Biology subjects

Furuike, Y.

Publications and source records attributed to Furuike, Y..

8 recordsLinked to original sources

bioRxiv2026

Local and non-local impacts of intra-hexamer interactions on temperature compensation of KaiC

A hexameric clock protein KaiC exhibits a 24-hour phosphorylation cycle with a unique property termed temperature compensation. The period is kept constant over physiological temperatures through compensatory coordination of underlying elementary reactions. The temperature-compensated ATPase activity of KaiC is one such key reactions that potentially contribute to maintaining a constant circadian period. We identified four amino acid residues responsible for the temperature compensation in an N-terminal ATPase domain of KaiC. D82 and K172 were located in a primary site, and the ATPase activity of each alanine mutant showed a positive correlation with rising temperature. N62 and E69 constituted a secondary site, where each alanine replacement resulted in a negative correlation with the temperature. The primary site exerts a compensatory regulation over the ATPase cycle locally within the N-terminal domain. The secondary site prevents the ATPase activity from becoming over-compensated by suppressing another compensatory regulation mediated through a non-local interaction with a C-terminal domain of KaiC. Therefore, any imbalance between the local and non-local compensatory regulations in KaiC affects the temperature dependence of its phosphorylation rhythm.

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↗

bioRxiv2026

Molecular Shape Evolution of Cyanobacterial Circadian Clock Protein KaiC

A primitive form of clock protein KaiC has diverged into autonomous or passive time-measuring system in prokaryotes under selective pressures of day-night environmental changes caused by the rotation of Earth. However, the timing of such functional diversification and its structural basis remain unknown. Here we traced molecular shape evolution of older KaiCs by using X-ray solution scattering and structure prediction techniques. The result shows that the oldest ancestral KaiC emerged approximately 3.1 billion years ago as a moderately expanded and asymmetric double-ring hexamer, and subsequently evolved over a period of approximately 1 billion years into a compact and symmetric hexamer that is essential for achieving the self-sustained rhythmicity in extant cyanobacteria. In parallel with this compactification, the oldest KaiC branched into an oligomer composed of two hexamers approximately 0.5 billion years after its emergence. This is the direct experimental result demonstrating the early appearance of the prototypical dodecamer predicted by Kern and colleagues. It appears that this prototypical dodecamer gained a higher enzymatic activity during the next 0.4 billion years or so, and was passed down to non-cyanobacterial lineages as the passive timer capable of responding rapidly to environmental cues. Consequently, geological fluctuations over approximately 1 billion years since the earliest KaiC appeared caused the molecular shape of ancient KaiCs to evolve dramatically along the two distinct pathways. Significance StatementMolecular shape analysis has shed light on the 3-billion-year evolutionary history of clock protein KaiCs. The earliest KaiC emerged as a less compact and asymmetric hexamer, and later diverged along two distinct pathways. One led to the formation of a compact and symmetric hexamer as the core component of the self-sustained and temperature-compensated circadian oscillator found in modern cyanobacteria. The other reached the oligomerization of two hexamers as a prototype of the environmentally responsive timer found in non-cyanobacterial species. In the billion years following the birth of the oldest KaiC, ancient KaiCs adapted environmental alterations by dramatically evolving their molecular shape along the two directions.

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↗

bioRxiv2024

The priming phosphorylation of KaiC is activated by the release of its autokinase autoinhibition

KaiC, a cyanobacterial circadian clock protein with autokinase activity, catalyzes the dual phosphorylation of its own S431 and T432 residues in a circadian manner in the presence of KaiA and KaiB. Priming phosphorylation at T432 is a key step that promotes secondary phosphorylation at S431. Although KaiA binding is considered essential for KaiC phosphorylation, the mechanisms underlying the activation and inactivation of priming phosphorylation remain elusive. We found that the priming phosphorylation proceeds even in the absence of KaiA, but is autoinhibited within KaiC, which decreases the rate constant to 0.019 h-1. The autoinhibition of KaiC and the mechanism underlying the release from autoinhibition by KaiA were examined by KaiC structural analysis, and by classical molecular dynamics and quantum mechanics / molecular mechanics simulations. We found that the side chain of T432 adopts two rotamers in dephosphorylated KaiC, one of which places T432 in a position suitable for a nucleophilic attack on the terminal phosphate of adenosine triphosphate (ATP). However, the nucleophilicity of T432 was insufficient to overcome an energy barrier of approximately 22 kcal mol-1 because the catalytic function of a nearby base, E318, was self-suppressed by hydrogen bonding to positively charged R385. Biochemical assays of KaiC mutants showed that the autoinhibition of KaiC autokinase activity is attenuated by conferring T432 high nucleophilicity through the KaiA-assisted release of R385 from E318 to E352. During the circadian cycle, R385 switches interacting partners to inactivate/activate the autokinase function and to ensure the unidirectionality of the KaiC phosphorylation cycle. Significance StatementKaiC, a central player in the circadian clock system of cyanobacteria, undergoes an ordered phosphorylation cycle in the presence of KaiA and KaiB. To elucidate the mechanism underlying the rhythmic regulation of the KaiC autokinase, we performed structural analyses, computational simulations, and biochemical assays of KaiC and its mutants. The results indicate that KaiC is essentially an autoinhibited autokinase, and the autoinhibition of primary phosphorylation at its T432 residue is attenuated by conferring it high nucleophilicity against the terminal phosphate of adenosine triphosphate. KaiA contributes to releasing the autoinhibition of KaiC in a morning phase by switching the interacting partners of R385 from a catalytic glutamate E318 to E352, as well as ensuring unidirectionality of the KaiC phosphorylation cycle.

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↗

bioRxiv2021

Cross-scale Analysis of Temperature Compensation in the Cyanobacterial Circadian Clock System

Clock proteins maintain constant enzymatic activity regardless of temperature, even though thermal fluctuation is accelerated as temperature increases. We investigated temperature influences on the dynamics of KaiC, a temperature-compensated ATPase in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated very slightly in a temperature-dependent manner. Our mutation studies revealed that internal motions of KaiC include several contributions of opposing temperature sensitivities. To take advantage of this balancing effect, the motional frequency of local dynamics in KaiC needs to exceed [~]0.3 ps-1. Some of the mutation sites may be in a pathway through which the motional frequency in the C-terminal domain of KaiC is fed back to the active site of ATPase in its N-terminal domain. The temperature-compensating ability at the dynamics level is likely crucial for circadian clock systems, into which the clock proteins are incorporated, to achieve reaction- or even system-level temperature compensation of the oscillation frequency.

biophysics↗