Biology subjects

Ito-Miwa, K.

Publications and source records attributed to Ito-Miwa, K..

5 recordsLinked to original sources

bioRxiv2026

Nucleotide-driven KaiC dynamics coordinate the core properties of the cyanobacterial circadian clock

Circadian clocks generate stable [~]24-h rhythms with a defined period, temperature compensation, and entrainment to external cues that set phase. However, the molecular reactions that generate these features are not fully understood. In cyanobacteria, timekeeping is driven by the hexameric ATPase KaiC, which consists of two homologous domains, CI and CII, and whose enzymatic turnover underlies rhythmic phosphorylation. Here we identify ADP release as the rate-limiting step in the KaiC ATPase cycle and demonstrate that the KaiC ATPase nucleotide cycle integrates core properties of the circadian clock. Across KaiC period mutants, increased ADP occupancy is associated with longer periods. Elevated temperature shifts KaiC toward an ADP-bound state, offsetting the thermal acceleration of ATP hydrolysis. KaiB reinforces this ADP-bound conformation by inhibiting nucleotide exchange, thereby strengthening inhibition of CI ATPase activity of KaiC and tuning oscillation amplitude in a temperature-dependent manner. In contrast, KaiA accelerates ADP-to-ATP exchange within the KaiB-KaiC complex, thereby stimulating CI ATPase activity and promoting KaiB-KaiC dissociation prior to KaiC phosphorylation. Phosphorylation begins only after this transition, indicating that KaiC nucleotide-bound state sets the phase of the phosphorylation cycle. Collectively, these results establish the nucleotide cycle of KaiC ATPase as a unifying mechanism that connects molecular reactions to the defining properties of the cyanobacterial circadian clock.

biochemistry↗

bioRxiv2026

Identification of a Third Period-tuning Site in Cyanobacterial Clock Protein KaiC

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.

biochemistry↗

bioRxiv2024

Evolutionary Origins of Self-sustained Kai Protein Circadian Oscillators

Light-dark cycles affect photosynthetic efficiency in autotrophic cyanobacteria; therefore, determining whether ancient cyanobacteria possessed a self-sustained circadian clock when oxygenic photosynthetic systems were established is an important issue in chronobiology. Here we examined the oscillation of the clock protein KaiC in modern cyanobacteria, as well as the function and structure of ancestral Kai proteins, to determine the evolutionary origin of the self-sustained Kai-protein oscillators. The results show that the oldest double-domain KaiC in ancestral bacteria lacked the factors functionally and structurally essential for rhythmicity. The ancestral Kai proteins had acquired these factors through molecular evolution that occurred around Global Oxidation and Snowball Earth events, and were eventually inherited as a self-sustained circadian oscillator by the most recent common ancestor of cyanobacteria capable of oxygenic photosynthesis. This autonomous Kai protein oscillator was further inherited by most freshwater and marine cyanobacteria present today as an autotrophic basis for time-optimal acquisition and consumption of energy from oxygenic photosynthesis.

biochemistry↗

bioRxiv2021

Regulation Mechanisms of the Dual ATPase in KaiC

KaiC is a dual ATPase, with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to ATP is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB. Significance StatementKaiC, a core clock protein in the cyanobacterial circadian clock system, hydrolyzes ATP at two distinct sites in a slow but ordered manner to measure the circadian time scale. We used biochemical and structural biology techniques to characterize the properties and interplay of dual ATPase active sites. Our results show that the N-terminal and C-terminal ATPases communicate with each other through an interface between the N-terminal and C-terminal domains in KaiC. The dual ATPase sites are regulated rhythmically in a concerted or opposing manner dependent on the phase of the circadian clock system, controlling the affinities of KaiC for other clock proteins, KaiA and KaiB.

biochemistry↗

bioRxiv2021

Elucidation of Master Allostery Essential for Circadian Clock Oscillation in Cyanobacteria

Spatio-temporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and ADP release from its N-terminal domain during ATPase cycle. An engineered KaiC-protein oscillator consisting of a minimal set of the identified master allosteric events exhibited mono-phosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple post-translational biochemical circadian oscillators. One Sentence SummaryCoupling between a phospho-switch and KaiC ATPase-dependent nucleotide exchange drives the cyanobacterial circadian clock.

biophysics↗