Chronobiologists identify a key circadian clock mechanism in cyanobacteria

Researchers have identified a key mechanism involved in setting the circadian clock of cyanobacteria – a model organism for study by chronobiologists due to the fact that the organism has one of the first circadian systems to evolve, and thus sets up light how our own systems of this type work.

An article describing the results appeared in the Proceedings of the National Academy of Sciences the 4and May 2022.

Chronobiologists – researchers who study the timing processes, including circadian clocks, of organisms – have long been interested in cyanobacteria (or blue-green algae) as a model organism for investigation, and its protein KaiC as a particular.

The KaiC protein forms a key part of the master clock in cyanobacteria, and regulation of the genes that produce this protein and others with which it interacts is crucial for maintaining the bacterium’s circadian rhythm, and therefore when to engage in its fundamental life processes such as photosynthesis and cell division. Further elucidation of the functioning of the system thus sheds light on the functioning of circadian clocks in the living world.

KaiC is an ATPase, an enzyme that initiates (catalyzes) the chemical reaction that splits a phosphoryl group (an ion containing phosphorus and oxygen) from adenosine triphosphate (ATP) using a water molecule, a process which releases energy which can then be harnessed to power actions through living beings.

But KaiC is a special type of ATPase in that it has a dual domain structure, with an active site (location on an enzyme where the chemical reaction takes place) in one domain and another active site in the other . The circadian clock system of cyanobacteria is governed by a slow and orderly, but also very complex, coordination of the two sites.

To do this, the KaiC protein uses two types of ATP molecules to produce various chemical reactions and thus govern the circadian rhythm. ATP molecules attach to a Walker motif – a looping structure in proteins associated with phosphate binding – present in both domains, called N-terminal C1 and C-terminal C2. The ATP molecule bound to the C1 domain is the main source of the ATP hydrolysis reaction whose rate determines the speed of the clock system. In the presence of the sister proteins KaiA and KaiB of KaiC, the ATP molecules bound not only in the C1 domain but also in the C2 domain are activated and then periodically inactivated.

“In recent years, this C1/C2-ATPase interaction of KaiC has become an important research target to achieve a better understanding of the circadian clock system in cyanobacteria,” said Shuji Akiyama, a biophysicist at the Institute of Science. Molecular Studies from National Institutes of Japan. of Natural Sciences, and co-author of the study, “because it is closely related to the properties of oscillation, period tuning, and adjustment of the clock system to compensate for the effects of temperature changes.”

Much research has explored the biochemistry and structure of KaiC ATPase, but the precise mechanisms of its activation and inactivation have so far remained unknown.

The researchers used biochemical and structural biology techniques, including amino acid substitutions in KaiC itself, to characterize the properties and interaction of the ATPase dual active sites. They also performed crystal structure analysis of KaiC to visualize the activated and inactivated forms of ATP and catalytic water molecules in the C1 domain.

They found that N-terminal and C-terminal ATPases communicate with each other through an interface between N-terminal and C-terminal domains in KaiC. Dual ATPase sites are rhythmically regulated in a concerted or opposite manner depending on the phase of the circadian clock system, to control the assembly and disassembly cycle of the other clock proteins, KaiA and KaiB. The results suggest that activation of dual KaiC ATPases through an autocatalytic mechanism (a product of one reaction then becomes a catalyst for the same reaction) contributes to sudden dawn disassembly of protein complexes built overnight.

This is crucial for resetting “subjective night”, or what the body’s clock predicts of the length of night, and then moving the entire system forward through its cycle.

Many structural details of the C2-ATPase remain unclear even after the researchers’ analysis, in part because they were unable to identify some catalytic water molecules involved, suggesting other areas for research. to refine their understanding of the clock system. The researchers were also amazed at the enormous range of C2-ATPase activity, which can be suppressed down to zero. The physiological significance of this is also the next important research target for scientists.

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