Life science : Structural Biology

Real-time Small-angle X-ray Scattering Observation of Assemblyand Disassembly Dynamics of Cyanobacterial Periodosome)(Phosapp ,24Fraction of Phosphorylated Kai C6),app(MWWeight-Averaged Molecular Weight (kDa)

Circadian clocks are endogenous timing systems
phosphorylated KaiC fraction (Phosapp), whereas theenabling a variety of living organisms to adapt daily
period of MWapp was nearly identical to that of Phosapp
alternations of environments on earth. Cyanobacterium(23.8 h). The phase delay of π/2 implies that Phosapp
Synechococcus elongatus PCC 7942 is one of theis closely related to the time derivative of MWapp, andsimplest organism known to possess a self-sustainedthat there is a relationship between reactant andcircadian oscillator composed of three clock proteinsproduct. The current observations therefore suggest
termed KaiA, KaiB, and KaiC [1]. KaiA enhances thethat the assembly/disassembly of the Kai
autophosphorylation of KaiC, whereas KaiB induces
periodosome is not under steady-state conditions, but
the autodephosphorylation of KaiC by antagonizing
evidently driven by the phosphorylation status of KaiC.
KaiA activity. Recently, Nakajima et al. succeeded inTo investigate the relationship between the Kaireconstructing the KaiC phosphorylation cycle in vitro
complex structure and KaiC phosphorylation, we
solely by incubating KaiA, KaiB, and KaiC in theconducted titration SAXS experiments [4,5] for binarypresence of ATP [2]. This pioneering work suggests
mixtures of KaiA and KaiC, and KaiB and KaiC.
that the Kai proteins are assembled and disassembled
Titration data sets of both binary pairs were fitted
into heteromultimeric Kai complexes (periodosome) touniquely to a theoretical curve for a 1:1 binding
effect a circadian switching of KaiC activity fromscheme. The dissociation constant ( K d ) of the
autophosphorylation to autodephosphorylation and vice
phosphorylated KaiA: PKaiC complex (4.7 μM) wasversa.
approximately 40-fold larger than that of theIn order to elucidate the oscillatory mechanism,
dephosphorylated KaiB:NPKaiC complex (0.12 μM),
much attention has been focused on the structure ofindicating a greater affinity of KaiB for NPKaiC than ofthe Kai periodosome. To date, the crystal structures
KaiA for PKaiC.
of the individual Kai proteins have already been
On the basis of the binding scheme and Kd, thedetermined independently [3]. However, a potential
scattering curves of the binary complexes wererelationship between the assembly/disassembly
estimated by global fitting on the titration data set.
dynamics and the KaiC phosphorylation cycle is
Low-resolution envelopes were restored from thestill poorly understood because of the difficulty indeconvoluted SAXS curves (Figs. 2(a) and 2(c)), ontounraveling the underlying mechanisms solely from thewhich the crystal structure of each Kai protein was
static crystal structure of individual clock components.
uniquely superimposed (Figs. 2(b) and 2(d)). Our
Although it is important to characterize Kai complexes
complex models clearly demonstrate that KaiA binds aalong a reaction coordinate, a blind search for
crystallization conditions of oscillatorytransient complexes is not necessarily
1.4498a promising strategy.
To take a structural snapshot of the1.2467
Kai periodosome, we followed the1.0436
assembly/disassembly dynamics of aternary mixture containing KaiA, KaiB,0.8
405
and KaiC in real-time using small-angle
0.6373
X-ray scattering (SAXS) at beamline
BL45XU [4]. As shown in Fig. 1,
0.4343weight-averaged molecular weight0.2311( MW app ) estimated from forward
scattering intensity of measured SAXS
2800.0curves increased dramatically upon
01224364860
69
mixing three Kai proteins and peaked
Time (h)at 9 h. After a monotonous decrease
Fig. 1. Assembly/disassembly dynamics of ternary mixture
from 9 to 18 h, MW app revealed acontaining KaiA, KaiB, and KaiC in the presence of ATP.
Weight-averaged molecular weight (MWapp, blue open circle)
robust oscillation with a period ofis
phase-delayed by π/2 relative to phosphorylated KaiC
app
was
24.4 h. Interestingly, MW
fraction (Phosapp, red open square). The solid lines represent
phase-delayed by approximately 6.6 hsine-wave functions fitted to the experimental data.
(6.6/24.4 = ~π/2) relative to thebroad interface ranging from the C2 domain to the tailof P KaiC. Interestingly, KaiB shares a common
docking site of the C2 domain with KaiA.
These binary interactions offer a reasonable
explanation for the assembly/disassembly of the Kai
periodosome in a phosphorylation-dependent manner.
Upon mixing three Kai proteins, KaiA immediately
binds KaiC to form the KaiA:KaiC complex, thereby
enhancing the autophosphorylation of KaiC. KaiB
then approaches the KaiA:PKaiC complex to inhibit
KaiA activity. Because of the greater affinity of KaiB
for the common docking site of the C2 domain thanKaiA, KaiB can displace KaiA of the KaiA: P KaiC
complex, and thereby switching KaiC activity from(a)autophosphorylation to autodephosphorylation.
It must be noted that the Kai periodosome
assembles and disassembles robustly also in vivo [1,2].
This is astonishing because assembly/disassembly
processes under physiological conditions should beaffected to some extent by crowding and higherviscosity. From the crystal structure [3], dual
phosphorylation sites of KaiC are localized to theC2 domain. Thus, the resistances against crowdingand viscosity will be achieved in the Kai complexes
by controlling the timing of assembly/disassembly ofKaiA and/or KaiB though phosphorylation-dependent
structural changes of the C2 domain of pacemaking
KaiC.

(b)

KaiC

KaiC
C1
C2
Tail

KaiA

KaiA

(c)(d)

KaiC

KaiC
C1
C2

KaiB

KaiB

TailFig. 2. Low-resolution models of Kai complexes. (a) Low-resolution envelope ofKaiA:PKaiC complex restored from SAXS data. (b) Superimposed crystal structures ofKaiA and KaiC to low-resolution model of KaiA:PKaiC complex. (c) Low-resolution
envelope of KaiB:NPKaiC complex restored from SAXS data. (d) Superimposed crystal
structures of KaiB and KaiC to low-resolution model of KaiB:NPKaiC complex.

References[1] M. Ishiura et al.: Science 281 (1998) 1519.
[2] M. Nakajima et al.: Science 308 (2005) 414.
[3] C.H. Johnson and M. Egli: Nat. Struct. Mol. Biol. 11(2004) 584.
[4] S. Akiyama, A. Nohara, K. Ito and Y. Maéda: Mol. Cell
29 (2008) 703.
[5] S. Yamada et al.: J. Mol. Biol. 362 (2006) 123.

Shuji Akiyama a,b,*, Atsushi Nohara c, Kazuki Ito band Yuichiro Maéda d,ea PRESTO, Japan Science and Technology Agency
b SPring-8 / RIKEN

c Division of Biological Science, Nagoya University

d Structural Biology Research Center, Nagoya University
e ERATO Actin Filament Dynamics Project*E-mail: akishu@spring8.or.jp

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