KaiC A-loops Affects KaiA and KaiB Binding

Circadian oscillators of Cyanobacteria, are comprised of three proteins, KaiA, KaiB, and KaiC. By simply mixing these proteins with ATP, a 24-hour oscillation can be generated that functions as the basis of a biological clock. By studying this simple system, our lab focuses on how biological time keeping is produced and maintained at the molecular level.

In the KaiABC clock system, the circadian oscillation is generated in KaiC, which exhibits phosphorylation and dephosphorylation profiles over a 24-hour period. KaiC is a ring-like hexametric protein composed of two domains, CI and CII domains.  The last 30 C-terminal residues of each monomer named ‘a-loops’ are the KaiA binding site.  The a-loops are situated within the hexametric protein at certain phosphorylation states and are exposed at other phosphorylation states.  Initially, CII domains of KaiC interact with KaiA to produce the phosphorylation signal over the first 12-hour period. Then the phosphorylation signal at CII domains is transmitted to CI domains, which subsequently interact with KaiB and KaiA forming a dephopshorylation complex in the next 12 hours to complete the oscillation.  Therefore, we hypothesize that the a-loops affect KaiA and KaiB binding.

From our preliminary data based on thrombin proteolysis assay, we observed that the a-loops were exposed in phosphorylation states and buried in the dephosphorylation states.  To further test our hypothesis, we designed KaiC mutations that expose the a-loops when they would normally be buried.  We will then perform experiments to test our hypothesis.