Unravelling circadian rhythm with cyanobacteria

Early bird or night owl? The circadian rhythm – the internal biological “clock” that controls when we sleep and wake, among other things – is an object of fascination, and sometimes frustration, for many of us. But humans are far from alone – organisms from plants to microbes all have an internal clock that shapes their day-to-day rhythms as the Earth spins on its axis.

Now, scientists from Japan’s National Institutes of Natural Sciences have unveiled a detailed look at how the circadian rhythm functions in cyanobacteria – photosynthetic bacteria that typically live in both salt and freshwater.

Cyanobacteria operate life’s simplest known circadian clock, controlled by three proteins called KaiA, KaiB and KaiC. A circadian clock is important for these tiny creatures because they photosynthesise, which requires sunlight and produces oxygen, during the day, and fix nitrogen at night.

The circadian rhythm is created by KaiC adding and removing phosphate chemical groups to itself over the course of a roughly 24-hour cycle. KaiA helps KaiC add phosphate groups, while KaiB counteracts the activity of KaiA. Other proteins track signals from the environment, such as light, and translate them to the Kai proteins in order to fine-tune the clock.

Cyanobacteria circadian rhythm concept illustration showing the earth's rotation, cyanobacteria cellular rhythm all last 24 hours
Illustration of the basis of the cyanobacterial circadian clock. Credit: NINS/IMS.

At the start of a cycle, KaiA binds to KaiC and stimulates KaiC to add phosphate groups to itself. As the cycle progresses, KaiB also binds to KaiC, and KaiC begins to remove its phosphate groups. Once this is done, KaiC and KaiB separate and the cycle begins again.

In the process, KaiC uses energy stored in a molecule called adenosine triphosphate (ATP). By breaking a bond in ATP, the energy is released to drive the other chemical reactions. Therefore, the addition and removal of phosphate groups (known as phosphorylation and dephosphorylation, respectively) and the consumption of energy by ATP hydrolysis are the key chemical reactions at play in the cyanobacteria’s circadian cycle.

As KaiC binds to different proteins and adds and removes phosphate groups, its shape changes slightly in a process known as allostery.

In the new paper, the research team used protein crystallography to study allostery in KaiC at the atomic level. KaiC was crystallised in eight different states to understand allosteric changes through the cycles of phosphorylation and ATP hydrolysis.

The team found that the two cycles were closely coupled with each other, like two gears in a machine. The two processes are connected through changes in hydrogen bonds within KaiC.

Cyanobacteria circadian rhythm concept an illustration showing kaic and the molecular reactions in atp hydrolysis, hyrdogen-bond regulation and phosphorylation
Illustration of the paper’s key findings linking ATP hydrolysis and the phosphorylation cycle by KaiC via hydrogen bond regulation. Credit: NINS/IMS.

“Because proteins are composed of a vast number of atoms, it is not easy to understand the mechanisms of their complicated but ordered functions,” says author Yoshihiko Furuike, an assistant professor at the National Institutes of Natural Sciences’ Institute for Molecular Science.

“We need to trace the structural changes of proteins patiently.”

The findings may have application to understanding circadian clock proteins throughout the tree of life, including mammals and plants.

“The logic behind the relationship between KaiC dynamics and clock functions can be applied to other studies on various organisms,” says Furuike.

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