MOLECULE OF THE MONTH:

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CIRCADIAN CLOCK PROTEINS

Counting the Hours
Molecular processes occur so fast that is it difficult to imagine a 24hour clock that works at the molecular level. But surprisingly, different organisms have evolved many different ways of doing this. Animal cells use a complex collection of proteins (with fanciful names like Clock, Cryptochrome, and Period) that are rhythmically synthesized and degraded each day. The 24-hour oscillation of the levels of these proteins is controlled by a series of interconnected feedback loops, where the levels of the proteins precisely regulate their own production. A much simpler system has been discovered in cyanobacteria. It is composed of three proteins, KaiA, KaiB and KaiC, that together form a circadian clock. At the beginning of the cycle, KaiA (at the top, PDB entry 1r8j) stimulates the large KaiC hexamer (center, PDB entry 2gbl), which then adds phosphate groups to itself. Then, as KaiC fills itself up with phosphates, it binds to KaiB (bottom, PDB entry 1r5p), which inactivates KaiA and allows the phosphates to be slowly removed. As the number of phosphates drops, KaiB falls off and KaiA can start the cycle again. 1r8j

Our cells contain tiny molecular clocks that measure out a 24hour circadian rhythm. This clock decides when we get hungry and when we get sleepy. This clock can sense when the days are getting longer and shorter, and then trigger seasonal changes. Our major clock is housed in a small region of the brain, called the suprachiasmic nuclei. It acts as our central pacemaker, checking the cycles of light and dark outside, and then sending signals to synchronize clocks throughout the rest of the body.

2gbl

1r5p

About the RCSB PDB Molecule of the Month

Using selected molecules from the PDB archive, each feature includes an introduction to the structure and function of the molecule, a discussion of its relevance to human health and welfare, and suggestions for viewing and accessing further details. The RCSB PDB Molecule of the Month is read by students, teachers, and scientists worldwide at www.pdb.org. This January 2008 edition was written and illustrated by David S. Goodsell (RCSB PDB and The Scripps Research Institute).

Synchronize Your Watches

These clocks have a period of about 24 hours, but as you can imagine, they are not exact. So cells have a way of synchronizing their clocks with the outside world. The clock in our brain is synchronized by exposure to light. Light is sensed by the retina, and signals are sent into the brain to modify the timing of the circadian oscillations. If you have traveled across several time zones, you have experienced this synchronization. For the first day or two, you experi-

ence jet lag because your clock is synchronized with the old schedule. But gradually, the bright light of day (blue light seems to work best) shifts your clock to bring you into alignment with the local time.

CIRCADIAN CLOCK PROTEINS

RCSB Protein Data Bank
The Protein Data Bank (PDB) is the single worldwide repository for the processing and distribution of 3D structure data of large molecules of proteins and nucleic acids. The RCSB PDB is operated by Rutgers, The State University of New Jersey and the San Diego Supercomputer Center and the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California, San Diegotwo members of the Research Collaboratory for Structural Bioinformatics (RCSB). It is supported by funds from the National Science Foundation, the National Institute of General Medical Sciences, the Office of Science, Department of Energy, the National Library of Medicine, the National Cancer Institute, the National Institute of Neurological Disorders and Stroke and the National Institute of Diabetes & Digestive & Kidney Diseases. The RCSB PDB is a member of the worldwide PDB (wwPDB; www.wwpdb.org).

Sleepy Molecules
The small hormone melatonin is produced selectively at night, and circulates through the blood to coordinate our nightly activities, such as sleep. Treatment with melatonin may be used to change this cycle artificially, for instance helping to shift cycles that are out of phase during jet lag. The daily rise and fall of melatonin is caused by changes in the levels of the enzyme serotonin N-acetyltransferase, shown here from PDB entry 1cjw. This enzyme adds a few atoms to the neurotransmitter serotonin, then a second enzyme converts it into melatonin. In this structure, the enzyme is caught in the process of performing the reactionthe large green molecule bound in the active site is similar to the intermediate formed as the acetyl group is added to serotonin. 1cjw

Exploring the Structure

The KaiC clock protein (PDB entry 2gbl) is composed of six identical subunits that form a barrelshaped structure. The protein is shown from side here, with two subunits removed to show the tunnel that runs through the middle. The phosphates that tick off the hours of each day are added to a serine and a threonine on each subunit. They are buried deep inside the tunnel, near the binding site for ATP, shown here in green. KaiA stimulates KaiC to add these phosphate groups to itself, and KaiB blocks the action of KaiA, allowing KaiC to remove these phosphates from itself. The speed of these reactions are all calibrated so that the whole process takes 24 hours to complete.

References:
1r8j: S. Ye, I. Vakonakis, T. R. Ioerger, A. C. LiWang, J. C. Sacchettini (2004) Crystal structure of circadian clock protein KaiA from Synechococcus elongatus. J.Biol.Chem. 279, 20511-20518 2gbl: R. Pattanayek, D. R. Williams, S. Pattanayek, Y. Xu, T. Mori, C. H. Johnson, P. L. Stewart, M. Egli (2006) Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods. Embo J. 25, 20172028 1r5p: R. G. Garces, N. Wu, W. Gillon, E. F. Pai (2004) Anabaena circadian clock proteins KaiA and KaiB reveal a potential common binding site to their partner KaiC. (2004) Embo J. 23, 1688-1698 1cjw: A. B. Hickman, M. A. Namboodiri, D. C. Klein, F. Dyda (1999) The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme

complex at 1.8 A resolution with a bisubstrate analog. Cell(Cambridge,Mass.) 97, 361-369 M. J. Rust, J. S. Markson, W. S. Lane, D. S. Fisher and E. K O'Shea. (2007) Ordered phosphorylation governs oscillation of a three-protein circadian clock. Science 318, 809- 812. J. Arendt and D. J. Skene (2005) Melatonin as a chronobiotic. Sleep Medicine Reviews 9, 25-39. D. Bell-Pedersen, V. M. Cassone, D. J. earnest, S. S. Golden, P. E. Hardin, T. L. Thomas and M. J. Zoran (2005) Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nature Reviews Genetics 6, 544-556. S. L. Harmer, S. Panda and S. A. Kay (2001) Molecular bases of circadian rhythms. Annual Review of Cell and Developmental Biology 17, 215-253.