Circadian rhythms are endogenous biological programs that time metabolic and/or behavioral events to occur at optimal phases of the daily cycle. They have three diagnostic characteristics: (i) In constant conditions, the programs """"""""free-run"""""""" with a period that is close to, but not exactly, 24 hours in duration; (ii)in an appropriate environmental cycle (usually a light/dark and/or temperature cycle), the rhythm will take on the period of the environmental cycle, i.e., they will entrain; (iii) the period of the free-running rhythm is nearly the same at different constant ambient temperatures within the physiological range, i.e.they are temperature-compensated. One of the fascinations of circadian rhythms is to explain how a biochemical mechanism can keep time so precisely over such a long time constant (~24h) at different ambient temperatures. Cyanobacteria are the simplest organisms that display circadian rhythms and provide a model system for the circadian clock. The long-term goal of this proposal is a structural characterization of the circadian clockwork of Synechococcus elongatus. Close to 80% of the genes of 5. elongatus are regulated with a circadian rhythm. Three relevant loci have been identified by genetic screens: kaiA, kaiB and kaiC. The corresponding proteins physically associate and autoregulate gene expression to produce circadian molecular cycling. Thus, cycling gene expression and autoregulation appear to always provide the molecular foundation for circadian rhythms. In S. elongatus, inactivation of any single kai gene abolished the circadian rhythms and reduced kaiBC-promoter activity.Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Therefore, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, KaiA sustains the oscillation by enhancing kaiC expression and KaiB is an anagonist of KaiA. Thus, KaiC plays a role as a 'state variable' of the circadian oscillator and emerges as a key component of the circadian clockwork. Remarkably and of importance for the specific aims proposed here, it was shown very recently that the KaiABC clock keeps time independent of de novo transcription and translation. As part of the dissection of the fundamental mechanism of the cyanobacterial clock, we have determined the three-dimensional structure of the KaiC protein by X-ray crystallography.
The specific aims of this proposal are: (1) A structure-based mutational analysis of KaiC; (2) The determination of X-ray crystal structures of selected KaiC mutants; (3) The crystal structure determination of the complex between KaiC and KaiA; and (4) The crystal structure determination of the complex between KaiC and KaiB. Because circadian rhythms are evolutionarily convergent, insights gained from the structural analyses of Kai proteins may provide clues as to the general mechanism of controlling sleep-wake cycles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073845-03
Application #
7390235
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2006-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
3
Fiscal Year
2008
Total Cost
$283,192
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Egli, Martin (2015) Structural and biophysical methods to analyze clock function and mechanism. Methods Enzymol 551:223-66
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
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

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