In a new study, Japanese structural biologists from the National Institutes of Natural Sciences in Okazaki, Japan, and their collaborators from SOKENDAI, Nagoya, and Osaka Universities, tackled the complex workings of the simplest of all biological clock mechanisms in the cyanobacterium (Synechococcus elongatus) and provided insights on its regulation.
The study was published in an article in the journal Science Advances (“Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria”).
The rhythmic orderliness of complex biological phenomena in space and time depends on diurnal molecular clock systems. Oscillations in a variety of cellular activities are generated by clock mechanisms through allosteric cooperation, where protein components are chemically modified at multiple locations in a programmed manner to alter their binding characteristics with other clock proteins.
The stability of the interactions between different clock proteins regulates delays in feedback loops (which otherwise would be near instantaneous) and the length or period of oscillatory activities.
Understanding allosteric structural changes in clock proteins across the phases of oscillation that occur through post-translational modifications such as phosphorylation of clock proteins, therefore, is critical in understanding basic biology and designing molecular tools that are in phase with inherent biological rhythms.
“The cyanobacterial circadian clock is the simplest circadian clock system known to date in terms of the number of components, but mechanistically, it is a very complex system involving the ATPase cycle and a four-state phosphorylation-cycle,” the authors noted.
In the cyanobacterial clock, two adjacent amino acid residues—the 431st serine (S) and 432nd threonine (T)—in the core clock protein called KaiC are phosphorylated in a characteristic cyclical pattern (ST → SpT → pSpT → pST → ST) called the P-cycle, in the presence of other clock proteins, KaiA and KaiB.
Although the P-cycle of KaiC has been studied in vitro, in vivo, and in silico, its allosteric regulation is unclear, largely because all known KaiC structures share nearly identical conformations at the phosphorylation sites, in the presence and absence of phosphoryl modifications.
“Because proteins are composed of a vast number of atoms, it is not easy to understand the mechanisms of their complicated but ordered functions. We need to trace the structural changes of proteins patiently,” said lead author Yoshihiko Furuike, PhD, assistant professor at the Institute for Molecular Science, National Institutes of Natural Sciences, Japan.
To study the structural basis for its allosteric oscillatory regulation, the team studied the atomic structures of the KaiC clock protein, by screening thousands of crystallization conditions that allowed them to cover its transitions through the entire P-cycle. The investigators then crystallized the KaiC hexamer in eight separate states and sorted them from the fully phosphorylated (KaiC-pSpT) to the fully dephosphorylated (KaiC-ST) so that fractional changes in the crystal phase reflect changes seen during the circadian cycle in solution.
Phosphorylation of KaiC couples with ATP hydrolysis to determine the speed of the clock. Crystallizing KaiC in eight distinct states allowed the team to observe the cooperativity between the two gears of the clock mechanism: phosphorylation and ATP hydrolysis.
They found the minimal set of allosteric events in KaiC that resulted in an oscillatory nature of the complex, involved a bidirectional switch between a coil and a helix conformation that depended on the phosphorylation of Ser431 in the carboxyl terminal of KaiC and the release of adenosine 5′-diphosphate from its amino terminal.
“An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a mono-phosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators,” the authors noted.
The investigators also designed several KaiC mutants by replacing key amino acid residues to identify the minimal requirements of an oscillatory unit by extracting the core allostery from the complex cycle.
“This will serve as a research tool for further elucidation of mechanisms, such as period determination, temperature compensability, and entrainment, and will provide design principles for the simplest posttranslational biochemical oscillator that oscillates with a temperature-compensated circadian period,” the authors noted.
In future studies, Furuike said, his team’s goal would be to visualize all cyanobacterial clock proteins during oscillation at an atomic level and describe the moment that rhythm arises from chaos.
Beyond cyanobacterial rhythms, the research team envisions that their findings could have wider applications in understanding rhythms in other bacteria, plants, insects, and mammals that have their own clock proteins with distinct sequences and structures. Furuike believes the relational logic between KaiC dynamics and clock functions identified in the current study can be used to probe periodicity in other organisms.