title: 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 ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) 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 ATP-binding 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.
authors.affiliation: Center for Gene Research, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya, 464-8602, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hayashi
authors.forename: Noriyo
authors.initials: N
authors.lastname: Itoh
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Uzumaki
authors.forename: Ryo
authors.initials: R
authors.lastname: Iwase
authors.forename: Yuka
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Hisanori
authors.initials: H
authors.lastname: Yamakawa
authors.forename: Megumi
authors.initials: M
authors.lastname: Morishita
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Onai
authors.forename: Shigeru
authors.initials: S
authors.lastname: Itoh
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ishiura
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002940:Circadian Rhythm; D056925:Circadian Rhythm Signaling Peptides and Proteins; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D004355:Drug Stability; D007700:Kinetics; D049449:Luminescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114271:KaiC protein, cyanobacteria; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases