Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock

Area of Science:

• Biochemistry
• Circadian Biology
• Molecular Biology

Background:

• The KaiA, KaiB, and KaiC proteins of Synechococcus elongatus form a 24-hour post-translational oscillator crucial for circadian rhythm.
• KaiA stimulates KaiC phosphorylation, while KaiB antagonizes KaiA and induces subunit exchange, but the precise mechanisms remain unclear.

Purpose of the Study:

• To elucidate the mechanism of KaiA-stimulated KaiC phosphorylation using a specific KaiC mutant (A422V).

Main Methods:

• Crystal structure analysis of the A422V KaiC mutant.
• Molecular dynamics simulations comparing wild-type and mutant KaiC.
• Analysis of in vivo data on phase resetting and phosphorylation amplitude.

Main Results:

• The A422V KaiC mutant shows altered phosphorylation at S431 and a subtle conformational change.
• Molecular dynamics simulations reveal increased 422-loop mobility in the mutant and differences in phosphorylation-associated regions.
• Evidence suggests an allosteric mechanism where KaiA binding to the A-loop influences the 422-loop, promoting kinase activity.

Conclusions:

• The A422V mutation provides insights into the KaiA-mediated KaiC phosphorylation mechanism.
• A relay system involving the A-loop and 422-loop, propagating to neighboring subunits, supports a concerted allosteric model for KaiC phosphorylation.