Circadian Clock Protein KaiC

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Contents 1 Introduction 2 KaiC - KaiA - KaiB System 3 Biological Importance 4 References

Introduction

Biological Circadian Clocks are self-sustaining oscillators that function on a rhythmic cycle of or around 24 hours. They exhibit a very precise time constant and temperature compensation, which allows the system to run at a steady rate independent of temperature fluctuations ^[1]. They are found in almost all organisms, the simplest of which are cyanobacteria, which have been extensively studied in order to determine the mechanism of the fine-tuned biological process of circadian rhythmicity. In most eukaryotes, a region of the brain called the superchiasmic nuclei detects light and dark cycles, then relays the message to biological clock systems that maintain rhythmicity within the body. Conversely, cyanobacteria have a fairly modest system comprised of three proteins: KaiC, KaiA, and KaiB. The system is based around the central protein KaiC which exhibits ATP binding, inter-subunit organization, a scaffold region for Kai protein complex formation, a location where critical mutations are found, and an evolutionary link to other well-known proteins ^[2]. In order to devise an explanation for the mechanism of biological oscillators, we need to characterize the structure, function, and interactions among molecular components. To study these, scientists begin with analyzing cyanobacteria such as Synechococcus elongatus, since it is the smallest organism that expresses rhythmic clock properties.

KaiC - KaiA - KaiB System

The system by which the protein complex maintains a precise time constant and temperature compensation is achieved by the cooperation of a three-protein network. In a nutshell, KaiC requires ATP so that it can autophosphorylate, while KaiA enhances this phosphorylation and KaiB antagonizes it. KaiC is the central clock protein, which has autokinase and autophosphorylase activity. Yet in the presence of ATP alone, KaiC cannot perform the autophosphorylation function. It requires two other proteins, KaiA and KaiB, the genes of which are found in the same cluster on the chromosome ^[2]. Although KaiC phosphorylates itself, the presence of KaiA and KaiB are essential to rhythmicity. KaiA stimulates KaiC autophosphorylation, possibly by stabilizing the phosphorylated form of KaiC. On the other hand, KaiB antagonizes the process possibly by enhancing KaiC dephosphorylation or interfering with the binding of KaiA to KaiC. Even in the presence of very high ATP, KaiB still prompts KaiC to dephosphorylate ^[3]. Therefore, we can see that ATP alone cannot trigger KaiC to maintain global rhythmicity. The mechanism is not well understood and is the focus of further study. Many possibilities have been proposed one of which is shown on the left. Here, the mechanism proposed involves KaiC altering the CII domain to attract KaiA. KaiA then binds KaiC to trigger autophosphorylation. KaiC alters the CII domain again to bring KaiB to the molecule the latter of which binds the KaiA-KaiC complex. KaiC is dephosphorylated by this interaction, and KaiA and KaiB dissociate from KaiC. The entire process starts again for a new circadian cycle ^[4].

spinqualitylabelsall models Structure of KaiC (PDB entry 1TF7) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Contents 1 KaiC Homohexameric Complex 2 KaiC Autophosphorylation Sites 3 KaiA <-> KaiC Interaction Site 4 KaiB <-> KaiC Interaction Site KaiC Homohexameric Complex Intramolecular interactions of ATP binding site The structure of KaiC resembles a double donut formation with a central pore through the center. Conceptually, it is comprised of two subunits, which appear as the two donuts. Yet, technically, the protein is divided perpendicular to the conceptual donut subdivision, which can be visualized as six that form a barrel-shaped structure. The entire molecule is made of 519 amino acid residues in each monomer and spans 100 Å in diameter. The waist of the molecule (the region between CI and CII) has a diameter of 62 Å. The that runs through the molecule is wider at the CI region, 22 Å, and narrows to a nearly that appears to be secured by six residues. The basic, polar nature and physical divergence of this CII pore in comparison to the CI end indicates the possibility of conformational change, such as closing and opening [2]. Although it is not believed to be directly associated with the autophosphorylation process, it may be a binding site for KaiA and/or KaiB. There are 12 molecules of bound at the interface between monomers. Three potential phosphorylation sites have been identified within 10 Å of the . KaiC Autophosphorylation Sites The phosphorylation sites on the KaiC protein are essential to the system. This is because phosphorylation status corresponds to clock speed. The protein predominantly phosphorylates on threonine and serine residues, whose specific identification is not completely resolved. Nonetheless, three potential phosphorylation sites have been identified within 10 Angstroms of the ATP binding region in the CII domain [3]. The key autophosphorylation site is . When this residue is mutated, there is no circadian rhythm at all. The process is believed to demonstrate a transfer of the of ATP from one CII subunit to the T432 site on an adjacent subunit. A region of each hexamer that is notable regarding the phosphorylation of KaiC is the P Loop. This zone is recognized as site for binding and hydrolysis of ATP. Along with the T432 site, evidence shows a shuttling of phosphates between residues of the . Based on RecA structure/function similarity, a conserved glutamate in CI is proposed to activate water for an attack on the gamma-phosphate of ATP in order to release the phosphate to be shuttled. (difference between ATP binding and phosphorylation residues) (Key residues = T432, S431, T426) (show 3D image of these sites and highlight bonds/interactions) The site within the A and B monomer and its interactions are displayed in the image on the left which shows hydrogen bonds and inteeractions between molecules. KaiA <-> KaiC Interaction Site KaiA is a protein that stabilizes the phosphorylation status of KaiC. This enhancer is hypothesized to bind to the interface of the two donut-shaped KaiC subunits, CI and CII. This area, known as the "waist" of the molecule, expresses head-to-tail orientation between CI and CII which regulates the essential phosphorylation by interacting with distant areas on the surface of the hexamer. Two possible sites are shown to present complementarity between KaiC and KaiA. On KaiC, these regions are located at the waist of the molecule and at the CII pore, or "dome" of the molecule. KaiB <-> KaiC Interaction Site (show 3D image of KaiC site for KaiB binding and highlight key residues in interaction -weak? strong? ions in site? how does it stabilize dephosphorylation/destabilize phosphorylation/destabilize KaiA?)

Biological Importance

In cyanobacteria, the KaiC system is vital to survival because of it's role in global gene regulation: nearly all promoters in a cyanobacteria are under circadian control. Correlating with a circadian clock system enhances fitness of any organism in a rhythmic environment. [function is important to whole life cycle] (1) - Structure similarity with RecA and DnaB. RecA is a DNA recombinase and DnaB is a DNA helicase, so the observation that there is similarity between these molecules imply possible direct interactions with DNA. The folds of each monomer resemble those of RecA, where eight α-helices surround the twisted β-sheet made up for seven strands. The similarity indicates nucleotide binding on the carboxy side of the β-sheet ^[2].

References