Roles of Two ATPase-Motif-containing Domains in Cyanobacterial Circadian Clock Protein KaiC

Abstract

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATP␥S binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiC WT ) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATPbinding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.Circadian rhythms, 24-h biological oscillations of metabolic and behavioral activities observed ubiquitously in prokaryotes and eukaryotes, are endogenously regulated by circadian clocks. Several clock and clock-related genes from Drosophila, Neurospora, Arabidopsis, mice, and cyanobacteria have been cloned and analyzed (1).Cyanobacteria are the simplest organisms that exhibit circadian rhythms. We previously cloned and analyzed the kaiABC circadian clock gene cluster in the cyanobacterium Synechococcus sp. strain PCC 7942 (hereafter called Synechococcus) that is essential for the generation of circadian rhythms in cyanobacteria. The gene cluster consists of two

Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC

“…To investigate the molecular basis of these phenomena, we first examined the effect of KaiA on the KaiC-bound nucleotides that serve as substrates in the reversible phospho-transfer reaction. We reconstituted KaiC hexamers from monomers in the presence of 2 mM [α-32 P]ATP; this concentration of ATP is ∼60-fold higher than the dissociation constant of ATPγS (34 μM), a nonhydrolyzable analog of ATP (7). We incubated these hexamers at 30°C, with or without KaiA, and plotted the ratio of KaiC-bound ATP to total KaiC-bound nucleotides as a function of time over an 8-h reaction.…”

Orderly wheels of the cyanobacterial clock

“…In full-length KaiC, ATP binding in the CI domain is the principal force that assembles the protein into active multimeric particles (9). This information suggests that destabilization of KaiC's hexameric structure is required for KaiB binding, helping to explain why exchange of monomers between KaiC particles occurs at the same time in the circadian cycle when KaiB interaction is strongest (12,18).…”

“…KaiC is an unusual relative of the AAA+ superfamily of ATPases with very slow enzymatic activity (7,8). The protein forms hexameric rings in the presence of ATP in which each KaiC monomer consists of two homologous catalytic domains, CI and CII (9). The KaiC hexamer thus appears as a "double doughnut," with the CII ring atop the CI ring.…”

Crystal structure of KaiC‐like protein PH0186 from hyperthermophilic archaea Pyrococcus horikoshii OT3

“…3 KaiCI is responsible for the ATP induced hexamerizaion of KaiC, while the KaiCII is flexible and responsible for the phosphorylation of KaiC. 3 The autophosphorylation of KaiC is stimulated by KaiA, whereas KaiB antagonizes the effects of KaiA on KaiC autophosphorylation. 4 Recently, KaiC homologues have been found in almost all species of archaea such as Pyrococcus and Sulfolobus.…”