Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.
Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. The opposing spherical regions of the dumbbell-shaped structures correspond to the N-terminal and C-terminal domains of KaiC. Previously, we hypothesized that the N-terminal domain of KaiC is responsible for the ATP-induced hexamerization of KaiC while the C-terminal domain is responsible for the phosphorylation of KaiC (Hayashi et al. 2004, J. Biol. Chem. 279, 52331-52337). Here, we tested that hypothesis using the purified protein of each domain. We prepared N-terminal and C-terminal domain proteins (KaiCN and KaiCC, respectively), examined their function by analyzing their ATP- or 5'-adenylylimidodiphosphate (AMPPNP; an unhydrolyzable ATP analog)-induced hexamerization, interactions with KaiA, and phosphorylation, and we demonstrated the following: (1) KaiCN had higher ATP- or AMPPNP-induced oligomerization activity than KaiCC. (2) KaiCc had phosphorylation activity as KaiCWT whereas KaiCN had no activity. (3) KaiCC interacted with KaiA whereas KaiCN did not. (4) The interactions of KaiCC with KaiA did not require that KaiC has a hexamer structure. (5) The interactions of KaiCC with KaiA enhanced the phosphorylation of KaiCC. Furthermore, we presented evidence for the intersubunit phosphorylation of KaiC. KaiCCatE2-, which lacks KaiC phosphorylation activity due to mutations of the catalytic Glu residues, was phosphorylated when it was co-incubated with KaiCC. We propose that the KaiC hexamer consists of a rigid ring structure formed by six N-terminal domains with hexamerization activity and a flexible structure formed by six C-terminal domains with intersubunit phosphorylation activity.
Hayashi
Fumio
F
Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan.
Iwase
Ryo
R
Uzumaki
Tatsuya
T
Ishiura
Masahiro
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001683:Biological Clocks / Q000502:physiology; D002940:Circadian Rhythm / Q000502:physiology; D056925:Circadian Rhythm Signaling Peptides and Proteins; D000458:Cyanobacteria / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114271:KaiC protein, cyanobacteria; D021122:Protein Subunits