Assembly and Disassembly Dynamics of the Cyanobacterial Periodosome Shuji Akiyama, Atsushi Nohara, Kazuki Ito, Yuichiro Maéda  Molecular Cell  Volume 29, Issue 6, Pages 703-716 (March 2008) DOI: 10.1016/j.molcel.2008.01.015 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Time Courses of SAXS Parameters and the Phosphorylated Fraction of KaiC6 Red circles, a ternary mixture of KaiA2 (0.15 mg/ml), KaiB4 (0.15 mg/ml), and KaiC6 (0.60 mg/ml); blue squares, a binary mixture of KaiA2 (0.15 mg/ml) with KaiC6 (0.60 mg/ml); and green triangles, a binary mixture of KaiB4 (0.15 mg/ml) with KaiC6 (0.60 mg/ml). (A) Time-dependent changes of apparent forward scattering intensity, I(0)app. Red line, I(0)app = 2838 + 182 × sin{2π (t − 7.3) / 24.4}; green line, I(0)app = 2270 − 2090 × exp(−0.18 × t) + 1610 × exp(−0.13 × t). (B) Time-dependent changes of apparent radius of gyration, Rgapp. The red line represents a fitting with Rgapp = 57.8 + 0.55 × sin{2π (t − 7.3) / 24.2}. (C) Time-dependent changes in the phosphorylated fraction of KaiC6 (Phosapp). Filled green triangles represent the time course of Phosapp in the absence of both KaiA2 and KaiB4. The red line, Phosapp = 0.41 + 0.19 × sin{2π (t − 0.67) / 23.8}; the blue line, Phosapp = 0.89 − 0.22 × exp(−2.7 × t); and the green line, Phosapp = 0.04 + 0.63 × exp(−0.13 × t). (D) Retention of the π/2 phase delay in a short-period KaiC6 mutant (KaiC6S157P) carrying S157P substitution. A small aliquot of a concentrated ATP solution was added to the system at IT 54 hr (black arrows). Purple circles and black squares represent I(0)app (left axis) and Phosapp (right axis), respectively, of a ternary mixture containing KaiA2 (0.15 mg/ml), KaiB4 (0.15 mg/ml), and KaiC6S157P (0.60 mg/ml). For comparison, I(0)app and Phosapp of the wild-type ternary mixture are presented as purple and black dashed lines, respectively. Error bars were generated from three separate experiments. Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Titration Experiments of Binary Complexes with SAXS (A) Phosphorylated fraction of KaiC6 (Phosapp) in the presence of KaiA2 (squares) or KaiB4 (circles). (B) Plots of raw forward scattering intensity, cI(0)app, against total concentration of KaiA2 (squares) or KaiB4 (circles). The solid lines correspond to the least-squares fitting to the data using a 1:1 binding scheme (see details in text). The resulting parameters are as follows: K(0)KaiA2=30.06×106, K(0)KaiB4=10.81×106, K(0)KaiC6=767.5×106, K(0)KaiA2:KaiC6=1185×106, and K(0)KaiB4:KaiC6=935.3×106. Error bars were generated from five separate measurements. (C) SAXS curves of KaiA2 (red dots), PKaiC6 (blue dots), and the KaiA2:PKaiC6 complex (green dots). The red, blue, and green lines represent theoretical curves of low-resolution models of KaiA2, PKaiC6, and the KaiA2:PKaiC6 complex, respectively, shown in Figure 3A. (D) SAXS curves of KaiB4 (red dots), NPKaiC6 (blue dots), and the KaiB4:NPKaiC6 complex (green dots). The red, blue, and green lines represent theoretical curves of the low-resolution models of KaiB4, NPKaiC6, and the KaiB4:NPKaiC6 complex, respectively, shown in Figure 3B. The black line corresponds to a theoretical SAXS curve calculated from the crystal structure of KaiB4 (Hitomi et al., 2005) with the program CRYSOL (Svergun et al., 1995). Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Low-Resolution Models of Binary Complexes Restored from SAXS Data Smooth envelopes for the bead models of KaiA2 (orange), KaiB4 (pink), PKaiC6 (cyan), and NPKaiC6 (lime) were calculated by using the SITUS package (Wriggers and Chacon, 2001). Red and purple ribbons correspond to superimposed crystal structures of KaiA2 (Ye et al., 2004) and KaiB4 (Hitomi et al., 2005; Iwase et al., 2005), respectively. The crystal structure of KaiC6 (Pattanayek et al., 2006) superimposed on the envelopes of PKaiC6 and NPKaiC6 is presented as blue and green ribbons, respectively. (A) Orthogonal views of the low-resolution envelope of the KaiA2:PKaiC6 complex. (B) Orthogonal views of the low-resolution envelope of the KaiB4:NPKaiC6 complex. (C) A potential binding interface between KaiA2 and PKaiC6. For clarity, only the C2 domains (blue ribbon) and the C terminus 30 residues of KaiC6 (yellow ribbon) are displayed. (D) Period-modulating KaiA2 mutations mapped onto its association interface with PKaiC6. Seventeen out of twenty-nine mutations (Nishimura et al., 2002; Uzumaki et al., 2004) are highlighted in the CPK mode. (E) A potential binding interface between KaiB4 and NPKaiC6. For clarity, only the C2 domains and tails of KaiC6 are illustrated (green ribbon). (F) Period-altering KaiB4 mutations mapped onto its association interface with NPKaiC6. Four out of six critical mutations (Ishiura et al., 1998; Iwase et al., 2005) are emphasized by the CPK mode and residue labeling. (G) Competitive binding to KaiC6. The molecular shape of the KaiA2:PKaiC6 complex is superimposed on the KaiB4:NPKaiC6 complex. KaiB4 (purple) sterically collides with KaiA2 (orange). (H) A hypothetical ternary complex assuming KaiB4 docking in close contact with the N-terminal pseudo-receiver domain of bound KaiA2. (I) A putative ternary complex assuming KaiB4 docking in close contact with the C-terminal domain of bound KaiA2. All graphical presentations were prepared with VMD (Humphrey et al., 1996). Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 In Vitro Assembly/Disassembly Dynamics Revealed by Hybrid Simulation of SAXS and Phosphorylation Parameters (A) Feasible reaction scheme for in vitro oscillation of the Kai proteins. Models I and II, n = 1; model III, n = 4; and model IV, n = 2 (see details in the text). (B and C) Hybrid simulation of raw forward scattering intensity, cI(0)app, and phosphorylated KaiC6 fraction, Phosapp. Error bars were generated from three separate experiments. Broken, dotted, thin, and thick lines correspond to least-squares fits using models I, II, III, and IV, respectively, shown in Figure 5. Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Four Different In Vitro Models Evaluated by Hybrid Simulation Upper and lower panels indicate fractional changes of KaiC6 and concentration profiles of the Kai proteins, respectively (see details in the Supplemental Experimental Procedures). KaiA2, KaiB4, and KaiC6 particles are schematically illustrated by using orange plums, blue diamonds, and green barrels, respectively. fP shown in parenthesis is a phosphorylation parameter ranging from zero (full dephosphorylation) to one (full phosphorylation) dependent on the degree of the KaiC6 phosphorylation. (A) Model I. The fractional change of associated KaiC6 was taken from the previous work (Kageyama et al., 2006). (B) Model II. The fractions of the associated KaiC6 were optimized by tuning their amplitude and baseline while retaining the original phase in model I. (C) Model III. The association order of the Kai complexes was optimized by using the original amplitude, baseline, and phase in model I. (D) Model IV. Both the fractional change of associated KaiC6 and the association order were optimized simultaneously while retaining the original phase in model I. Model IV best describes the robust circadian oscillations of Phosapp and I(0)app (Figures 4B and 4C). Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Rhythmic Changes of KaiC6 Phosphorylation in Intracellular Environments Mimicked by Additions of Cosolvents (A) In vitro oscillation of phosphorylated KaiC6 fraction (Phosapp) as a function of glycerol concentration. Black circles, 0%; red circles, 9%; blue circles, 19%; and green circles, 26%. (B) Dependence of in vitro rhythm of Phosapp on Ficoll 70 concentration. Black circles, 0%; red circles, 9%; blue circles, 17%; and green circles, 23%. The solid lines represent sine-wave functions fitted to the experimental data. The estimated parameters are plotted in Figure 7 and Figure S2. (C and D) Rapid jumps in Ficoll 70 concentration from 0% to 19% (a jump in relative viscosity from 1.0 to 8.2). A ternary mixture containing KaiA2 (0.15 mg/ml), KaiB4 (0.15 mg/ml), and KaiC6 (0.60 mg/ml) was incubated at 30°C (black circles). At IT 24 or 36 hr (black arrows), the solvent condition was changed by a 3-fold dilution of the ternary mixture with a buffer containing concentrated Ficoll 70 (blue circles) or with a Ficoll 70-free buffer (red circles). The solid and dashed lines represent sine-wave functions fitted to the experimental data. Red line, period (Tcycle) = 23.6 ± 0.17 hr and phase (ϕ) = −0.42 ± 0.31 hr; blue line, Tcycle = 23.7 ± 0.43 h and ϕ = −0.90 ± 0.94 hr; red dashed line, Tcycle = 23.5 ± 0.37 hr and ϕ = −0.19 ± 0.91 hr; and blue dashed line, Tcycle = 23.9 ± 0.38 hr and ϕ = 1.71 ± 0.89 hr. Error bars were originated from three separate experiments. Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Viscosity and Osmolality Dependencies of In Vitro Oscillatory Parameters Circles and squares correspond to the estimates in the presence of glycerol and Ficoll 70, respectively. The line represents linear fitting to overall data on glycerol and Ficoll 70. (A) Osmolality dependence of the oscillatory period (R = −0.910). The inset represents an expanded view around the physiological cytoplasmic osmolality. (B) Viscosity dependence of the oscillatory period (R = 0.313). (C) Viscosity dependence of the second peaking time correlated to the oscillatory phase (R = 0.832). (D) Dependence of the logarithm of oscillatory frequency on osmotic pressure (R = 0.878, 1 atm = 0.1 MPa). Error bars were originated from three separate experiments. Molecular Cell 2008 29, 703-716DOI: (10.1016/j.molcel.2008.01.015) Copyright © 2008 Elsevier Inc. Terms and Conditions