ½T
KTS + ½TKTS h

(Equation 54)

dS
;
dTS

(Equation 55)

KmTS ð½TÞ = KmTS

KmTS =

KT + ½T
;KTS + ½T

dT
;
dTS

(Equation 56)(Equation 57)

dS + dTS ½T
KTS + ½T;= KiTSdTSS ½T
½T

KiTS h(Equation 53)

nTS
;
dTS

kcatTS hKiTS ð½TÞh(Equation 52)

dTS
:
dTSS

(Equation 58)(Equation 59)

The term [S]/KiTS([T]) in Equation 49 represents the inhibition effect for higher substrate concentration and is known for thesubstrate inhibition (Cook and Cleland, 2007). We denote ATP saturated kinetic parameters (such as kcatTS) by omitting the ATP concentration from the ATP dependent kinetic parameters (such as kcatTS([T])). By putting Equations 33 and 34 into Equation 52, wehave thatKT =ðkdT + kD ÞkdD= KmT :
kaT ðkD + kdD Þ(Equation 60)

Thus, KT is identical to Km of the ATPase activity (KmT; Equation 24) in the current model.
If we do not incorporate the binding between enzyme-ADP complex and substrate (kaDS = 0) as models for S0 / S1 (Figures 1C
and S3G), Equations 42 and 45 lead to dTSS = dDSS = 0. In this case, Equation 49 is described by the Michaelis-Menten model
d½P kcatTS ð½TÞ½E0 ½S
=:dt
KmTS ð½TÞ + ½S

(Equation 61)

Thus, the substrate inhibition term is present due to the binding between enzyme-ADP complex and substrate (Cook and Cleland,2007) in the current model.
We consider ATP saturated kinetic parameters for S0 / S1. By putting Equations 30, 34, and kTS = 0 into Equation 51, we have that
kcatTS
kdD
kaTS kDP
1
kaTS kDP==:
KmTS kD + kdD kDP + kdTS 1 + aD kDP + kdTS

(Equation 62)

By putting Equations 30, 38, and kTS = kaDS = 0 into Equation 54, we have that
kcatTS =kdD kDP kdDP
1
=
;
1
1
kDP kdDP + kdD kdDP + kdD kDP 1++
kdD kDP kdDP

(Equation 63)

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e11

kDP + kdTS
kaTS kDP
KmTS = ð1 + aD Þ
:
1
1
1
+
+
kdD kDP kdDP

(Equation 64)

We note that Equations 62, 63, and 64 hold for both models in Figures 1C and S3G. If the rate limiting step of ATPase is the forward
reaction (aD < < 1; Equation 23), aD do not contribute to kcatTS/KmTS in Equation 62. On the other hand, the rate limiting step of ATPaseis the ADP dissociation (aD >> 1), the temperature dependence of aD largely contribute to kcatTS/KmTS. Therefore, we consider theestimation of aD. From Equations 53, 54, 30, and 38, we can show inequalities
kcatTS ð½TÞ%kcatTS %kdD :(Equation 65)

kcatT
kcatTS ð½TÞ  kcatT(Equation 66)

By using Equations 22 and 65, the inequalityaD %holds. Thus, the upper bound of aD can be evaluated with kcat values of ATPase and phosphorylation activities. We note that theinequality Equation 66 holds for S0 to S1 and S1 to S2 models. Therefore, the upper bound of aD can be tightly estimated by using
the maximum value of kcat for S0 to S1 or S1 to S2 reactions.
By using results for S1 to S2 (kcat = 681 min-1; Figure 2A) and ATPase (kcat = 2.32 min-1; Figure S1D) at 35 C, Equation 66 leads to
aD < 0.0035. Thus, the rate limiting step of the ATPase is not the ADP dissociation but the forward reaction. The result was also
confirmed by the perturbation analysis of kcat for the ATPase (Figure S4A). Under the condition aD < < 1, Equation 62 can be further
simplified for the magnitude relation between kdTS and kDP as
8kaTS ; for aD << 1 and kdTS << kDP
kcatTS <z kDP;(Equation 67)
KmTS :; for aD << 1 and kdTS >> kDP
KdTS

KdTS h

kdTS
:
kaTS

(Equation 68)

For kdTS < < kDP, the temperature independency of kcatTS/KmTS is accounted for by the temperature independence of the association rate between enzyme-ATP complex and substrate (kaTS). For kdTS >> kDP, the temperature independency of kcatTS/KmTS is
explained by cancelling the change of the reaction rate (kDP) with the change of the affinity between enzyme-ATP complex and substrate (KdTS).
Rate Equation for the Dephosphorylation due to the ATPase Activity without Initial ADP and Substrate
The term –nTP[T][P] in Equation 28 represents the dephosphorylation activity by adding ATP and product (for simplicity, we use theterm product for [P] that is a product of the phosphorylation and the substrate of the dephosphorylation). Without ATPase activity(kD = 0), the equality nTP = 0 holds in Equation 31. Therefore, the dephosphorylation activity is caused by using ADP produced
from ATP by the ATPase activity. Without initial ADP and substrate ([D] = [S] = 0), Equation 27 is expressed as the Michaelis-Menten
model
d½S kcatTP ð½TÞ½E0 ½P=;dt
KmTP ð½TÞ + ½P

(Equation 69)

kcatTP ð½TÞnTP ½T
kcatTP ½T
h=;KmTP ð½TÞ d1 + dT ½T KmTP KT + ½T

(Equation 70)

kcatTP nTP
h;
KmTP dT(Equation 71)

where we defined

kcatTP ð½TÞhnTP ½T:dP + dTP ½T

Rate Equation for the Dephosphorylation without Initial ATP and Substrate
Without initial ATP and substrate ([T] = [S] = 0), Equation 27 is expressed as the Michaelis-Menten model

e12 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

(Equation 72)

d½S kcatDP ð½DÞ½E0 ½P
=;
dt
KmDP ð½DÞ + ½P

(Equation 73)

kcatDP ð½DÞnDP ½D
kcatDP ½Dh=;KmDP ð½DÞ d1 + dD ½D KmDP KD + ½D

(Equation 74)

kcatDP nDPh;
KmDP dD(Equation 75)

where we defined

KD hkcatDP ð½DÞh

d1
;
dD

(Equation 76)

nDP ½D½D
= kcatDP;dP
dP + dDP ½D
+ ½D
dDP

nDP
;
dDP

(Equation 78)

dD
:
dDP

(Equation 79)

kcatDP h

KmDP =(Equation 77)

By putting Equation 33 and Equation 35 into Equation 76, we have that
KD =

kdD
= KdD :
kaD

(Equation 80)

Thus, KD is identical to the dissociation constant between enzyme and ADP (KdD).
Rate Equation for the Phosphorylation with Initial ADP and Product
For S0 to S2 reaction, S2 were produced almost linearly with respect to the progression of time (Figures S3C and S3D). In these conditions, produced ADP and peptide do not largely affect the reaction rate. For S1 to S2 reaction, on the other hand, nonlinearities wereobserved (Figures S3C and S3D). Although nonlinearities for lower substrate concentrations are considered as a result of depletion of
the substrate (e.g., over 50% conversion at 25 min for [S1]0 = 20 mM), nonlinearities were observed even for higher substrate concentrations (e.g., less than 20% conversion at 25 min for [S1]0 = 320 mM). This indicates produced ADP and peptide affect the reaction
rate. To consider effects of products, we discuss the rate equation with initial ADP and product.
First, we consider the numerator term n([T],[D],[S],[P]) in Equation 27. Its sign determines the direction of the reaction. To quantify
contributions of two negative terms in Equation 28, we represent n([T],[D],[S],[P]) as
½P
½D½P
;(Equation 81)
nð½T; ½D; ½S; ½PÞ = nTS ½T½S 1  aTP  aDP½S
½T½S

nTP kcatTPnTP dT KmTP
aTP h
=
=
;
nTS nTS kcatTS
dT KmTS(Equation 82)

nDP dD kcatDP 1
nDP dD d1 KmDP KD
=
=
:
aDP h
nTS nTS dT kcatTS 1
dT d1 KmTS KT(Equation 83)

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e13

To relate aTP and aDP in Equations 82 and 83 to kinetic parameters, we used Equations 51, 52, 71, 75, and 76. The equilibrium condition d[P]/dt = 0 is achieved for n([T],[D],[S],[P]) = 0. We denote equilibrium concentrations of [T], [D], [S], and [P] as [T]eq, [D]eq, [S]eq,
and [P]eq, respectively. By solving n([T]eq,[D]eq,[S]eq,[P]eq) = 0 for Equation 81, we have that
½Deq
kcatTP
kcatDP½Peq½Deq ½Peq KmTP ½Peq KmDP ½Peq KD
aTP
+ aDP
=
+
= 1:
½Teq
½Seq
½Teq ½Seq kcatTS
kcatTS
½Seq
½Seq
KmTS
KmTS
KT

(Equation 84)

The relation between equilibrium concentrations and kinetic parameters for a reversible reaction is known as the Haldane relationship(Cook and Cleland, 2007). Therefore, Equation 84 corresponds to the Haldane relationship in our model.
If initial concentrations [T], [D], [S], and [P] satisfy following conditions
½P
<< 1;
½S

(Equation 85)½D½P
<< 1;½T½S

(Equation 86)

aTP

aDP
Equation 81 can be approximated asnð½T; ½D; ½S; ½PÞznTS ½T½S:(Equation 87)

If these conditions are satisfied, Equation 27 can be represented by the substrate inhibition model
d½P
=
dt

kcatTS ð½T; ½D; ½PÞ½E ½S
KmTS ð½T; ½D; ½PÞ 0

;
½S
½S
1+
1+
KmTS ð½T; ½D; ½PÞKiTS ð½T; ½D; ½PÞ(Equation 88)

where we defined the apparent kcat/Km that depends on ATP, ADP, and product as
kcatTS ð½T; ½D; ½PÞ kcatTS ð½TÞhKmTS ð½T; ½D; ½PÞ KmTS ð½TÞ

1
;
½D
½P
½D½P
1+++
KcD ð½TÞ KcP ð½TÞ KcD ð½TÞKcDP

(Equation 89)
d1 + dT ½T
½T= KD 1 +;KcD ð½TÞhdD
KT(Equation 90)

d1 + dT ½T;
KcP ð½TÞhdP + dTP ½T

(Equation 91)

KcDP hdD:dDP(Equation 92)

KcD([T]), KcP([T]), and KcDP represent competitive inhibition constants by adding ADP, product, and both, respectively. By comparing
Equations 79 and 92, we can show the identity
KcDP = KmDP :(Equation 93)

Thus, the competitive inhibition constant by addition of ADP and product (KcDP) is identical to the ADP saturated Km for the dephosphorylation (KmDP) in the current model. We also introduced the apparent kcat as
kcatTS ð½T; ½D; ½PÞhkcatTS ð½TÞe14 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

1
;
½D
½P
½D½P
1+++
KuD ð½TÞ KuP ð½TÞ KuD ð½TÞKuDP

(Equation 94)

dS + dTS ½T
KuD ð½TÞh;dDS(Equation 95)

dS + dTS ½T;
KuP ð½TÞhdTSP ½T

(Equation 96)

dDS
KuDP h:dDSP(Equation 97)

KuD([T]), KuP([T]), and KuDP represent uncompetitive inhibition constants for adding ADP, product, and both, respectively. The
apparent substrate inhibition constant is defined as
dS + dTS ½T + dDS ½D + dTSP ½T½P + dDSP ½D½P
KiTS ð½T; ½D; ½PÞh:dTSS ½T + dDSS ½D

(Equation 98)

In some cases, KcDP or KuDP can be represented by the dissociation constant between enzyme-ADP complex and product
kdDP:
KdDP hkaDP(Equation 99)

Without the dephosphorylation activity, by putting Equation 35, Equation 41, and kTS = 0 into Equation 92, KcDP can be expressed as
KcDP = KdDP :(Equation 100)

Without the dephosphorylation activity and the binding between enzyme-ADP complex and substrate, by putting Equation 40,
Equation 46, and kTS = kaDS = 0 into Equation 97, KuDP can be expressed as
KuDP = KdDP :(Equation 101)

For S0 / S1 models in Figures 1C and S3G, the dephosphorylation activity and the binding between enzyme-ADP and substrate are
missing. Therefore, identities Equations 100 and 101 hold for S0 / S1 models.
We noted the following table provided Bayes estimators of aTP and aDP (Equations 82 and 83) for S1 to S2 reaction. Data are mean ±SD (sampled from 108 step MCMC simulation with 10,000 step interval).
25 C
aTP
aDP

35 C

< 1.88 3 10

6

< 9.38 3 106
4

(1.24 ± 0.22) 3 103

(6.87 ± 0.82) 3 10

We also noted the following table provided Bayes estimators of magnitudes of dephosphorylation activity (Equations 85 and 86) for
S1 to S2 reaction. ATP, substrate, ADP, and product concentrations correspond to the product inhibition assay in Figures 2B andS2B. Data are mean ± SD (sampled from 108 step MCMC simulation with 10,000 step interval).

[T] = 640 mM, [S] = 20 mM, [D] = [P] = 320 mM
25 C
aTP[P]/[S]aDP[D][P]/([T][S])< 3.00 3 10

35 C
5

(5.49 ± 0.65) 3 10

< 1.51 3 104
3

(9.93 ± 1.73) 3 103

Since values are smaller compared to 1, dephosphorylation activities in product inhibition assays are ignorable.
Chemical Screening
Chemical screening was performed by using Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich) consistsof 1259 chemicals. Each chemical was transferred twice into eight 384-well plates filled with phosphorylated peptide (2pS) (20 mM)
and ADP solution (1 mM). Next, DCKId solution (1 mM) was dispensed to each 384-well plate, n = 2. Then each plate was incubatedat 35 C for 3 hr. After that, stop solution were dispensed to each 384-well plate to stop enzyme reaction. All dispensing steps
described above were performed by using Biomek FX (Beckman Coulter). To quantify dephosphorylated form of peptide, mobility
shift assay was done. After normalization of all assay data, fold change against negative control (DMSO) was calculated. Finally,

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e15

we selected chemicals that fold change was less than mean - 3 SD. We performed second chemical screening against it discovered
chemicals.
To evaluate chemicals, we performed second screening both kinase assay and dephosphorylation assay. For kinase assay, eachchemical (10 mM) was added to the reaction buffer (1 mM DCKId, 25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA,
1 mM ATP and 20 mM *CKItide (KRRRALSVASLPGL), respectively) at 35 C. Kinase reaction was stopped by using a stop solution(20 mM Tris-HCl pH7.5, 100 mM NaCl, 5 mM EDTA) at 30 min. These solutions were analyzed by mobility shift assay in 384-well plates(see also ‘‘ATP-dependent Kinase Assay’’ section). For dephosphorylation assay, each chemical (10 mM) was added to the reaction
buffer (1 mM DCKId, 25 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 1 mM ADP and 20 mM CKItide(2pS)(KRRRALpSVApSLPG), respectively) at 35 C. Dephosphorylation reaction was stopped by using a stop solution (20 mM Tris-HCl
pH7.5, 100 mM NaCl, 5 mM EDTA) at 30 min. These solutions were analyzed by mobility shift assay in 384-well plates (see also‘‘Dephosphorylation Assay’’ section). Since N-arachidonylglycine and MRS 2211 sodium salt hydrate were not available at the second screening stage, they were not included in Figure 4D.
Docking Simulation of ATA and Its Derivative
Our approach to docking ATA molecule to the potential binding sites of DCKId utilized four main steps: (1) structure preparation of
DCKId for docking, (2) binding site prediction, (3) docking of ATA molecule, and (4) clustering and re-scoring according to the calculated binding free energy. An initial structure of mouse CKId was modeled from the chain A of rat CKId (PDB: 1CKJ) (Longenecker
et al., 1996). Amino acid sequences of mouse CKId and rat CKId is identical. To incorporate the ATP, the N-lobe (1-86 residues)
was superposed to ATP and Mg2+ complex of fission yeast CK1 (PDB: 1CSN) (Xu et al., 1995). The mouse CKId structure with
ATP and Mg2+ was refined for docking simulations using the Protein Preparation Wizard (Sastry et al., 2013) Script within Maestro.
For small molecule, ionization and energy minimization were performed by the OPLS3 force field in the LigPrep Script in the Maestro(Schrödinger, LLC, New York, NY, USA). These minimized structures were employed as input structures for docking simulations.
Next, putative druggable sites on DCKId-ATP complex were detected and represented by small dummy atoms using the grid-basedenergy calculation (Halgren, 2007, 2009) incorporated in the SiteMap program with shallow binding site mode (Schrödinger, LLC,
New York, NY, USA). As a result, we obtained five binding regions (Figure 4H). In this work, we searched for ATA binding sites otherthan a known ATP binding site. Docking simulations were performed using the Glide (Friesner et al., 2004; Halgren et al., 2004) SP
docking program (Schrödinger, LLC, New York, NY, USA). Up to 100 docking poses of ATA molecule were generated in a grid box
defined by each potential binding site position from previous step. We obtained 500 poses of ATA molecule in total. After the docking
simulations were completed, nine representative poses of cluster center from 500 poses on all potential binding sites were selectedby clustering analysis with average linkage method using Conformer Cluster Script in the Maestro. RMSD cutoff of clustering analysis
was 10 Å. Finally, ligand binding free energy of the representative poses was calculated using the MM-GBSA (Schrödinger, LLC, New
York, NY, USA) (Figure 4I). The docking procedure of NS-05199909 was identical to ATA. For NS-05199909, ten representative poses
were selected (Figure S5E).
Basic Residues around Phosphate Analogs
To identify basic amino acid residues that could interact with a phosphate group, we used two structures that bind phosphate analogs. One is the rat CKId with tungstate ions (WO42-), which contains 2 chains A and B (PDB: 1CKJ) (Longenecker et al., 1996). The
other is the human CKId with sulfate ions (SO42-), which contains 4 chains A, B, C, and D (PDB: 3UYS) (Long et al., 2012). There are 4
binding sites among 6 chains (two chains from 1CKJ and four chains from 3UYS). We labeled them as site 1, 2, 3, and 4 (Figure 5A). In
the following table, we show which chain binds the phosphate analog in the specified site.
1CKJ (Rat CKId with WO42-)

3UYS (Human CKId with SO42-)

Site 1

Chain A and B

Chain A, B, C, and D

Site 2

Chain B

Chain A and B

Site 3Chain A and B

Chain B

Site 4

Nothing

Chain A, C, and D

We note that all chain bind a phosphate analog in site 1. On the other hand, the phosphate analog in site 4 is only contained inchains of 3UYS.
Since phosphate analogs are negatively charged, we searched basic amino acid residues (histidine, arginine, and lysine) whose
minimum distance to a phosphate analog in the specified site is less than 10 Å. Identified residues are shown in Figures 5A and
5B. Mean and SD of minimum distances between identified basic residues and the phosphate analog in the specified site is shownin Figure 5B. The sample number in the computation of mean and SD corresponds to the number of chains which bind the phosphate
analog in the specified PDB ID (e.g., n = 2 for site 1 of 1CKJ and n = 1 for site 2 of 1CKJ).

e16 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

Plasmid Construction
Per2 and CKId plasmid used in this study were subcloned in the pMU2 vector (Ukai et al., 2007) as described in a previous study(Narumi et al., 2016) and expressed under the CMV promoter as a Flag-tag fusion to their N terminus. CKId mutagenesis with inverse
PCR followed by self-ligation of blunt-end product was performed using Mighty Cloning Reagent Set (Takara Bio, Japan). Primer sequences used CKId mutagenesis were summarized in Table S1.
Cultivation of Mouse ES Cells
ES cells were cultured without feeder cells as described in a previous report (Ode et al., 2017). Before beginning cultivation, surface of
BD PURECoat amine dishes were exposed to medium which are containing LIF plus 6-bromoindirubin-30 -oxime (BIO) (Sato et al.,2009) for more than 5 hr at 37 C with 5% CO2. ES cells were cultured without feeder cells in iSTEM medium (Clontech Laboratories
A Takara Bio Company) at 37 C with 5% CO2.
Targeting into Rosa26 Locus
The ROSA26::P(CAG)-CKId knockin mouse strains were established in our laboratory. All of the required elements, including P(CAG)CKId-polyA from pMU2-CKId (wild-type and K224D mutant, see ‘‘Plasmid Construction’’ section) and the Puromycin resistance gene
cassette (P(PGK)-PuroR-polyA), were inserted into pENTR-1A (Invitrogen). The resulting sequence (pENTR-P(CAG)-CKId-polyAP(PGK)-PuroR-polyA) was then inserted into the ROSA26 targeting vector (Abe et al., 2011), which contains the Reading Frame
Cassette B from the Gateway Conversion System (Invitrogen), using the Gateway system. In the resulting targeting vector, the insertion cassette was in the same orientation as the ROSA26 gene.
C-terminal-truncated (+63) TALENs (Miller et al., 2011) that bind target sequence (50 - CTGCAACTCCAGTCTTTCTAGAAGATGGGCGGGAGTCTTCTGGGCAGGCTTA-30 , TALEN binding sequences are indicated with italics) were designed using TALE-NT(Doyle et al., 2012).
Purified 0.9 mg of the targeting vector and 1.2 mg each of the TALEN-L and TALEN-R expression vector were co-transfected into
4 3 105 cells of C57BL/6 mouse ES cells, HK3i, (Kiyonari et al., 2010) by using Xfect Transfection Reagent (Clontech Laboratories A
Takara Bio Company). The homologous recombined, puromycin resistant ES cell clones were isolated for further culture andexpansion.
The screened for successful homologous recombination and the confirmation of genome integrity were performed as previously
described (Susaki et al., 2014) by PCR using the following primers (Hokkaido System Science): 1) forward primer annealing to theregion upstream of the 50 homologous arm: 50 -TGCTGGCCTACTGCTGCCTCGATCTTAC-30 , 2) reverse primer annealing to the region downstream of the 50 homologous arm: 50 -AGGACAACGCCCACACACCAGGTTAGC-30 , 3) forward primer annealing to the region upstream of the 30 homologous arm: 50 -CGTGGTGGAGCCGTTCTGTGAGACA-30 , 4) reverse primer annealing to the region
downstream of the 30 homologous arm: 50 -GGTGAAATGCTTGACTCCTAGACTT-30 , 5) reverse primer annealing to between 50 homologous arm and the CAG promoter: 50 -GGCTTTTAGTAAGCGAATTCGGTACC-30 , and 6) forward primer annealing to the polyA region of the puromycin resistance gene: 50 -TCCATCAGAAGCTGGTCGATC-30 . The copy number of the inserted cassette wasconfirmed with quantitative PCR assay (Susaki et al., 2014).
The selected ES cell clones were injected into 8-cell-stage ICR embryos to generate 100% ES cell-derived chimeras (Kiyonari
et al., 2010).
Locomotor Analysis of CKId Mutant Mice
Either 6, 10, or 20 weeks old male WT-KI mice were maintained under LD cycles (light 12 hr, dark 12 hr) for 16 days. Then the mice
were released into DD (constant dark) environment, and maintained for 21 days. The mice were resynchronized with LD cycle environment (light 12 hr, dark 12 hr) for 16 days, before they were released into LL (constant light) environment (18 days). K224D-KI miceof same ages were also exposed to the similar sequence of light conditions, that is LD for 21 days, DD for 18 days, LD for 14 days, andthen LL for 16 days. The circadian period of each light condition was estimated by using whole data obtained in the corresponding
condition. The LL data of non-significant circadian rhythmicity (chi-square test) were excluded. The behavior was monitored by an
infrared monitoring system (NS-AS01, Neuroscience), and data were analyzed with ClockLab software (Actimetrics).
Luminescence Monitoring of SCN Slice
The mice were maintained in 24 hr light dark cycle (12 hr dark, 12 hr light) before sampling. The 7-day old pups of mice carrying luciferase reporter gene with mutated or wild-type Csnk1d gene inserted into ROSA26 locus were anesthetized on crushed ice anddecapitated. Mice with intact ROSA26 locus were also used as a control. Brain was rapidly removed and trimmed down to a small
block containing the medial basal hypothalamus. The coronal brain slices of 300 mm thickness were obtained from the block by using
a McIlwain tissue chopper. The slices were then transferred into cooled Hanks buffer, and the one slice including suprachiasmatic
nucleus (SCN) was selected under a stereoscopic microscope. The extra region outside SCN were trimmed out. The SCN slice wasthen placed on Millicell-CM (PICMORG50, Millipore) in a 35 mm Petri dish which was sealed with silicone grease. The SCN slice wascultured with 1.2 mL of DMEM/F12 (12400-024, Thermo Fisher Scientific) supplemented with serum-free growth supplements (Nakamura et al., 2002). The measurement of bioluminescence from each SCN slice was performed with a photomultiplier tube (PMT)
detector assemblies (LM-2400, Hamamatsu Photonics) for 7 days either at 37 C or 30 C, followed by a medium exchange andMolecular Cell 67, 783–798.e1–e20, September 7, 2017 e17

another 7 days measurement at the complementary temperature (i.e., if a SCN slice was first measured at 37 C, the same slice wasmeasured at 30 C in the following measurement). Graphs of oscillations were plotted by Mathematica 9 (Wolfram). The detector assemblies were maintained in an incubator without CO2 gas, and interfaced with computers for continuous data-acquisition. Photons
were counted for 1 min with 15 min intervals.
The rhythmicity period length and oscillation phase of the SCN were determined as described previously (Ukai-Tadenuma et al.,2011), with a slight change. Briefly, bioluminescence time course data beginning 24 hr after measurement-start were used for analysis in order to distinguish endogenous circadian oscillation from acute effects of culture preparation. Bioluminescence data weredetrended using the trend curve calculated by smoothing spline method. Then, autocorrelation of the detrended bioluminescence
time courses data was calculated within the range of 14-34 hr to determine the circadian period of oscillation. The average timecourse and its standard deviation were calculated by aligning time courses from SCN slices in a same genotypic group according
to their oscillation phases.
Q10 was calculated with the formula

Q10 =

R2
R1

 10

T2 T1;

where T1 and T2 are the measurement temperatures, R1 and R2 are wave-numbers of the observed luminescence at the corresponding temperature (Barrett and Takahashi, 1995).
For the timelapse imaging, samples were prepared as described above. Sealed 35-mm culture dishes were placed on the stage ofa macrozoom microscope (MVX10; Olympus) in a dark hood. The culture dishes were kept at approximately 30 or 37 C in a heatedchamber (Tokai Hit) on the microscope stage. Bioluminescence was imaged using a 1.6 3 Plan Apochromat objective (NA 0.24;
Olympus) with 2.5 3 zoom and transmitted to an electron-multiplying charge-coupled device (EM-CCD) camera cooled to 80 C(ImagEM C9100-13, Hamamatsu Photonics). The dimension of an image is 512 3 512 pixels, and each pixel is 16 bits. Exposure
time was 540 s with EM gain of 300 3. Images were transferred at 690 kHz to minimize readout noise, and recorded using MetaMorphsoftware (Molecular device). Subsequent image processing including noise-reduction, intensity extraction and construction of themulti-slice movie was performed by Fiji/ImageJ software (Schindelin et al., 2012).
Multiple Sequence Alignment
The multiple sequence alignment was performed by Clustal Omega (Sievers et al., 2011) in UniProt (http://www.uniprot.org/align/)
and was rendered by ESPript (Robert and Gouet, 2014) (http://espript.ibcp.fr/ESPript/ESPript/) (Figure 7A).
Protein Preparation for X-ray Crystallography
The cDNAs encoding mouse CKId (residues 1–294) and CKI homolog 1 derived from yeast (residues 62–355) were amplified by
PCR and subcloned into a pET28a(+) derivative vector, which has a glutathione S-transferase-encoding sequence inserted afteran N-terminal polyhistidine tag. The constructs contained a tobacco etch virus (TEV) protease recognition site at the junction between GST and the protein sequences. The recombinant proteins were expressed in E. coli Rosetta2 (DE3) (Merck Millipore) cells
in LB broth at 37 C until the OD600 reached 0.6, at which point the temperature was shifted to 15 C. Protein expressions were
induced by addition of isopropyl-b-D-thiogalactopyranoside to a final concentration of 200 mM. The cultures were grown for an
additional 20 hr and harvested by centrifugation. Cell pellets were resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing
500 mM NaCl and 20 mM imidazole. Cells were lysed by sonication and clarified by centrifugation. The cell lysates were loaded
onto a HisTrap HP column (GE Healthcare), and CKId and CKI homolog 1 was respectively eluted with 50 mM Tris-HCl buffer(pH 8.0) containing 500 mM NaCl and 500 mM imidazole. Then, the CKId and CKI homolog 1 proteins were dephosphorylated
and the N-terminal histidine-GST tags were cleaved by incubating with l phosphatase (Cosmo Bio) and TEV protease (Kapust
et al., 2001) overnight at 4 C. The dephosphorylated proteins were further purified by cation exchange chromatography, followedby size-exclusion column chromatography using a HiLoad Superdex 200 16/60 (GE Healthcare). The CKId protein stored in 50 mM
Tris-HCl buffer (pH 7.5) containing 200 mM NaCl, 5 mM b-octyl glucoside, 1 mM EDTA and 1 mM DTT, and the CKI homolog 1
protein stored in 10 mM MOPS buffer (pH 7.0) containing 100 mM NaCl, 100 mM EGTA and 2 mM DTT was subjected to
crystallization.
Crystallization and Structure Determination
Crystallization was performed by the sitting-drop vapor-diffusion method under 20 C by mixing equal volumes (200 nl) of D294CKId(13.5 mg/ml) and a reservoir solution of 100 mM sodium citrate buffer (pH 5.0) containing 25 mM (NH4)2SO4 and 7% (w/v) PEG 8000.
The crystals were soaked with 2 mM of ADP for 24 hr in mother liquor. Reservoir solution for CKI homolog 1 (10 mg/ml) was 200 mM
sodium malonate buffer (pH 7.0) containing 20% (w/v) polyethylene glycol 3350. The crystals were briefly soaked in a cryoprotectant
drop composed of the reservoir solution supplemented with 20% glycerol and then flash-cooled in liquid nitrogen for X-ray diffraction
data collection. The datasets were collected at the beamline BL26B2, SPring-8 (Harima, Japan), and were indexed, integrated, andmerged using the HKL2000 program suite (Otwinowski and Minor, 1997). The crystal structures were determined by molecularreplacement using MOLREP (Vagin and Teplyakov, 2010) with the apo form structure of CKId (PDB: 3UYS) as the search model.

e18 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

Model building was accomplished with Coot (Emsley et al., 2010), and structural refinement was performed with REFMAC (Murshudov et al., 1997) and PHENIX (Adams et al., 2010). The structural models in the figures were depicted using PyMOL version 1.8 software (Schrödinger, LLC).
Multiple Structural Alignment
Multiple structural alignment was performed by Dali server (Holm and Rosenström, 2010) with the ‘‘Expand gaps’’ option.
Following structures were used: Rat CKId (PDB: 1CKJ and 1CKI); Human CKId (PDB: 3UYS); Human CKIε (PDB: 4HOK); Fission
yeast CK1 (PDB: 1CSN); Human CKIg3 (PDB: 4G17); Human TTBK1 (PDB: 4BTJ); Human VRK2 (PDB: 2V62); Human AurA (PDB:
4DEE); Human CDK2 (PDB: 1HCK). If a PDB file includes multiple chains, the chain that had the highest Dali score (Z-score) from
1CKJ:B structure was used. Secondary structures (a helix, 310 helix, turn, bend, b strand, b bridge, and no secondary structure
assigned) were labeled based on the ‘‘DSSP Legend’’ of the corresponding PDB ID in the PDB website (http://www.rcsb.org/).
Labels of secondary structure elements such as b7 and aD were based on previous studies (Longenecker et al., 1996; Xuet al., 1995). In Figure 7D, WO4, SO4, MG, PO4, and EDO represent tungstate ion WO42-, sulfate ion SO42-, magnesium ion
Mg2+, phosphate ion PO43-, and 1,2-ethanediol, respectively. The binding sites in ‘‘CKId WO4/SO4 site’’ are shown (see also
Figure 5A) if the minimum distance between the amino acid and a phosphate analogs (WO42-/ SO42-) is less than 10 Å among
chain A and B of 1CKJ (Longenecker et al., 1996) and chain A-D of 3UYS (Long et al., 2012). Underlined site indicates the contact
residue (minimum distance is less than 3.5 Å).
Molecular Dynamics Simulations
For MD simulations, the chain B of rat CKId (PDB: 1CKJ) (Longenecker et al., 1996) was used as an initial structure. The modelingprocedure of the initial structure of mouse CKId (apo form; Figure S9A) and ATP bound structure (Figure 7E) is described in ‘‘Docking
Simulation of ATA and Its Derivative.’’ The ADP binding structure (Figure 7E) was modeled by removing the g-phosphate from the ATPbound structure. Protonation states of histidine were determined from neighboring atoms as follows: 46 HID, 50 HIP, 120 HIP, 126
HIP, 162 HIP, 164 HIP, 185 HIP, and 278 HIE, where HID, HIE, and HIP indicate d nitrogen is protonated, ε nitrogen is protonated, andboth nitrogens are protonated, respectively. The first methionine (Met1) was excluded and Glu2-Leu293 residues were used for MD
simulations.
MD simulations were conducted by MD package Amber 10 (Case et al., 2008). The ff99SB force field parameter (Hornak et al.,€schweiler, 2010; Lindorff-Larsen et al., 2010) was used for proteins. Force field of2006) with additional modifications (Li and Bru
ATP and ADP (Meagher et al., 2003) were obtained from AMBER parameter database (http://research.bmh.manchester.ac.uk/
bryce/amber). Truncated octahedron periodic boundary condition was applied. TIP3P water molecules were added with 15 Å bufferfrom the protein surface. To neutralize the system, Cl- atoms were included. Electrostatic interactions were evaluated by smooth particle mesh Ewald (Essmann et al., 1995) with 10 Å direct space cutoff. After performing energy minimizations with 1000 steps, 100 ps
MD simulations were conducted at 1 atm and 25 C or 35 C. Production MD simulations were performed for 1.5 ms with the constantvolume condition at 25 C or 35 C. Langevin dynamics with collision frequency 1.0 ps-1 and 2 fs time step was used for MD simulations. Movies S2 and S3 were created by using VMD (Humphrey et al., 1996).
Root mean square fluctuation (RMSF) for each Ca atom (Figures 7E and S9A) and minimum distances between residues (Figures
S9D–S9G) were computed with PTRAJ and CPPTRAJ programs (Roe and Cheatham, 2013), respectively. Potential energy principal
component analyses (PEPCA; Figures S9D–S9I) (Koyama et al., 2008, 2011) were performed with a C++ program written by us. Residue based potential energies were computed without cutoff distance. PCA was implemented by diagonalization of the centered
Gram matrix (Koyama et al., 2008) with 1500 structures.
Alanine Scanning of CKId around K224
In structural analyses (Figures 7F and S8F) and MD simulations (Figures 7E and S9A), CKI specific domain K217-Q223 (Figure 7D)
showed large fluctuations. Furthermore, fluctuations of aF helix (K221-S234, Figure 7D) showed temperature sensitivity in MD simulations (Figures 7E and S9A). To understand the contribution of each residue to the temperature compensation, we performed
alanine scanning of these residues:
K217A, (A218), (A219), T220A, K221A, R222A, Q223A, K224A, Y225A, E226A, R227A, I228A, S229A, E230A, K231A, K232A,
M233A, S234A.
We also performed the alanine scanning of interaction residues of K217-S234. Since the 1st principal component (PC1) of PEPCA
corresponds to the conformational change of aF helix for DCKId-ADP at 35 C (Figures S9D and S9E) and DCKId-ATP at 25 C (Figures
S9F and S9G), we selected residues including K217-S234 from the top 20 components of the 1st eigenvector (bar graphs in Figures
S9D–S9G). As a result, we performed following alanine mutations:
K45A, D128A, K130A, D149A, L173A, T176A, (A177), R178A, (A180), H185A, D194A, E197A, Y201A, W213A, Q214A,
T235A, R256A.
For MD simulations of DCKId-ADP at 25 C and DCKId-ATP at 35 C, large conformational changes of aF helix were not observed(Figure 7E). To check whether interaction residues with K217-S234 specific to these conditions could change the temperature dependency, we also performed PEPCA in these conditions (Figures S9H and S9I). By comparing the top 20 components of the 1st

Molecular Cell 67, 783–798.e1–e20, September 7, 2017 e19

eigenvector (Figures S9D–S9I), there is only one specific interaction, that is the vdW interaction between L216 and R227 found in
DCKId-ADP at 25 C (Figure S9H). We also created the alanine mutation of the residue:
L216A.
We confirmed that the mutation did not induce the large change of Q10 (Figure 7G).
Dual-luciferase assay of these CKId mutants are shown in Figure 7G. See also ‘‘Transfection and Luciferase Assay’’ and ‘‘Plasmid
Construction’’ sections.
Design of a Temperature-compensated Kinase
TTBK1 was deficient in K217 to Q223 regions of CKId (Figure 7D; PDB: 4BTJ; Xue et al., 2013). We also note that contact residues ofphosphate analogs in site 1 of CKId are R178, Q214, G215, and K224 (‘‘CKId WO4 / SO4 site’’ in Figure 7D). Since R178 of CKId andthe corresponding residue of TTBK1 (R204) is conserved (Figure 7D), introduction of residues corresponding to Q214, G215, andK224 into TTBK1 is expected to recognize a phosphate group. With these considerations, we rationally designed TTBK1 mutant(rMT; reconstituted mutant type) by replacing 5 amino acid residues (R240-D244) of TTBK1 with 11 amino acid residues (Q214K224) (Figure 7H).
The double strand DNA of TTBK1(WT) and TTBK1(rMT) were synthesized by DNA synthesis (Thermo Fisher Scientific). HumanTTBK1(WT) (residues 1-343) and TTBK1(rMT) were amplified by PCR and subcloned into a pGEX-6P vector. The recombinant protein
was expressed in E. coli Rosetta2 (DE3) cells in LB broth at 30 C until the OD600 reached 0.4, at which point the temperature wasshifted to 37 C. Protein expression was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentrationof 100 mM. The cultures were grown for an additional 20 hr and harvested by centrifugation. Cell pellets were resuspended in 50 mM
Tris-HCl buffer (pH 7.5) containing 300 mM NaCl, 1 mM EDTA, 1mM DTT, 10% glycerine and cOmplete mini (Roche). Cells were lysedby sonication and clarified by centrifugation on ice. TTBK1(WT) and TTBK1(rMT) were purified from the cell lysate by GST-tag. Purified proteins were incubated with l phosphatase 5 hr at 25 C. The dephosphorylated proteins were further purified by HiTrap HP
column (GE Healthcare). Elution buffer used was 50 mM Tris-HCl buffer (pH 8.0) containing 600 mM NaCl and 1 mM DTT. Quality ofprotein was confirmed by SDS-PAGE and UV spectra.
QUANTIFICATION AND STATISTICAL ANALYSIS
Sample numbers of mean and SEM/SD are described in figure legends. Two-tailed Welch’s t tests were performed by Microsoft
Excel 2013.
DATA AND SOFTWARE AVAILABILITY
The accession numbers for CKId structure complexed with ADP and CKI homolog 1 structure are PDB: 5X17 and 5X18, respectively.
Raw images of SDS-PAGE analysis have been deposited to Mendeley Data and are available at http://dx.doi.org/10.17632/
8v6y744r3t.1.

e20 Molecular Cell 67, 783–798.e1–e20, September 7, 2017

Molecular Cell, Volume 67

Supplemental Information

Temperature-Sensitive Substrate and ProductBinding Underlie Temperature-CompensatedPhosphorylation in the ClockYuta Shinohara, Yohei M. Koyama, Maki Ukai-Tadenuma, Takatsugu Hirokawa, Masaki
Kikuchi, Rikuhiro G. Yamada, Hideki Ukai, Hiroshi Fujishima, Takashi
Umehara, Kazuki Tainaka, and Hiroki R. Ueda

D

40
80
Elution Time (sec)

35˚C

80
60
40
20
0

E

0

5

10 15 20 25
Time (min)n = 3 (± SEM)

0.2
0.1

100

S0 → S2

40
20
5

10
0

5

10 15 20 25
Time (min)n = 3 (± SEM)

10 15 20 25 30
Time (min)n = 3 (± SEM)

ATP (μM)
100
50
15
25
10
10
5
0

30

25˚C

0

5

10 15 20 25
Time (min)n = 3 (± SEM)

4

0.08

60

0

20

0

25˚C

80

0

30

20
Produced ADP (μM)

0.3

015 20 25 30 35 40 45 50
Temperature (˚C)
n = 3 (± SEM)

[S0]0 = 20, 40, 80, 160, 320, 640 μM
S0 → S2

0.4

120

ATPase activity (T → D)
35˚C
ATP (μM)
100
40 50
25
30 10
50

0.06

*

0.04

0.00

1.0
0.5
35˚C
25˚C

0.0

30

0

20

40 60 80 100
ATP (μM)n = 3 (± SEM)

60

2

40
20

0
25˚C 35˚C
Q10 = 1.65 ± 0.39
n = 3 (± SEM)

0
25˚C 35˚C
Q10 = 2.70 ± 0.54
n = 3 (± SEM)

25˚C 35˚C
Q10 = 1.68 ± 0.11
n = 3 (± SEM)

T→D

n.s.

n.s.

3

1

0.02

1.5

80

Km (μM)0

Substrate[S0]0 = 20 μM
S0 → S2

kcat (min-1)

100

Product

Produced product (μM)

C

[S0]0 = 20, 40, 80, 160, 320, 640 μM

Product (μM)[T]0 = 20 μM
835˚C

S0 → S1[T]0 = 40 μM[T]0 = 80 μM[T]0 = 160 μM

S0 → S1

S0 → S1

S0 → S1

25

15

6

20

10

4

0
5
10
Time (min)

15

5
10
Time (min)

15

30
20

10

5
0

40

20

10

0
0

50

30

15

5

2

40

[T]0 = 320 μM
60 S0 → S1

10

0

0

0

5
10
Time (min)

0
0

15

Initial velocity (μM/min)

Produced product (μM)

Initial velocity (μM/min)

Fluorescence intensity (a.u.)

Mobility shift assay

100
80
60
40
20
0

0.5

Produced ADP (μM)

Time course sampling
Reaction Stop

Initial velocity (μM/min)

B

Kinase Reaction

kcat/Km (min-1 μM-1)A

510Time (min)

15

0

5
10
Time (min)S0 → S1
4.0 [ATP] (μM)
320160
3.0 80
40
20
2.0

35˚C

1.0
0.0
0 100 200 300 400 500 600
Substrate (μM)

15[S0]0 = 20, 40, 80, 160, 320, 640 μM

Product (μM)

5

25˚C

S0 → S1

10[T]0 = 80 μM
S0 → S1

20

30
25

30
20

6

2

4

1

2

5

5

0

0

0

0

5
10
Time (min)

15

10

0

5
10
Time (min)15[T]0 = 320 μM
40 S0 → S1

S0 → S1

15

8

3

15

[T]0 = 160 μM

20

4

0

10

0

5
10
Time (min)

15

10
0
0

5
10
Time (min)

15

0

5
10
Time (min)S0 → S1[ATP] (μM)
2.5 320160
2.0 8040
1.5 20

25˚C

1.0
0.5
0.0

15

0 100 200 300 400 500 600
Substrate (μM)

G

15
10
5
0

K1

R2

R3

R4

L6

S7

V8 S10 L11 P12 G13 L14

n = 3 (± SD)

12*CKItide(S7D)KRRRALDVASLPGL
S0 → S1

0.04

10
8
6
4
2

35˚C
25˚C

0
0

200
400
600
Substrate (μM)
n = 3 (± SEM)

Figure S1: Shinohara et al.

0.03
*
0.02
0.01
0
25˚C 35˚C
Q10 = 1.51± 0.17
n = 3 (± SEM)

160
140
120
100
80
60
40
20
0

8000

n.s.

25˚C 35˚C
Q10 = 1.52 ± 0.32
n = 3 (± SEM)

6000
Km (μM)

20

14

kcat (min-1)

KRRRALSVASLPGL

Alanine scanning

Initial velocity (μM/min)

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14

kcat/Km (min-1 μM-1)

F
Conversion (%)[T]0 = 40 μM
S0 → S1

Initial velocity (μM/min)[T]0 = 20 μM

n.s.

4000
2000
0
25˚C 35˚C
Q10 = 1.02 ± 0.10
n = 3 (± SEM)

Figure S1. Temperature-sensitive binding of a substrate to an enzyme underlies thetemperature compensation of CKIδ-dependent single-serine phosphorylation, related to
Figure1.
(A) Workflow of the phosphorylation assay by using the mobility shift assay system.
(B) Initial velocity of a two-serine substrate *CKItide (KRRRALSVASLPGL) (20 μM) to a doublephosphorylated product CKItide(2pS) (KRRRALpSVApSLPGL) at 20°C to 45°C. Data are mean ±SEM (n = 3).
(C) Time course of products of ΔCKIδ-dependent phosphorylation on a two-serine substrate*CKItide (20 to 640 μM) at 35°C (left) and 25°C (right). Their substrate saturation curves are shownin Figure 1A. Data are mean ± SEM (n = 3).
(D) Time course of produced ADP (upper left and middle), their substrate saturation curves (upper
right), and kinetic parameters (lower) of ΔCKIδ-dependent ATPase activity (10 to 100 μM ATP)without a peptide substrate at 25°C and 35°C.
(E) Time course of products (left 5 columns; 20 to 320 μM ATP) and their substrate saturation
curves (right column) of ΔCKIδ-dependent phosphorylation on a single-serine substrate*CKItide(S10A) (KRRRALSVAALPGL) (20 to 640 μM) at 35°C (upper) and 25°C (lower). For
substrate saturation curves, data are initial velocity ± SE estimated from the linear fitting for the
initial velocity (n = 1).
(F) Phosphorylation activity of a series of alanine mutants of *CKItide by ΔCKIδ. Data are mean ±SEM (n = 3).
(G) Substrate saturation curves (left) and kinetic parameters (right) of ΔCKIδ-dependent
phosphorylation on a single-serine substrate *CKItide(S7D) (KRRRALDVASLPGL) at 25°C and
35°C.
For (D) and (G), data are mean ± SEM (n = 3); *p < 0.05 and n.s. (not significant); two-tailed Welch's
t-test. Solid line and shade represent mean ± SEM of curves of Michaelis-Menten model withestimated parameters.

05

10 15 20 25 30
Time (min)n = 3 (± SEM)**

5

10 15 20 25
Time (min)n = 5 (± SEM)

30

[S1]0 = 20 μM

ADP (μM)
320
15 160
80

25˚C

10
5

0.2
0
n=3
(± SEM)

80

160
320
ADP (μM)n = 5 (± SEM)

ΔCKIδ + ADP

80

25˚C

0.00

-0.10
0.01
μcal/sec-0.20

Time (min)
0 10 20 30 40 50 60

0.00

-0.01

0.5

1.0
1.5
2.0
Molar Ratio

05

10 15 20 25
Time (min)n = 5 (± SEM)

2.5

3.0

30

Product (μM)

1.0

0.9

0.8

80 160 320
ADP (μM)n = 5 (± SD)

-0.20
0.05

-0.80

5

10 15 20 25 30
Time (min)n = 5 (± SEM)

S1 → S2[S1]0 = 20 μM

ADP + S2 (μM)
320
15 160
80

25˚C

10

0

0

5

10 15 20 25
Time (min)n = 5 (± SEM)

30

1.0

0.9

0.8
80 160 320
ADP + S2 (μM)
n = 5 (± SD)

1.0

0.9

0.8

80 160 320
ADP + S2 (μM)
n = 5 (± SD)

E
N = 1.18 sites
Kd = 2.6 μM
ΔH = -771.3 cal/mol
ΔS = 23.0 cal/mol/deg

Time (min)
0 10 20 30 40 50 60

0.00

-0.60

0

5

0.00

-0.50

10

20

-0.10

-0.40

35˚C

15

0

80 160 320
ADP (μM)n = 5 (± SD)

35˚C

-0.30

[S1]0 = 20 μM

ADP + S2 (μM)
25 320
160
20 80

5
0.8

ΔCKIδ + ADP

-0.70

-0.03

0.0

0

160
320
ADP + S2 (μM)
n = 5 (± SEM)

N = 1.18 sites
Kd = 2.4 μM
ΔH = -276.9 cal/mol
ΔS = 24.8 cal/mol/deg

-0.02

-0.30

0

S1 → S2

20

0.4

Dkcal mol-1 of injectant

00.9

S1 → S2

Coefficient of determination (R2)5[S1]0 = 20 μM
25˚C

0.6

0.0

10

30

Coefficient of determination (R2)

30

15

1.0

-0.05
-0.10

S2 → S1
20
Conversion (%)

0

35˚C

Product (μM)

0.8

10 15 20 25
Time (min)n = 3 (± SEM)

20

[S1]0 = 20 μM

ADP (μM)
25 320160
20 80

μcal/sec

Initial velocity (μM/min)

1.0

5

40

kcal mol-1 of injectant

B

0

60

S1 → S2

Coefficient of determination (R2)

20

30

25˚C

Coefficient of determination (R2)40

S1 → S2

Product (μM)

60

0

80

35˚C

S1 → S2

Product (μM)

Product (μM)

80

C[S1]0 = 20, 40, 80, 160, 320 μM

Product (μM)

A

10

-0.15
-0.20

0.0

0.5

1.0

1.5
2.0
Molar Ratio

2.5

Figure S2: Shinohara et al.

3.0

0

25˚C

30˚C
35˚C
Temperaturen = 3 (± SEM)

Figure S2. Temperature-sensitive and ADP-dependent binding of a product to an enzyme
underlies the temperature compensation of CKIδ-dependent multi-site phosphorylation,
related to Figure 2.
(A) Time course of products of phosphorylation on a single-phosphoserine substrate CKItide (20to 320 μM) at 35˚C (left) and 25˚C (right). Their substrate saturation curves and kinetic parameters
are shown in Figure 2A. Data are mean ± SEM (n = 3).
(B) Initial velocities of CKItide-phosphorylation at 25˚C with initial ADP or mixture of ADP and
CKItide(2pS). Data are mean ± SEM (n = 3 for [ADP] = [S2] = 0 μM and n = 5 for otherwise); *p <0.05; two-tailed Welch's t-test.
(C) Time course of products of phosphorylation on a single-phosphoserine substrate CKItide (20μM) at 35˚C (upper) and 25˚C (lower) with initial ADP (left) or mixture of ADP and CKItide(2pS)(right). The coefficient of determination (R2) for the linear fitting to estimate the initial velocity
became closer to 1.0 (i.e. the dynamics of the reaction became more linear) in the presence of
higher dose of ADP or mixture of ADP and CKItide at the beginning of the reaction. Data are mean
± SEM (n = 5) for the time course of product and mean ± SD (n = 5) for the coefficient ofdetermination. Initial velocities are shown in Figures 2B and S2B.
(D) ITC assay for the binding of ADP to ΔCKIδ at 25˚C (left) and 35˚C (right).
(E) Temperature dependency of dephosphorylation activity by ΔCKIδ on CKItide(2pS). Data aremean ± SEM (n = 3).

kaTS0
kaDS1
kaTS1
kaDS2

35˚C
kdT

2x107

4x107
6x107
MCMC step

25˚C
Phosphorylation (S0 → S2)[S0]0 = 20, 40, 80, 160, 320 μM[S2] (μM)[S2] (μM)

30
200
10 15 20
Time (min)30
20

Enzyme complex ratio

10 15 20
Time (min)10 15 20
Time (min)Phosphorylation (S0 → S2)[S0]0 = 20 μM
25˚C

30
20

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0
0

10 15 20 25
Time (min)[S0]0 = 20 μM
35˚C

1.0
0.8

0.6

0.6

0.4

0.4

0.2

0.2

5

10 15 20
Time (min)5log10 (Reaction rate ( min-1))

4

5

10 15 20
Time (min)5

10 15 20
Time (min)[S0]0 = 320 μM

14
12
10
8
6
4
2
0

25

5

5

F

10 15 20
Time (min)

1.0
0.6

0.4

0.4

0.2

0.2

0.0

0.0
5

1.0
0.8

0.6

0.6

0.4

0.4

0.2

0.2

5

10 15 20
Time (min)

6
3
0
-3

kDS1

6
3
0
-3

kDS2

6
3
0
-3

kTS1

6
3
0
-3

12

2x107

4x107
6x107
MCMC step

8x107

108

0

2x107

4x107
6x107
MCMC step

8x107

108

Dephosphorylation (S2 → S1)[S2]0 = 20, 40, 80, 160, 320 μM

ATPase (T → D)
[T]0 = 10, 25, 50, 100 μM
15

8
6
4

25

5

10 15 20
Time (min)

25

10
5
0

0

10 15 20
Time (min)

5

0

25

4
2

20
10
0

0

5

10 15 20
Time (min)025

S0 → S1

kaD[D]
kdD
ED

EDS1

H

5

Figure S3: Shinohara et al.

25

30

kaDS1[S1] kdDS1

10 15 20
Time (min)[S1]0 = 320 μM

10 15 20
Time (min)40

6[S1]0 = 320 μM

55ATPase (T → D)
[T]0 = 10, 25, 50, 100 μM

Dephosphorylation (S2 → S1)[S2]0 = 20, 40, 80, 160 μM

G

25

0.0
10 15 20 25
0
5 10 15 20 25
Time (min)Time (min)[E]/[E]0 [ET]/[E]0 [ED]/[E]0 [EDS1]/[E]0 [ETS1]/[E]0 [EDS2]/[E]0

0

kD

0

0

0

0.8

0.0

6
3
0
-3

0

0

0.8

1.0

kTS1

2

5

0.6

10 15 20 25
Time (min)[S1]0 = 20 μM
35˚C

6
3
0
-3

10

10

0.8

0

kDS2

Phosphorylation (S1 → S2)[S1]0 = 20 μM[D]0 = [S2]0 = 80, 160, 320 μM

25Phosphorylation (S1 → S2)[S1]0 = 20 μM
25˚C

6
3
0
-3

108

0
0

25

8x107

kDS1

108

15

10

1.0

8x107

Phosphorylation (S1 → S2)[S1]0 = 20 μM[D]0 = [S2]0 = 80, 160, 320 μM

0Phosphorylation (S1 → S2)[S1]0 = 20 μM[D]0 = 80, 160, 320 μM

25

0.0
0
5 10 15 20 25
10 15 20 25
Time (min)Time (min)[E]/[E]0 [ET]/[E]0 [ED]/[E]0 [ETS0]/[E]0 [EDS1]/[E]0 [ETS1]/[E]0 [EDS2]/[E]00

60[S0]0 = 320 μM

0

5

0.8

0.0

8

0
0

1.0

4x107
6x107
MCMC step

15

40

0.8

2x107

10

Phosphorylation (S1 → S2)[S1]0 = 20, 40, 80, 160, 320 μM

250

Phosphorylation (S1 → S2)[S1]0 = 20 μM[D]0 = 80, 160, 320 μM

25

0

E

1.0

5

10

0

4x107
6x107
MCMC step00[S2] (μM)[S2] (μM)

40

2x107

2

50

50

10

Enzyme complex ratio

20

25

60

5

kdTS1

6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3

0

12

30

35˚C
Phosphorylation (S0 → S2)[S0]0 = 20, 40, 80, 160, 320 μM

0

kdDS1

Phosphorylation (S1 → S2)[S1]0 = 20, 40, 80, 160, 320 μM

0
5

kdTS0

108

10

10

1.0

8x107

40

0

kdD

kdDS2
0

40

D

108

[S2] (μM)

50

8x107

[S2] (μM)

C

4x107
6x107
MCMC step

Enzyme complex ratio

log10 (Association rate ( μM-1min-1))

kaD

2x107

6
3
0
-3

[D] (μM)

5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4

kaT

0

kD

[D] (μM)

B

kdDS2

log10 (Reaction rate ( min-1))

kaDS2

kdTS1

[S1] (μM)

kaTS1

kdDS1

[S1] (μM)

kaDS1

kdTS0

[S2] (μM)

kaTS0

kdD

6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3
6
3
0
-3

[S2] (μM)

kaD

kdT

Enzyme complex ratio

log10 (Association rate ( μM-1min-1))

kaT

25˚C

log10 (Dissociation rate ( min-1))

5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4
5
2
-1
-4

log10 (Dissociation rate ( min-1))

A

10 15 20
Time (min)E

kaT[T]
kdT
kaTS1[S1]
ET
ETS1
kdTS1
kD
kdTS0 kaTS0[S0]

kDS1

ETS0

S1 → S2

E
kaT[T]
kaD[D]
kdT
kdD
kaDS1[S1]
EDS1
ED
ET
kD
kdDS1
kaDS2[S2] kdDS2
EDS2

I

5

S→P
EDS

kTS1

kDS2

kdTS1 kaTS1[S1]ETS1EkaD[D]
kaT[T]
kdD kdTkaTP[P]
ED
ET
ETP
kD
kdDS
kdTP
kaDP[P] kdDP
kdTS kaTS[S]kaDS[S]

EDP

kTS
kDP

ETS

25

Figure S3. Bayes estimation of rate parameters in a computational model of temperaturecompensated CKIδ-dependent multi-site phosphorylation, related to Figure 3.
(A and B) Sampling of 16 rate parameters by Markov chain Monte Carlo (MCMC) simulations at
25°C (A) and 35°C (B). MCMC simulations were performed for 108 steps. The distribution of 10,000
rate parameter sets were sampled from 108 step MCMC simulation with 10,000 step interval. Some
rate parameters such as kaT and kdT fluctuate similarly. However, these data are not identical. We
confirmed that dissociation constants (such as kdT/kaT) fluctuated.
(C-D) Experimental (points) and simulated (curves) time course at 25°C (C) and 35°C (D). In eachexperiment, three independent datasets were used for the MCMC simulation. Bold curves
represent mean time courses obtained by MCMC simulations. Shaded curves represent the region
for mean ± SD of simulated time courses (sampled from 108 step MCMC simulation with 10,000
step interval).
(E-F) Mean time courses of ratios of enzyme complexes for S0 to S2 (E) and S1 to S2 (F) reactions.
(G-I) Computational models of reactions from S0 to S1 (G), from S1 to S2 (H), and the unified
reaction (I).

A

2

40
30

WT at 35˚C

kdD

kD

kdT

0

kdD

kD

10
WT

kD

kdT

kaT

50

20

1

WT at 25˚C

1/2-fold at 35˚C 2-fold at 35˚C

Dephosphorylation (S2 → S1)

300

20

200

WT at 25˚C

WT at 35˚C

kDS2

kdDS2

kaDS2

kTS1

kDS2

kdD

kdTS1

kaD

kD

WT

kdT

*

0

kTS1

kDS2

*

kdTS1

100

kdDS1

kdDS2

kDS2

kaD

kaDS2

0

kTS1

0.0

kdTS1

10

kdD

0.1

WT

Km (μM)

30

kaD

0.2

400

kD

kcat (min-1)

0.3

40

kdT

Q10 (WT) = 5.03 ± 0.71

WT

0.4
kcat/Km (μM-1 min-1)

60

3

0

Q10 (WT) = 1.17 ± 0.24

kaT

0.05

B

70

Km (μM)

kcat (min-1)

0.10

0.00

Q10 (WT) = 2.40 ± 0.21

4

0.15

WT

kcat/Km (μM-1 min-1)

5

WT

ATPase activity (T → D)
0.20 Q10 (WT) = 2.04 ± 0.28

1/2-fold at 35˚C 2-fold at 35˚C

60
50
40

WT at 35˚C
1/2-fold at 35˚C
2-fold at 35˚C

30
20

Q10 of [S2] for S1 → S2

WT

2.0

1.0

1/2-fold at 35˚C

0.5

0.5
5

10 15 20
Time (min)1.0

5

10 15 20
Time (min)

25

kaTS1
2-fold at 35˚C 2.0

1.5

1.5

1.5

1.0

2-fold at 35˚C 1.0

kdTS1

5

1/2-fold at 35˚C

0.5

0.5
0

5

10 15 20
Time (min)25

5

10 15 20
Time (min)25

1/2-fold at 35˚C5

10 15 20
Time (min)

1.0

25
kTS1

2.0

1/2-fold at 35˚C

2-fold at 35˚C

1.0

5

10 15 20
Time (min)25

5

10 15 20
Time (min)

25

kdDS2

2.0

2-fold at 35˚C

1/2-fold at 35˚C

0.5
0

5

10 15 20
Time (min)Figure S4: Shinohara et al.

25

1/2-fold at 35˚C

0.5
0

1.0

0.5
0

1.0

2-fold at 35˚C

1/2-fold at 35˚C 1.5

1.5

2-fold at 35˚C
1.5

0.5
0

kdDS1

2.0

1/2-fold at 35˚C
1.5

2-fold at 35˚C

0.5
0

kdDS2

25

kDS2

2.0

1/2-fold at 35˚C

1.0

10 15 20
Time (min)

kaDS1

2.0

2-fold at 35˚C
1.5

0.5
0

2.0

kdD

2.0

1.0

2-fold at 35˚C

0.5
0

25

1/2-fold at 35˚C

1.5

0.8

kaDS2
kaD

2.0

2-fold at 35˚C

1.0

kTS1

kdTS1

kaTS1

kDS1

kdTS0

kaT

2.0

1.4 1.51.5

0Q10 of [S2] for S1 → S2

kaDS1

Phosphorylation (S1 → S2)[S1]0 = 20, 40, 80, 160, 320 μM

kaTS0

kdD

kaD

kD

kdT

0

kaT

10
WT

RMSRE (%) of [S2] for S1 → S2

C

0

5

10 15 20
Time (min)

25

0

5

10 15 20
Time (min)25

Figure S4. A computational model recapitulated the importance of substrate and productbinding in temperature-compensated CKIδ-dependent multi-site phosphorylation, related to
Figure 3.
(A and B) Perturbation analysis of kinetic parameters for ATPase activity (A) anddephosphorylation from S2 to S1 (B) by changing each rate parameter value 2-fold or 1/2-fold at
35˚C. A label * indicates that the mean value could not be determined precisely (the SD value wasgreater than the mean).
(C) Perturbation analysis of the product concentration ([S2]) from S1 to S2 phosphorylation bychanging each rate parameter value 2-fold or 1/2-fold at 35˚C. The upper panel shows RMSRE
from experimental product concentration by the perturbation. Lower panels show the ratio (Q10) of
perturbed mean time course of [S2] at 35˚C to unperturbed one at 25˚C. The shaded region (pink)indicates that the reaction is temperature-compensated (0.8 ≤ Q10 ≤ 1.4) in this region.
For (A), (B), and the upper panel in (C), data are mean ± SD (sampled from 108 step MCMC
simulation with 10,000 step interval).

4.0
3.0
2.0

5.0

1.0

O

0.0

T→D

1

2

3.0
2.0
1.0
0

5

3

4
5
6
Plate Number

7

8

10 15 20 25
Time (min)n = 3 (± SEM)

30

T→D

0.20

35˚C

0.15

Concentration (μM)
25
20
10
5
15
DMSO
10
5

OH

0

NS-05199909

ATP
A(-17.9)

0

5

C3(-11.3)

10 15 20 25
Time (min)n = 3 (± SEM)

30

1.0

[S0]0 = 20 μM
S0 → S2

0.8
0.6
0.4
0.2
0

35˚C
25˚C
0

5 10 15 20 25
NS-05199909 (μM)
n = 3 (± SEM)

Lys130 NS-05199909(C1)C5(-25.7)

0.10

C4(-25.3)

0.05
0.00

[S0]0 = 20 μM

E[T]0 = 100 μM
0
0.5
1.0
3.0
5.0
10
25

OH

HO

-2.0

35˚C ATA (μM)

O

HO

-1.0

[T]0 = 100 μM

4.0

0.0

OH

Initial velocity (μM/min)

6.0

25

O

Initial velocity (μM/min)

5.0

B

O

2.0

Product (μM)

6.0

2.0
3.0
4.0
5.0
6.0
Log 2 (Normalized activity)

Produced ADP (μM)

DC
Log 2 (fold change)

Log 2 (Normalized activity)A0

5 10 15 20 25 30
ATA Concentration (μM)
n = 3 (± SEM)

B(-6.7)D(-7.8)C1(-35.4)

C2(-28.9)
E2(-28.2)
E1(-24.6)

Figure S5: Shinohara et al.

Arg178

Lys224Gly21530

Figure S5. Identification of ATA that inhibits ADP-dependent product binding of CKIδ bycomprehensive chemical screening and putative ATA-binding site of CKIδ by in silico
docking simulation, related to Figure 4.
(A) Chemical inhibitor screening against ΔCKIδ-dependent dephosphorylation activity by using
LOPAC1280 chemical library. Scatter plot (left panel) was constructed by calculating the normalized
value among different 384-well plates. Box plot (right panel) was constructed by calculating theratio between dephosphorylation activities with or without chemicals, which was represented as afold change in a logarithmic scale with base 2. Chemicals that exceed the 3 SD (dashed red line)
indicated as purple circles.
(B) ΔCKIδ-dependent ATPase activity (100 μM ATP at 35˚C) without a peptide substrate wasinhibited by ATA in a dose-dependent manner. Data are mean ± SEM (n = 3).
(C) Chemical structure of NS-05199909, which is similar to ATA structure (Figure 4C).
(D) NS-05199909-concentration dependency of ΔCKIδ-dependent phosphorylation on *CKItidewas enhanced by NS-05199909 in a dose-dependent manner. Data are mean ± SEM (n = 3).
(E) Putative NS-05199909 binding sites identified by in silico docking simulation. The binding freeenergy (kcal/mol) was indicated in parentheses (left panel). The detailed structure of putative NS05199909-binding site C1 with the lowest binding free energy was indicated in the right panel.

0

Relative Activity at 37°C (WT = 1)

HEK293T Cells

27˚C

F

P(CMV)-Luc + P(CMV)-CKIδ mutant
12
Slope = 2.0
site 1
site 2
D, E mutants

10
8

Slope = 1.0

6
4
2
0

0

1
2
3
4
5
6
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

K224D mutant [E]0 = 0.005 μM
35˚C
S1 → S2

20
15
10

5

0

0

5

0

5

10 15 20 25
Time (min)n = 4 (± SEM)

K224E mutant
S1 → S230[E]0 = 1 nM
35˚C

4
3
2

0

5

0

5

10 15 20 25
Time (min)n = 4 (± SEM)

0

5

10 15 20 25
Time (min)n = 3 (± SEM)

30

2
0

R178 mutants
0

1
2
3
4
5
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

P(CMV)-Luc + P(CMV)-CKIδ mutant
10
Slope = 2.0
site 3site 4
D, E mutants

8
6

Slope = 1.0

4
2
0

0

K224E mutant
S1 → S2

4
3
2

0

0

5

[E]0 = 1 nM
25˚C [S1]0 (μM)
320
160
80
40
20

10 15 20 25
Time (min)n = 3 (± SEM)

1
2
3
4
5
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

1.5

30

300
250

**

1.0
0.5

10

P(CMV)-Per2-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

8
6

Slope = 1.0

4
2
0

E
10

0

8
6

Slope = 1.0

420

H185 mutants
0

n.s.

200
150
100

4

25˚C
35˚C
Q10 = 1.18 ± 0.14
n = 4 (± SEM)

150
*

3
2
1
0

0

25˚C
35˚C
Q10 = 1.67 ± 0.18
n = 4 (± SEM)

25˚C
35˚C
Q10 = 1.62 ± 0.27

n.s.
100

Figure S6: Shinohara et al.

n = 3 (± SEM)

50

0

H185 mutants
1
2
3
4
5
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

P(CMV)-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

50
0

30

1

1

4

K224D mutant [E]0 = 0.005 μM
25 S1 → S2
25˚C
[S1]0 (μM)
320
20
160
15
80
40
10
20

5

G
Product (μM)

1
2
3
4
5
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

Product (μM)

Product (μM)

250Product (μM)

37˚C

K224 mutants

Slope = 1.0

Relative Activity at 37°C (WT = 1)

2

6

Relative Activity at 37°C (WT = 1)

4

Transfection

C

Slope = 1.0

8

Km (μM)

6

P(CMV)-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

Km (μM)

Csnk1d mutant

810kcat/Km (min-1 μM-1)

P(CMV)Luc

P(CMV)-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

kcat/Km (min-1 μM-1)

P(CMV)

10

Relative Activity at 37°C (WT = 1)

Plasmid DNA

D
Relative Activity at 37°C (WT = 1)

B
Relative Activity at 37°C (WT = 1)A

25˚C 35˚C
Q10 = 1.27 ± 0.29
n = 3 (± SEM)

1
2
3
4
5
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

Figure S6. Comprehensive genetic screening identified K224D and K224E mutants of CKIδthat impaired ADP-dependent product binding and temperature compensation of multi-site
phosphorylation, related to Figure 5.
(A) Workflow of the dual-luciferase assay to examine a series of CKIδ mutants for their effect on
temperature dependency of the stability of the LUC protein in HEK293T cells. See “Transfection
and Luciferase Assay” section.
(B) K224 (left) and R178 (right) mutation did not promote the degradation of LUC protein in atemperature-dependent manner. The relative CKIδ-dependent LUC destabilization activity at 27˚Cor 37˚C was calculated as the reciprocal of the normalized LUC value of a sample, and calibrated
so that that of a wild-type sample was set as 1.0.
(C) The aspartate D or glutamate E mutation of all other candidate residues in four sites did notpromote the degradation of LUC protein in a temperature-dependent manner.
(D and E) H185 mutation did not promote the degradation of PER2::LUC (D) or LUC (E) proteinsin a temperature-dependent manner. The relative CKIδ-dependent PER2::LUC or LUC
destabilization activity at 27˚C or 37˚C was calculated as the reciprocal of the normalized LUC
value of a sample, and calibrated so that that of a wild-type sample was set as 1.0.
For (B)-(E), data are mean ± SD (n = 3).
(F and G) Temperature and substrate-concentration dependency of the phosphorylation activity ona single-phosphorylated CKItide (KRRRALpSVASLPGL) by K224D (F) and K224E (G) ΔCKIδ
mutants. Kinetic parameters such as catalytic efficiency (kcat/Km) and the Michaelis constant of thesubstrate (Km) were estimated for 35˚C and 25˚C, respectively. For (F) and (G), data are mean ±SEM ((F) n = 4 and (G) n = 3); *p < 0.05, **p < 0.01, and n.s. (not significant); two-tailed Welch's ttest.

A

Targeting vector

P(CAG)

Flag

Csnk1d or Csnk1d mutant (K224D)

Right homology arm

Left homology arm

tctgcaactccagtctttctagaagatgggcgggagtcttctgggcaggcttaa
XbaI

1 kb

48

0

20

20

20

30

30

30

40

40

40

50

Days

10

50
60

48 (hr)

0

LD
DD

LD

50

LL

60

ROSA26::P(CAG)-CKIδ(K224D)Time of day
48
0

10

10

20

20

20

30

30

30

40

40

40

50
60

50
60

n.s.

26

19

20

21
22
23
Period (hr)24

25

48 (hr)
LD

DD
LD

50

LL

ROSA26 (+/+)
n = 32 (± SD)***

ROSA26 (+/WT)
n = 12 (± SD)

24
22

18
18

Time of day

60

ROSA26 (+/K224D)
n = 8 (± SD)

20

1

0

F
28

Period (hr)

2

48

30

ROSA26::P(CAG)-CKIδ(WT) n =9
ROSA26::P(CAG)-CKIδ(K224D) n = 14

3Time of day

10

E

5

0

Time of day

E3

Days

Time of day

104
Number of animals

C

10

60

D

E2

Days

ROSA26::P(CAG)-CKIδ(WT)Time of day
0
48
0

Days

Days

B

E1

Days

Chr6: ROSA26

WT(n = 9)K224D(n = 6)LD(± SD)

WT(n = 9)K224D(n = 6)
LL(± SD)

Figure S7: Shinohara et al.

-2

-1

0
1
Period difference (hr)2Figure S7. K224D mutation of CKIδ remarkably shortens behavioral circadian rhythm andsignificantly changed the temperature-dependency of circadian rhythm of SCN inmammalian circadian clocks, related to Figure 6.
(A) Schematic of structure of the targeting vector and TALEN-targeting site on the ROSA26 locus.
The FLAG-tagged CKIδ genes (wild type and K224D mutant) under the control of CAG promoter
were knocked into the ROSA26 locus of ES cells by using TALEN-based gene targeting. Genomic
DNA is shown as blue lines. Three of the exons (E1, E2, and E3) are indicated as blue boxes. The
short nucleotide sequence of the target site containing the TALEN recognition sites (labelled inmagenta) and an XbaI-recognition site sequence (tctaga) is shown. Corresponding regions for the
3’-terminal of left homology arm of the targeting vector and the 5’-terminal of right homology arm,
which are overlapped by 6 base pairs (tctaga: XbaI-recognition site sequence), are shown as green
lines.
(B and C) Locomotor activity of other individuals of wild-type CKIδ knock-in mice (WT-KI mice, B)
and K224D mutant knock-in mice (K224D-KI mice, C) in the light-dark (LD) cycle, constant light(LL), or constant darkness (DD) conditions.
(D) The distribution of circadian period of WT-KI (n = 9) and K224D-KI (n = 14) mice under DD
condition.
(E) Behavioral period of WT-KI and K224D-KI mice in the second LD and LL conditions. K224D-KI
mice showed markedly shorter circadian period (21.3 ± 1.7 hr) than WT-KI mice (24.2 ± 0.3 hr) in
LL condition while no significant circadian period difference was observed in LD condition. Data aremean ± SD (n = 9 for WT-KI mice and n = 6

for K224D-KI mice); *p < 0.05, **p < 0.01, and n.s.

(not significant); two-tailed Welch's t-test.
(F) The period difference in circadian rhythms between 30˚C and 37˚C in the suprachiasmatic
nucleus (SCN) from WT-KI or K224D-KI mice. To record endogenous circadian rhythms of SCN
slices, WT-KI or K224D-KI mice were mated with PER2::Luciferase (PER2::LUC) protein-fused
reporter mice (Per2Luc). Time course of luciferase activity in each long-term cultured organotypic
SCN slice was monitored by photomultiplier tubes (PMTs) over 6 days under 30˚C and 37˚C. Data
are mean ± SD (n = 32 for control mice without knock-in, n = 12 for WT-KI mice, and n = 8 forK224D-KI mice).

LengthIdentical positions

Similar positions

Q9DC28P23291
P23292
P39962
P29295

KC1D_MOUSE
KC11_YEAST
KC12_YEAST
KC13_YEAST
HRR25_YEAST

415
538
546
524
494

176
169
177
215

131
125
123
115

CKI homolog 1 (S. cerevisiae)[S0]0 = 20, 40, 80, 160, 320 μM
200
200
S0 → S235˚C
150

DS0 → S2

25˚C

150

100

100

50

50

0

5

10 15 20 25
Time (min)n = 3 (± SEM)

30

0

0

5

10 15 20 25
Time (min)n = 3 (± SEM)

260
160
110
80

CKI homolog 1 (S. cerevisiae)[S0]0 = 20 μM
0.7
S0 → S2
0.6

60
50
40

0.5

CKI homolog 1(S. cerevisiae)

0.4
0.3
30

0.2
0.1
0.0

30

B
kDa

35˚C
0

5 10 15 20 25 30
ATA Concentration (μM)
n=1

20

F

Structural alignment

CKI homolog 1 (S. cerevisiae)
Chain A
100
Chain B
120

217

CKIδ (1CKJ)

224

LPWQGLKAATKRQKYERISEKKMS

CKI homolog1 LPWQGLKAPNNKQKYEKIGEKKRS

80

B-factor(Å2)

0

E

Entry

60
40

286

279

20
0
50 100 150 200 250 300 350 400
Amino acid

CKI homolog1 (S. cerevisiae)
CKIδ (1CKJ chain B)
K221
R178
R240

K284

R222
N282
Q223

K286
K217

R222

T220

N283
K224

A280
K279

Q285
P281

90˚
R240

R178

N282
K286

K221

K224

T220
Q223
A218
A219

K217

<10

20 30 40 50 >60
B-factor (Å2)<10

20 30 40 50 >60
B-factor (Å2)

CKIδ (1CKJ)
Chain A
100 Chain B
120

B-factor(Å2)

Product (μM)

C

Accession

Initial velocity (μM/min)

A

80
60
40
20
0

0

50 100 150 200 250 300
Amino acid

Figure S8: Shinohara et al.

Figure S8. Temperature compensation of multi-site phosphorylation is evolutionary
conserved in CKI family, related to Figure 7.
(A) Pairwise sequence alignment between CKIδ from a mouse (Mus musculus, MOUSE) and CKI
homologs from a budding yeast (Saccharomyces cerevisiae, YEAST). Pairwise sequencealignments were performed by Clustal Omega program in UniProt (http://www.uniprot.org/align/).
(B) SDS-PAGE analysis of the purified budding yeast CKI homolog 1. The gel was stained with
Coomassie Brilliant Blue (CBB).
(C) Temperature and substrate-concentration dependency of CKI homolog 1-dependent
phosphorylation on a two-serine substrate, *CKItide (KRRRALSVASLPGL). Data are mean ± SEM(n = 3). See also Figure 7B.
(D) ATA-concentration dependency of ΔCKIδ-dependent phosphorylation on *CKItide at 35˚C (n =1).
(E) Structural comparison between CKIδ (Rattus norvegicus, 1CKJ) and CKI homolog 1 (S.
cerevisiae). Green and orange colors indicated CKIδ (Rattus norvegicus, 1CKJ) and CKI homolog
1 (S. cerevisiae), respectively.
(F) CKI-specific regions (green) located in K217-Q223 of CKIδ (Rattus norvegicus, 1CKJ; lower left
panel) and K279-Q285 of CKI homolog 1 (S. cerevisiae; upper left panel) were highly fluctuated in
X-ray crystal structures of apo-ΔCKIδ (lower right panel) or apo-CKI homolog 1 (upper right panel).
Structural fluctuation among the three-dimensional structural polymorphism was represented as its
B-factor for each Cα atom. See also Figure 7F.

Figure S9: Shinohara et al.
5

0

200

100

Lys171 - Glu189
His185 - Glu226
Arg259 - Asp261
Arg259 - Asp262
Arg127 - Lys171
His185 - Glu230
Arg168 - Glu189
His164 - Asp261
Arg178 - Lys224
His185 - Lys232
0.0
−100
0

−200
0.0
0.5
1.0
Time (μs)

0.51.0Time (μs)

0.15
0.10
0.05
0.00
-0.05
-0.10
-0.15
1.5

25

20

15

10

5

0

ΔCKIδ-ATP at 35˚C
vdW PEPCA

Ser283−Asp285
Lys171−Asp194

10

Asp149−Lys221
Glu197−Lys232

15

Ser181−His185
Lys45−Lys221

20

Lys130−Lys221

25

Glu226−Arg227

0.5
1.0Time (μs)
Ala180−Lys232

Asp110−Arg274
Arg222−Arg227
Arg127−Glu189

Arg127−Lys130
Asp194−Lys232

1.5

Glu33−Lys140

10

0.5
1.0
Time (μs)

Asp28−Arg69

8

0

His164−Asp261
Arg178−Lys224−100

Arg168−Glu189

2000

100

Glu226−Arg227

Glu197−Arg256

Asp110−Arg270
Arg227−Glu230
Lys232−Arg256
Arg178−Lys224
Arg127−Lys171
Arg127−Lys154

Lys154−Glu189
Arg127−Asp128

200

Lys154−Glu189
Ala180−Lys232

4000

S88

Asp128−Lys221

6000

ADP

His185−Lys232
Lys154−Lys171

ΔCKIδ-ATP at 25˚CElectrostatic PEPCA

0.0

Glu117−Arg270
His185−Glu226
Lys171−Glu189
Glu197−Lys232

ΔCKIδ-ADP at 35˚CElectrostatic PEPCA

Arg259−Asp262

Lys154 - Glu189
Arg127 - Asp128
Glu117 - Arg270
His185 - Glu226
Lys171 - Glu189
Glu197 - Lys232
Asp110 - Arg270
Arg227 - Glu230
Lys232 - Arg256
Arg178 - Lys224
0.0

−200

Lys171−Glu189

t = 1.3 μs

Arg127−Lys171
His185−Glu230
Arg168−Glu189

4 6
Index

10

L85

His185−Glu230

I

Y56

Arg127−Lys171
Glu34−Lys140
Glu83−Lys140

−0.2
2

8
1st eigenvector
component

Arg178

Lys154−Arg168

−0.1

0

ΔCKIδ-ADP

His164−Asp261
Lys154−Lys171
Arg127−Asp128

0.0

C

Asp110−Arg274

0

4 6
Index

His185−Glu226
Arg259−Asp261

0.1
PC1

Eigenvalue

Tyr244−Phe248
Gln214-Lys232

Ile125−Gln190
Arg222−Arg227

Met204-Tyr244

Ile228

Asp110−Arg270
Lys154−Glu189
Arg259−Asp262

8000

2

Lys224

1st eigenvector
componentG0

1st eigenvector
component

1.5
5000

Ala177−His185
His126−Leu152

0

PC1

Phe20−Gln48
Trp213−Leu216

15000

Ser283−Asp285
Arg127−Thr174

Eigenvalue

Gln223−Arg227
Tyr284−Tyr286
Ala180−His185

Glu230−Ser234

Lys224-Tyr225
Gln214−Thr235

ΔCKIδ-ADP at 35˚C

Lys171−Glu189
Glu117−Arg270
Arg259−Asp261

0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
10000

Lys221−Tyr225
Ile125−Gln190
Thr174−Thr176

ΔCKIδ-ADP at 25˚CElectrostatic PEPCA
Tyr244−Phe248

Ala177−Ser229

Met204−Leu240

Lys232−Thr235
Leu211−Gln214
Arg127-Lys154

Gln214Tyr156−Arg192
His164−Asp261

0.10
t = 0.3 μs

Met204−Tyr244

ΔCKIδ-ADP at 25˚C
vdW PEPCA
Ser181−Thr184
Trp213−Lys232

4

Tyr266−Arg270

-0.15
1.5

Lys232

Arg178−Gln2140Ile228Gln214
Lys232

Tyr284−Tyr286
Lys154−Gln190

5Trp213−Met233
Lys154−Gln190

E

Leu173−Tyr225

-0.10
RMSF
25˚C > 35˚C
25˚C < 35˚C

Arg127−Leu152

10
Ala177-Tyr225

0

Glu117−Arg270
Thr174−His185

15

1st eigenvector
component

20
1st eigenvector
component

2

Arg127−Lys154

25

Lys224

Asn143−Tyr286-0.05
Arg178

Trp213−Lys232

0.00
1.5

Arg127−Glu189
Ser181−Asp194
Asp110−Arg274

0.5
1.0Time (μs)
0.51.0Time (μs)

Asp128−Asp149
Asp22−Lys45

Ala177 - Tyr225
His185 - Ser229
Tyr201 - Lys232
Pro212 - Tyr244
Ala177 - Ile228
Arg178 - Ile228
Met204 - Phe248
Thr176 - Tyr225
Trp213 - Lys231
Glu197 - Arg256
0.0
0.0

Glu226−Arg227

10
Met204−Phe248

0

Thr176−Tyr225
Trp213−Lys231
Glu197−Arg256
Trp213−Ile228

5
0.15
0.10
0.05
0.00
−0.05
−0.10
−0.15

Arg127−Lys171
Thr220−Glu230

10

Glu197−Arg256

15

Leu173-Tyr225
Trp213−Ile228
Arg178−Gln214
Tyr201-Lys232
Trp213-Lys231

PC1

RMSF (Å)

35˚C
25˚C

His185−Glu230

15
10
5
0
−5
−10
Pro212−Tyr244

20

Ala177−Ile228
Arg178−Ile228

25

Ala177−Tyr225
His185−Ser229
Tyr201−Lys232

8
1.5

Arg227−Glu230
His185−Lys221
Lys38−Glu52

ΔCKIδ-ATP at 25˚C
vdW PEPCA

Asp110−Arg270
Arg127−Asp128
Lys221−Glu230
Lys221−Glu226
Lys43−Asp76

4 6
Index
0.5
1.0
Time (μs)
0.51.0Time (μs)

1st eigenvector
component

0.0
0.0

Glu117−Arg270

0.05

1st eigenvector
component

2
10
20
15
10
5
0
−5
−10
−15

Asn143−Tyr286
Thr44−His46

PC1

ΔCKIδ-ADP at 35˚C
vdW PEPCA

Phe20−Lys45

Leu173 - Tyr225
Trp213 - Ile228
Arg178 - Gln214
Tyr201 - Lys232
Trp213 - Lys231
Trp213 - Met233
Lys154 - Gln190
Ala177 - Tyr225
Lys224 - Tyr225
Gln214 - Thr235

Arg222−Arg227

50 100 150 200 250 300
Amino acidB

Minimum distance(Å)

H
0
4 6 8
Index

Leu216−Arg227
His164−Ile165
Ile125−Arg127

50
40
30
20
10
0
2

Gln190−Asp194

F
0

Ser191−Phe260
Tyr286−Phe288
Lys154−Gln190
Val42−Lys45
Glu117−Arg270

140
120
100
80
60
40
20
0
6

Minimum distance(Å)

Ser181−Asp194

0

Thr44−Pro47
Arg127−Lys154

D

Leu173−Tyr225
Arg127−Leu152
Arg178−Gln214
Phe20−Thr44

Eigenvalue

ΔCKIδ

Minimum distance(Å)

Eigenvalue

8

Minimum distance(Å)

1st eigenvector
component

A
K38

E83
E52

D149

G86
D132

0.2

0.1

−0.1

0.0

−0.2

1.5

0.3

0.2

0.1

−0.1

0.0

−0.2

1.5

ΔCKIδ-ATP at 35˚CElectrostatic PEPCA

0.2

0.1

-0.1

0.0

-0.2

-0.3

Figure S9. A CKI-specific domain around K224 can be a structural basis of temperaturecompensation of CKIδ-dependent multi-site phosphorylation, related to Figure7.
(A) 1.5 μs molecular dynamics (MD) simulation of an apo-ΔCKIδ protein at higher (35˚C) and lower(25˚C) temperatures. MD simulation revealed that CKI-specific region located from K217 to Q223
(green) around αF helix was highly fluctuated in apo-ΔCKIδ protein. Structural fluctuation during
MD simulation was represented as its Root Mean Square Fluctuation (RMSF) for each C α atom.
See also Figure 7E.
(B) The conformational change of αF helix observed in 1.5 μs MD simulation of ΔCKIδ-ADP
complex at 35°C. See also Movie S2.
(C) ADP-binding site identified by X-ray crystal structure analysis of ΔCKIδ-ADP complex.
(D-G) Analysis of conformational changes of ΔCKIδ-ADP complex at 35°C (D and E) and ΔCKIδATP complex at 25°C (F and G) by PEPCA. PCA of potential energies of van der Waals (vdW) (Dand F) or electrostatic (E and G) interactions between residues were performed. Eigenvalues
represent the magnitude of the conformational change due to interactions identified by thecorresponding eigenvector. PC1 represents the 1st principal component that discriminates
conformational states due to interactions by the 1st eigenvector. The bar graph shows the top 20
components (interactions) of the 1st eigenvector. The heat map represents the minimum distance
between two residues identified by the top 10 components of the 1st eigenvector. Interactions tostabilize conformational states with negative values of PC1 can be identified by interactions withnegative components of the 1st eigenvector. For example, PC1 in (D) shows the state transition
around 0.9 μs. The state corresponding to negative values is stabilized by negative components of
the 1st eigenvector such as vdW interaction between Leu173 and Tyr 225. We can confirm that
contacted two residues are detached from 0.9 μs as shown in the heat map.
(H-I) Top 20 components of the 1st eigenvector of PEPCA for ΔCKIδ-ADP at 25˚C (H) and ΔCKIδATP at 35˚C (I). In these MD simulations, the large conformational changes of αF helix were notobserved (Figure 7E).

P(CMV)-Luc + P(CMV)-CKIδ mutant
Slope = 2.0

4
Slope = 1.0

260
160
110
80
60
50
40

2

0

kDa

0

1
2
3
Relative Activity at 27°C (WT = 1)n = 3 (± SD)

C
Produced Product (μM)

6

T) T)
(rM (WK1 BK1
KIδ
B
ΔC
TT TT

30

250

S0 → S2[S0]0 = 320 μM

35˚C
25˚C

TTBK1 rMT

200
150
100

TTBK1 WT5003.0
Q10 (Initial velocity)

B
Relative Activity at 37˚C (WT = 1)

A

2.5
2.0
1.5

0.5
0

5

20

10
15
Time (min)n = 5 (± SEM)

0.0

20

TTBK1 WT TTBK1 rMT
n = 5 (± SEM)

0

2.0
1.5

1.25 ± 0.12

1.0
0.5

0

5

10 15 20 25
Time (min)n = 5 (± SEM)

30

0.0

TTBK1 WT TTBK1 rMT
n = 5 (± SEM)

250

S0 → S1

35˚C
200 25˚C

TTBK1 rMT

150
100

TTBK1 WT

50
0

3.0

[S0]0 = 320 μM
Q10 (Initial velocity)

TTBK1 WT

2

2.5

CKItide(S10A)*
2.27± 0.24

Produced Product (μM)

TTBK1 rMT

6
4

3.0

[S0]0 = 20 μM
Q10 (Initial velocity)

Produced Product (μM)

S0 → S135˚C
8 25˚C

1.53 ± 0.06

1.0

D
10

*
2.42 ± 0.25

CKItide(S10A)*
2.39 ± 0.26

2.5
2.0
1.5

1.41 ± 0.10

1.0
0.5

0

5

10
15
Time (min)n = 5 (± SEM)

Figure S10: Shinohara et al.

20

0.0

TTBK1 WT TTBK1 rMT
n = 5 (± SEM)

Figure S10. A CKI-specific domain around K224 can confer temperature compensation on
TTBK1,

which

shows

temperaturesensitivity

in

the

first-

andsecond-serine

phosphorylation, related to Figure 7.
(A) Dual-luciferase assay to examine a series of CKIδ mutants for their effect on temperaturedependency of CKIδ-dependent phosphorylation measured by the stability of the LUC protein in
HEK293T cells. The alanine mutations on αF helix or on its spatially adjacent regions did notpromote the degradation of LUC protein in a temperature-dependent manner. The relative CKIδdependent LUC destabilization activity at 27˚C or 37˚C was calculated as the reciprocal of thenormalized LUC value of a sample, and calibrated so that that of a wild-type sample was set as
1.0. Data are mean ± SD (n = 3).
(B) SDS-PAGE analysis of the purified wild-type TTBK1 (WT) and rationally designed TTBK1
mutant (rMT). The gel was stained with Coomassie Brilliant Blue (CBB).
(C) Temperature dependency of phosphorylation on a two-serine substrate *CKItide(KRRRALSVASLPGL) by wild-type TTBK1 (WT) and rationally designed TTBK1 mutant (rMT) at
higher substrate concentration (320 μM). Temperature-sensitive TTBK1 (WT) (Q10 = 2.42 ± 0.25)
was converted to significantly less temperature-sensitive TTBK1 (rMT) (Q10 = 1.53 ± 0.06). See
also Figure 7I.
(D) Temperature dependency of phosphorylation on a single-serine substrate *CKItide(S10A)(KRRRALSVAALPGL) by wild-type TTBK1 (WT) and rationally designed TTBK1 mutant (rMT) at
lower (left panels; 20 μM) and higher (right panels; 320 μM) substrate concentrations. Temperaturesensitive TTBK1 (WT) (Q10 = 2.27 ± 0.24 for 20 μM and Q10 = 2.39 ± 0.26 for 320 μM substrateconcentrations) was converted to significantly less temperature-sensitive TTBK1 (rMT) (Q10 = 1.25
± 0.12 for 20 μM and Q10 = 1.41 ± 0.10 for 320 μM substrate concentrations).
For (C) and (D), data are mean ± SEM (n = 5); *p < 0.05; two-tailed Welch's t-test.