Figure 1.
In the S. elongatus circadian clock the post-translational oscillator (PTO) is embedded in a transcription-translation feed loop (TTFL).
Key features of the oscillator are: (i) Mediation of global gene expression by rhythmic modulation of promoters including those driving the cluster of core clock protein genes, kaiA, kaiB and kaiC; (ii) modulation of global promoter activity by rhythmic DNA torsion and/or transcription factor activity (i.e. RpaA, signaled by the PTO output His kinase SasA); (iii) regulation of DNA topology and transcription factors by rhythmic phosphorylation and dephosphorylation of the KaiC homo-hexameric protein; (iv) robustness conferred by synchronization of KaiC hexamer status through monomer exchange in the PTO; (v) modulation of amplitude or phase setting by newly synthesized non-phosphorylated KaiC hexamers or monomers feeding into pre-existing hexamers; and (vi) a core PTO composed of KaiA, KaiB, KaiC and ATP, whereby KaiC has kinase, phosphatase (putative conformational changes between the two states are indicated by dark- and light-blue coloring of hexamers), and ATPase activities, KaiA enhances phosphorylation and KaiB antagonizes KaiA.
Figure 2.
SAXS envelopes for individual Kai proteins from S. elongatus.
(A) Envelope for KaiA with the crystal structure of the dimer (PDB ID 1R8J [23]; http://www.rcsb.org) modeled into it. The views are approximately perpendicular to the molecular dyad (left) and along it (right). Subunits of the domain-swapped dimer are colored in red and blue. (B) Envelope for KaiB with the crystal structure of the tetramer from Thermosynechococcus elongatus (PDB ID 2QKE [26]) modeled into it. The views are approximately along the dyad relating dimers (left) and perpendicular to it (right). (C) Envelope for KaiC hexamer (KaiC-aa mutant) with the crystal structure of wt-KaiC (PDB ID 3DVL [30]) modeled into it. The views are approximately perpendicular to the molecular sixfold rotation axis (left) and along it (right). The conformations of C-terminal tails depicted in the model are based on the one fully traced tail of subunit A in the crystal structure of wild type KaiC from S. elongatus refined to 2.85 Å [34]. Only two of the chains could be completely traced up to the C-terminal residue S519, and the conformations of individual tails are affected by the packing of hexamers in the crystal. ATP molecules are shown in space filling mode. The orientations of the crystallographic models inside the individual SAXS envelopes were optimized by rigid body refinement. The symbols indicate rotations of 90 degrees.
Table 1.
Overview of SAXS data for Kai proteins and their binary complexes.
Figure 3.
The complex was formed with S. elongatus KaiA and the KaiC-aa mutant. The shape of the protrusion above the KaiC barrel (magenta) is indicative of a single KaiA dimer (subunits colored in red and blue), bound to a KaiCII C-terminal peptide. The position of the KaiA dimer at some distance from the KaiCII surface is reminiscent of the ‘tethered’ model of the complex determined by EM [34]. The symbol indicates a rotation of 90 degrees.
Figure 4.
Crystal structure of the S. elongatus KaiC-ee mutant and KaiC conformational plasticity.
(A) The crystal structure of KaiC-ee reveals phosphorylation at S320 in the A and F subunits. The figure depicts a portion of the interface between the A (carbons green) and B (carbons gray) subunits and intra-subunit phosphorylation at S320[A] (top). Carbon atoms of E432[B] and E431[B] are highlighted in cyan (center) and hydrogen bonding interactions between E432[B] and S379[A], S381[A] and T415[A] (on the right) and between E431[B] and T426[B] (bottom) are shown as thin solid lines. The gamma phosphate (Pγ) of the ATP molecule bound between the A and B subunits is shown, as well as the distance (12.7 Å) between it and the phosphate of S320. (B) In vitro phosphorylation patterns of wt-KaiC (T426/S431/T432 = TST), and the KaiC-aaa (T426A/S431A/T432A), KaiC-aee (T426A/S431E/T432E) and KaiC-ee (T426/S431E/T432E) mutants. All mutants exhibit phosphorylation in the 32P assay (albeit at a much lower level than the wt protein), consistent with a new phosphorylation site outside the known triad T432, S431 and T426. (C) Calculated electrostatic surface potentials for KaiC (hypo-phosphorylated), KaiC-ee and P-KaiC (hyper-phosphorylated) (from top to bottom) with the hexamers viewed from their C-terminal ends. Markedly different polarizations might well contribute to KaiB's ability to distinguish between the hypo- and hyper-phosphorylated states and preferentially bind the hyper-phosphorylated state.
Table 2.
Distance1) relationships in the crystal structure of KaiC-ee.
Figure 5.
(A) Experimental scattering curves, pairwise function P(r) and Guinier plots (inset) for the KaiBC complex from S. elongatus (KaiC-ee mutant). Scattering curves: red with error bars = high concentration, 1.72 mg/mL; green with error bars = low concentration, 1.0 mg/mL; magenta line = FT of P(r) from GNOM [60]. The cyan curve corresponds to P(r) from GNOM. Inset: red with error bars = high conc.; green with error bars = low conc.; brown line = Guinier fit of data between the red bars (0.6/RG to 1.0/RG). (B) The SAXS envelope for KaiBC with an EM-based model for KaiBC [26] viewed in three different orientations. The model shows KaiC (magenta and cyan) with two KaiB dimers (green) bound on the CII side. ATP molecules are shown in space filling mode. To model the complete set of KaiC C-terminal peptides emerging from the CII of KaiC, sixfold rotational symmetry was applied to residues 499–519 from subunit A in the wt-KaiC crystal structure [34]. In the panel on the left, fogging was used to obscure the back of the model. The symbols indicate rotations of 90 degrees (left) and 45 degrees (right).
Figure 6.
EM model of the ternary KaiABC complex.
(A) Negative-stain electron micrographs of the KaiBC complex in the absence of KaiA (0×, top [26]), and in the presence of stoichiometric levels of KaiA (1×, middle) and with 4× the normal concentration of KaiA (bottom). (B) Calculated EM density of the ternary KaiABC complex viewed from the side (left) and along the central channel of the KaiC hexamer (right). The scale bar represents 50 Å. (C) Native PAGE assay of complex formation between KaiB-ΔKaiC and N-KaiA. The KaiB/ΔKaiC band is marked with “**” and is shifted down slightly compared to the KaiB band. The N-KaiA/KaiB/ΔKaiC band is marked with “***” and is shifted up slightly compared to the KaiB band. (D) Model of the KaiABC complex with the flipper-like protrusion, containing the KaiA monomer's N- (cyan) and C-terminal (blue) domains, and viewed from the side (left) and along the central channel in the KaiC hexamer (right). KaiB dimers and KaiC hexamer are colored green and magenta, respectively. The C-terminally truncated form of KaiC (ΔKaiC) is shown. Note the striking resemblance to the EM density in panel B. At the current resolution it is impossible to determine whether N-KaiA engages in an interaction with the KaiB dimer, or with KaiC, or both KaiB and KaiC at their binding interface. The symbols indicate rotations of 90 degrees.
Figure 7.
SasA-KaiC complex by SAXS and EM.
(A) Scattering curves I(q), pairwise function P(r) and Guinier plots (inset) for the SasA-KaiC complex (KaiC-ee mutant). Scattering curves: red with error bars = high concentration, 1.71 mg/mL; green with error bars = low concentration, 0.85 mg/mL; magenta = FT of GNOM scan P(r). The cyan curve corresponds to P(r) from GNOM [60]. Inset: red with error bars = high conc.; green with error bars = low conc.; brown line = Guinier fit of data between the red bars (0.5/RG to 1.2/RG). (B) SAXS envelope for the SasA-KaiC complex. (C) EM class average images for the SasA-KaiC complex (KaiC-aa mutant); red arrows point to extensive density above a third layer. (D) Calculated EM density for the SasA-KaiC complex viewed from the side, tilted by 45 degrees to show the CII half with the two N-SasA domains bound on either side of the rim, and along the central KaiC channel (left to right). The EM density reveals the location of the N-SasA domain bound to KaiC (the third layer in the images depicted in panel C). The scale bar represents 50 Å. (E) Three-dimensional model of the SasA-KaiC complex with superimposed SAXS envelope (gray) and EM reconstruction (cyan). Only one SasA dimer (red/yellow) is shown for clarity. The symbol indicates a rotation of 45 degrees (panel D) and 90 degrees (panel E).
Figure 8.
SasA and KaiB compete for the same binding site on KaiC.
(A) Negative stain electron micrographs of mixtures of SasA, KaiB and C-terminally truncated KaiC (ΔKaiC); red arrows indicate the location of bound SasA. (B) Native bandshift gel assay for the formation of the binary complex between KaiB and KaiC. Note that neither KaiB nor SasA binds to the KaiCI hexamer (rightmost lanes). Arrows indicate the positions of KaiB, KaiC, KaiBC and KaiCI. (C) Native bandshift gel assay for the formation of the binary complex between SasA and KaiC. Arrows indicate the positions of SasA and SasA-KaiC complex. (D) Competitive binding by SasA and KaiB to KaiC assayed by native PAGE. When increasing amounts of KaiB are added to a pre-existing binary SasA-KaiC complex (gel image on the left), KaiB appears unable to displace SasA. Conversely, when increasing amounts of SasA are added to a pre-existing binary KaiB-KaiC complex (gel image on the right), SasA infiltrates the complex and a band for the ternary complex appears (marked by an asterisk). Upon further increasing the SasA concentration, KaiB is completely displaced, resulting in the formation of the binary SasA-KaiC complex (compare boxed lanes in panel D to those boxed in panel C). Not only do KaiB and SasA interact with the same KaiCII regions, but binding between SasA and KaiC is considerably more tight than binding between KaiB and KaiC.
Figure 9.
Schematic illustration of PTO composition, protein-protein interactions and changes in the quaternary structures of Kai proteins in the cyanobacterial oscillator over a single 24-h period.
Binding of a KaiA dimer via its C-terminal domains causes a change in the KaiCII hexamer from the hypo- to the hyper-phosphorylated state, eventually triggering binding of a KaiB tetramer in the form of two separate dimers on either side of the KaiCII ring. KaiB binding is accompanied by KaiC subunit exchange and SasA dimer interacts with the PTO in a circadian fashion and competes with KaiB for binding to KaiC. Formation of the KaiBC complex results in release of the KaiA dimer from the C-terminal KaiCII tail and subsequently in reattachment via its N-terminal domain, leading to KaiA sequestration in a stable ternary KaiABC complex at the final stage of the clock cycle.