Many biological processes undergo daily (circadian) rhythms that are dictated by self-sustained biochemical oscillators. These circadian clock systems generate a precise ~24 h period in constant conditions (constant light and temperature) that is nearly invariant at different temperatures (temperature compensation). Circadian clocks also show entrainment to day and night, predominantly mediated by the daily light/dark cycle, so that the endogenous biological clock is phased appropriately to the environmental cycle. These properties - especially the period's long time constant and temperature compensation - are difficult to explain biochemically. Full understanding of these unusual oscillators will require knowledge of the structures, functions, and interactions of their molecular components. Mammalian clocks are exceedingly complex and require several interconnecting transcriptional, translational and post-translational feedback loops (TTFLs) to achieve gene expression with circadian periodicity. We study the components of the biological clock in the prokaryotic cyanobacterium, Synechococcus elongatus, which programs many processes to conform optimally to the daily cycle, including photosynthesis, nitrogen fixation, and gene expression. The endogenous circadian system in cyanobacteria exerts pervasive control over cellular processes including global gene expression. Indeed, the entire chromosome undergoes daily cycles of topological changes and compaction. Remarkably, the biochemical machinery underlying this circadian oscillator can be reconstituted in vitro with just three cyanobacterial proteins, KaiA, KaiB, and KaiC in the presence of ATP! These proteins interact to promote conformational changes and phosphorylation events that determine the phase of the in vitro oscillation. The high-resolution structures of these proteins suggest a racheting mechanism by which the KaiABC oscillator ticks unidirectionally. This post-translational oscillator may interact with a TTFL to generate the emergent circadian behavior in vivo. The conjunction of rigorous structural, biophysical, and biochemical approaches to this system will reveal molecular mechanisms of biological timekeeping. The KaiC homo- hexamer forms the central cog of the clock and is an auto-kinase and -phosphatase and an ATPase. The KaiA dimer enhances KaiC phosphorylation and KaiB dimers antagonize KaiA's action. We will dissect the mechanism of the KaiABC clock using hybrid structural techniques, including X-ray crystallography, electron microscopy (EM), small angle X-ray and neutron scattering (SAXS and SANS, respectively), a range of biophysical and biochemical approaches as well as functional assays in vivo and in vitro. The three specific aims are (1) Structure and function of phosphorylation site (P-site), phosphorylation loop and putative phosphatase active-site mutant KaiC proteins;(2) Structure determinations of KaiAC, KaiBC and KaiABC complexes using both wt and P-site mutant KaiCs for optimization of protein-protein interactions;and (3) Determination of the molecular origins of the clock's temperature compensation.

Public Health Relevance

A comprehensive structure-based program to dissect the mechanism of the cyanobacterial KaiABC circadian clock, a highly attractive target for biochemical and biophysical studies due to the fact that it can be fully reconstituted in vitro from three proteins in the presence of ATP;the kinase, phosphatase, ATPase, and potentially ATP synthase activities and protein-protein interactions of the core clock protein KaiC will be probed using a combined mutagenic, functional and hybrid structural (X-ray, EM, SAXS, SANS) approach and the molecular origins of temperature compensation, a hallmark of all biological clocks will be determined.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Flicker, Paula F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
Schools of Medicine
United States
Zip Code
Pattanayek, Rekha; Egli, Martin (2015) Protein-Protein Interactions in the Cyanobacterial Circadian Clock: Structure of KaiA Dimer in Complex with C-Terminal KaiC Peptides at 2.8 Å Resolution. Biochemistry 54:4575-8
Egli, Martin; Johnson, Carl H (2015) Biochemistry that times the day. Biochemistry 54:104-9
Egli, Martin (2015) Structural and biophysical methods to analyze clock function and mechanism. Methods Enzymol 551:223-66
Kraemer, Bradley R; Snow, John P; Vollbrecht, Peter et al. (2014) A role for the p75 neurotrophin receptor in axonal degeneration and apoptosis induced by oxidative stress. J Biol Chem 289:21205-16
Egli, Martin (2014) Intricate protein-protein interactions in the cyanobacterial circadian clock. J Biol Chem 289:21267-75
Johnson, Carl Hirschie; Egli, Martin (2014) Metabolic compensation and circadian resilience in prokaryotic cyanobacteria. Annu Rev Biochem 83:221-47
Pattanayek, Rekha; Xu, Yao; Lamichhane, Aashish et al. (2014) An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC. Acta Crystallogr D Biol Crystallogr 70:1375-90
Pattanayek, Rekha; Yadagiri, Kirthi Kiran; Ohi, Melanie D et al. (2013) Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator. Cell Cycle 12:810-7
Egli, Martin; Johnson, Carl Hirschie (2013) A circadian clock nanomachine that runs without transcription or translation. Curr Opin Neurobiol 23:732-40
Egli, Martin; Pattanayek, Rekha; Sheehan, Jonathan H et al. (2013) Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock. Biochemistry 52:1208-20

Showing the most recent 10 out of 22 publications