Circadian Clock Protein KaiC
Created by Callie Jenevein
The Circadian Clock Protein KaiC (PBD ID: 2GBL), mainly studied in the cyanobacteria Synechococcus elongatus, is a 519 amino acid residue protein involved regulating the biological circadian rhythm1. The name “kai” is used because it is the Japanese word for “cycle”1. It is a homohexamer involved in a complex with Circadian Clock Proteins KaiA and KaiB (collectively referred to as the “KaiABC complex”) to maintain this rhythm in approximately 24-hour cycles1,2. Biological clocks are important to all plants and animals. For example, the biological clock in humans triggers differences in body temperature and brain wave activity that correlate to the different demands of night and day 3. During the time of restfulness in the night, the human body requires growth and repair, while during the day, it is involved in a completely different set of processes and requires more energy. The rhythm also adjusts to changes in temperature and lengths of day and night, an important function in many animals that seasonally adopt certain crucial behaviors, such as migration, reproduction, or hibernation2.
KaiC is a member of the family RecA protein-like (ATPase-domain)4, and although it does not contain DNA-binding motifs in its sequence like other members of this family such as proteins RecA and DnaB, KaiC has been found to bind forked and single stranded DNA substrates5. RecA protein is a DNA recombinase and a homolog of the domains of KaiC protein (RecA is equally comparable to both the CI and the CII domains of KaiC monomer)5,6. The two proteins have comparable folds, and the central domains contain conserved regions on seven-strand beta sheets surrounded by eight alpha helices5. Like RecA, KaiC has DNA binding sites and has been shown to bind single stranded and forked DNA5. RecA is involved in genetic recombination and DNA repair, whereas KaiC is involved in the regulation of the circadian rhythm2,6. However, because of the homologies between the two proteins, scientists believe the function of KaiC may involve changes in DNA structure5.
Each monomer of the Circadian Clock protein KaiC has two domains, CI and CII, and in the hexameric form, the monomers are labeled chains A-F. In the presence of ATP, the protein forms hexameric rings resulting in an overall quaternary appearance as a “double-doughnut”2 with a central core that is partially constricted at one end5.
KaiC is an a/b protein, and its two domains have similar folds4,5. Both contain regions with Walker A (P loop) motifs, involved in ATP and magnesium ion binding, and Walker B motifs, involved in ATP hydrolysis and magnesium ion binding5. Chains A, B, E and F differ slightly from chains C and D; the former have 18 alpha helices and the latter have 167. All chains contain 25 beta strands that are mostly parallel4,7. Residues 14 to 247 make up the CI domain and residues 261 to 497 make up the CII domain, therefore residues 248 to 260 constitute the link between the two5. In scientific literature, KaiC is illustrated with the N-terminus of the CI domain at the bottom of the representation and the C-terminal of the CII domain at the top (the CII “doughnut” is atop the CI “doughnut”). When the hexamer forms, the central links between CI and CII domains on each monomer face the outside of molecule5. The central core formed by the double-doughnut conformation narrows in the CII domain and nearly seals at the C-terminal end due to six arginine (R488) residues, and scientists postulate that different conformations allow for the opening and closing of this end of the channel5, but not much else is said in scientific literature about this mechanism or its function.
ATP must be present in order for the protein to form its hexameric rings. Twelve ATP molecules, along with six magnesium ions (Mg2+ ), bind between domains CI and CII, thus in the hexameric form the ATP molecules appear to be wedged into the “waist” of the double-doughnut2,5. Specific ATP nucleobase recognition has only been found in the CI domain of the monomer, and it involves P loop residues T50, K52 and T53 as well as residues S89, K224, R226, H230, K232 and D2412,5. On the other hand, phosphorylation sites have been found in the CII domain but not the CI domain; T426, S431 and T432 have been identified as the CII phosphorylation sites2. E318 on CII activates S431 and T432 hydroxyl groups for nucleophilic attack on the γ-phosphate, and basic residues K294, K457 and R459 serve as stabilizing agents for the negatively charged phosphates, locking the ATP molecule in place8. The binding of magnesium ions (Mg2+ ) also contributes greatly to the secure hold the CII domain has on the γ-phosphates of ATP molecules5. Two Mg2+ ions are involved; the first helps direct the mentioned nucleophilic attacks by binding between the γ-phosphate and the S431/T432 residues, and the second plays a role in stabilizing the transition state and easing departure of the negatively charged oxygen8. The difference in binding mode between the two domains results in the CI binding pocket loosely bound to the γ-phosphate of ATP but tightly bound to its nucleobase, and the CII binding pocket tightly bound to the γ-phosphate but loosely bound to the adenine nucleobase5. Clearly, structural differences between the two KaiC domains at the primary level eventually determine functional complimentary. Though the domains bind ATP in different ways, both are necessary for hexamerization.
KaiC operates in a complex with Circadian Clock proteins KaiA and KaiB, collectively called the “KaiABC complex” 2. KaiA binds to KaiC to promote phosphorylation (and prevent dephosphorylation), and KaiB antagonizes the activity of KaiA9. Scientists are unclear whether KaiB also binds directly to KaiC, but it is believed that it either competes with KaiA for a binding site on KaiC or it has an inhibitory binding site on KaiA5,9. It is believed that KaiC has two binding domains for KaiA, one comprised of residues 206-263 near the waist of the double-doughnut and the second comprised of residues 418-519 at the domed-shaped “top” of the CII domain5. Mutations in residues R215, P248, R253, G421, Y442, G460, R468 and T495 were shown to significantly affect KaiA-KaiC interaction, so those residues probably play a crucial role in the binding of KaiA to KaiC5. The KaiA binding domain at the top of the KaiC CII domain has a convex shape, complimenting the concave shape of the KaiC binding domain of the KaiA protein5. Furthermore, both KaiA binding domains on KaiC show positive electrostatic surface potential whereas the KaiC binding domains on KaiA show negative electrostatic surface potential5.