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

Fumio Hayashi,Noriyo Itoh,Tatsuya Uzumaki,Ryo Iwase,Yuka Tsuchiya,Hisanori Yamakawa,Megumi Morishita,Kiyoshi Onai,Shigeru Itoh,Masahiro Ishiura

doi:10.1074/jbc.m406604200

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.

Highlights

Circadian rhythms, 24-h biological oscillations of metabolic and behavioral activities observed ubiquitously in prokaryotes and eukaryotes, are endogenously regulated by circadian clocks
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
To clarify the roles of these ATPase motifs, we prepared four mutant KaiCs, each carrying a mutation on Walker motif A or a pair of CatEs in each domain, and examined their ATP binding, hexamerization, stabilization, and phosphorylation in vitro

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The plasmids that express wild type KaiC derived from T. elongatus (hereafter called KaiCWT) and the KaiCK53H and KaiCK294H mutants were described previously (6). A reaction mixture (50 ␮l) containing 20 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 10 mM MgCl2, [35S]ATP␥S, and 12.5 pmol of KaiCs was incubated at 25 °C for 30 min. Assay for the Thermostability of Hexameric KaiCs by NativePAGE—To induce the hexamerization of KaiC, we incubated KaiC monomer (11 ␮M) with 0.5 or 20 mM AMPPNP in 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2 and 150 mM NaCl at 4 °C overnight, and confirmed the hexamerization by Native-PAGE as described previously (6). KaiC monomer (7.3 pmol in hexamer) was incubated with 1 mM ATP in 30 ␮l of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2 at 50 °C for various times in the presence or absence of 43.8 pmol of KaiA (in dimer).

TABLE II Hill plot analysis of the hexamerization of KaiCs

RESULTS

DISCUSSION