title: Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock.
abstract: Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Jenny
authors.initials: J
authors.lastname: Lin
authors.affiliation: Medical Scientist Training Program, Pritzker School of Medicine.
authors.forename: Justin
authors.initials: J
authors.lastname: Chew
authors.affiliation: Department of Chemistry, and.
authors.forename: Udaysankar
authors.initials: U
authors.lastname: Chockanathan
authors.affiliation: Department of Molecular Genetics and Cell Biology, Department of Physics, and Institute for Genomics and Systems Biology, University of Chicago, Chicago, IL 60637 mrust@uchicago.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rust
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D057906:Circadian Clocks; D002940:Circadian Rhythm; D000458:Cyanobacteria / Q000502:physiology*; D025461:Feedback, Physiological; D008954:Models, Biological; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D021122:Protein Subunits / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D010768:Phosphoserine
keywords: allostery; circadian rhythms; mathematical modeling