Structural basis of the day-night transition in a bacterial circadian clock

Abstract

Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post-adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

Fig. 1. Model of circadian formation of cyanobacterial clock protein complexes

During the day, KaiA binds to the C-terminal extensions of KaiC to enhance the autokinase activity of the CII domain of KaiC, leading to sequential autophosphorylation. Yellow and red circles represent phosphorylated S431 (pS) and T432 (pT), respectively. The sensor histidine kinase, SasA, is activated by KaiC. During the day, the hexameric CII ring is loose and unstacked from the hexameric CI ring. At dusk, phosphorylation of S431 tightens the CII ring, leading to withdrawal of the KaiA binding sites on CII. The tightened CII ring interacts and stabilizes the CI ring in its post-ATP hydrolysis state, which now has its KaiB binding sites exposed. The rare fold-switched state of KaiB, KaiB_fs, cooperatively binds CI, displacing SasA. KaiB_fs sequesters the autoinhibited conformation of KaiA, allowing autodephosphorylation of KaiC. KaiB_fs also binds and activates the phosphatase activity of CikA. Upon autodephosphorylation of S431, the CII ring loosens, causing unstacking of the rings. The CI ring returns to its pre-ATP hydrolysis state, letting go of KaiB, KaiA, and CikA to begin a new day.

Fig. 2. Fold-switched KaiB binds to the posthydrolysis state of the CI domain of KaiC

(A) Crystal structure of the KaiB_fs-cryst-CI_cryst complex at 1.8 Å. Orange, KaiB_fs-cryst; sky-blue, CI_cryst. (B) Secondary structures of KaiB_gs (PDB 2QKE, subunit A) and KaiB_fs-cryst bound to CI_cryst. Residues of KaiB_fs-cryst that interact with CI_cryst are highlighted in green. (C) Zoomed-in view of the boxed region in (A). Representative interacting residues in (B) are shown in green. Dashed lines: electrostatic KaiB_fs-cryst-CI_cryst interactions. (D) Superposition of CI structures before ATP hydrolysis. Sky-blue, CI_cryst; dark green, pre-ATP hydrolysis state of CI^Se (PDB 4TLC, subunit C), missing density for the B loop. Residue numbers in CI^Se are offset from those of T. elongatus CI by −1. (E) Superposition of CI structures after ATP hydrolysis. Sky blue, CI_cryst; magenta, post-ATP hydrolysis state of CI^Se (PDB 4TLA, subunit E). Please see fig. S1 and table S1 for construct details.

Fig. 3. KaiB assembles as a ring on the posthydrolysis state of KaiC

(A) The hexameric KaiB_fs-cryst*-KaiC_S431E complex at 3.87 Å. KaiC_S431E includes CII hexamer (dark cyan) and CI hexamer (sky blue). Orange, KaiB_fs-cryst*. (B) Zoomed-in view of the boxed region in (A) showing bound ADP highlighted by mesh representing the (F_obs − F_calc) omit maps contoured at 2.5σ. F_obs is the observed structure-factor amplitude, and F_calc is the calculated structure-factor amplitude. (C) Zoomed-in view of the boxed region in (A) showing KaiB_fs-cryst* interfaces in the KaiB_fs-cryst*-KaiC_S431E hexamer. Interfacial residues are shown in nontransparent mode. (D) Columbic electrostatic surface map of the interfacial residues between two subunits of KaiB_fs-cryst*. See fig. S1 and table S1 for construct details.

Fig. 4. The mechanism of KaiA autoinhibition is revealed by the KaiA_cryst-KaiB_fs-cryst-CI_cryst complex

(A) The ternary KaiA_cryst-KaiB_fs-cryst-CI_cryst complex at 2.6 Å. Orange, KaiB_fs-cryst; sky blue, CI_cryst; orchid, KaiA_cryst. (B) Zoomed-in view of the boxed region in (A). Dashed lines: electrostatic interactions. (C) Conformational changes of dimeric KaiA. NTD, N-terminal domain. Prime symbols denote the other protomer within the dimer. (Left) Crystal structure (PDB 5C5E) of KaiA^Se (purple) bound to KaiC CII^Se peptides (dark cyan). (Right) The KaiA_cryst-KaiB_fs-cryst-CI_cryst complex, with same coloring scheme as in (A). (Middle) Superposition of KaiA^Se (left) and KaiA_cryst in complex (right); only α5 helices and b6 strands are shown. (D) Fluorescence anisotropy of 6IAF-labeled CII peptides (0.05 μM). Open circles, 0 μM KaiA, titration with CI_cryst and KaiB_fs-cryst; triangles, 10 μM KaiA, titration with CI_cryst; diamonds, 10 μM KaiA, titration with KaiB_fs-cryst; squares, 10 μM KaiA, titration with equal molar of KaiB_fs-cryst and CI_cryst. Error bars, SD from triplicates. (E) Size-exclusion chromatography of ternary complex formation. Wild-type KaiA (green, dashed); wild-type KaiB (red, dashed); CI_cryst (orange, dashed); KaiA +KaiB+CI_cryst (green); L155A-KaiA+KaiB+CI_cryst (cyan); K158A-KaiA+KaiB+CI_cryst (blue); D212A-KaiA+ KaiB+CI_cryst (purple); D266A-KaiA+KaiB+CI_cryst (black); and N212A-D266A-KaiA+ KaiB+CI_cryst (red). (F) Bioluminescence rhythms from strains of S. elongatus: wt-kaiA^Se (blue), complemented with kaiA^Se (green), L156A-kaiA^Se (purple), and kaiA^Se knockout (red). L156A of kaiA^Se is analogous to L155A in kaiA. See fig. S1 and table S1 for construct details.

Fig. 5. Structure of the CikA_PsR-KaiB_fs-nmr complex reveals intermolecular contacts essential for output signaling

(A) Top, ensemble of the 20 lowest-energy NMR structures of the CikA_PsR-KaiB_fs-nmr complex. Orange, KaiB_fs-nmr; khaki, CikA_PsR. Bottom, zoomed-in view of boxed regions above using average minimized structure from the ensemble. Dashed lines, intermolecular electrostatic interactions. (B) Bioluminescence rhythms from S. elongatus. wt-cikA^Se (blue), complemented with cikA^Se (green), C644R-cikA^Se (purple), or cikA^Se knockout (red). C644R of cikA^Se is analogous to C630R of cikA. (C) Representative cell micrographs. wt-cikA^Se, complemented with cikA^Se, C644R-cikA^Se, or lacking cikA^Se. Red, membrane autofluorescence. Scale bars, 2.5 μm. (D) Scatter plots of cell lengths. wt-cikA^Se (blue) (n = 87), complemented with cikA^Se (green) (n = 110), C644R-cikA^Se (purple) (n = 353), or lacking cikA^Se (red) (n = 48). One-way analysis of variance of log₁₀-transformed cell length data produced P < 0.0001. ****, Bonferroni-corrected P values (< 0.0001) for pairwise comparisons to wt-cikA^Se (α = 0.05). (E) CikA_PsR and KaiA_cryst compete for the β2 strand of KaiB_fs. (Left) Orange, KaiB_fs-nmr (top) and KaiB_fs-cryst (bottom); khaki, CikA_PsR; orchid, KaiA_cryst; black lines, backbone-backbone or sidechain-backbone hydrogen bonds. (Right) Selected region of methyl-TROSY spectra of labeled CikA_PsR free (black), or mixed with wild-type KaiB+CI_mono (blue), or with wild-type KaiB+CI_mono+wild-type KaiA (red). See fig. S5, E and H, for full spectra. Also see fig. S1 and table S1 for construct details.

Fig. 6. Ground-state KaiB prevents daytime KaiA and CikA_PsR recruitment

(A) Superposition of the non–fold-switched region (residues 7 to 46) of KaiB_gs (blue) (PDB 2QKE, subunit A) with the ternary KaiA_cryst-KaiB_fs-cryst-CI_cryst structure. (B) and (C) are zoomed-in views of the boxed region in (A). Dashed lines, estimated distance between two atoms. (D) Superposition of the non–fold-switched region (residues 7 to 46) of KaiB_gs (blue) (PDB 2QKE, subunit A) with the CikA_PsR-KaiB_fs-nmr structure. (E) and (F) are zoomed-in views of the boxed region in (D). Dashed lines, estimated distance between two atoms.