Cracking the Biological Clock

A biochemical understanding of how organisms keep time may be closer at hand, thanks to a recent outpouring of structures of the clock proteins from primitive bacteria.

Humans and many other organisms contain biological clocks that control daily cycles of gene expression, physiology, and behavior. The simplest organisms known to exhibit such daily (circadian) rhythms are cyanobacteria, a widespread type of water-dwelling, photosynthetic bacteria commonly known as blue-green algae.

Cyanobacteria have caught the fancy of clock researchers, in part because their clockwork is much simpler than that found in humans and other animals. Just three proteins--the Kai proteins, named after the Japanese word for cycle--are required to keep time in these bacteria. Cyanobacteria also are an attractive model system because they can be genetically manipulated, making it easy to create strains with mutant clock proteins.

In recent months, this simple model system has gotten its biggest boost yet: a clear picture of what the three Kai proteins look like. "Every one of the central clock proteins of the cyanobacterial clock have now been described at the molecular level," enthuses biology professor Susan S. Golden of Texas A&M University. However, none of the clock proteins from higher organisms have been structurally characterized, she points out.

The growing wealth of structural data on cyanobacterial clock proteins makes it increasingly likely that clock researchers will be able to gain a comprehensive understanding of a biological clock, according to biological sciences professor Carl H. Johnson of Vanderbilt University. The sequences of cyanobacterial clock proteins are markedly different from human clock proteins, and there's no telling whether they share structural features, he admits. But "the hope is that the study of clock proteins and mechanisms in cyanobacteria will uncover general principles that will be applicable to circadian clocks in higher organisms," he adds.

The three Kai proteins form large complexes in cyanobacteria, with KaiC at the core. KaiC shares sequence similarities with enzymes that unwind DNA, which has led researchers to suspect that the cyanobacterial protein interacts with DNA to influence gene transcription. KaiC's phosphorylation status--the enzyme can phosphorylate itself in the presence of adenosine triphosphate--oscillates in a daily pattern and is thought to control the enzyme's activity. The other two Kai proteins are expected to help KaiC adjust for changes in the length of day and night by modulating KaiC's phosphorylation status, with KaiA enhancing KaiC phosphorylation and KaiB blocking it.

Clock researchers got their first glimpse of a Kai protein in 2002 when assistant professor of biochemistry and biophysics Andy C. LiWang, graduate student Ioannis Vakonakis, and coworkers at Texas A&M probed the structure of the N-terminal half of KaiA using nuclear magnetic resonance spectroscopy. From the protein's structural similarities to a signal transduction protein involved in chemotaxis, LiWang and Golden predicted that KaiA's N-terminal domain gathers signaling information about external cues such as light. The C-terminal half of KaiA then transmits this information to KaiC by binding to the enzyme and modulating its phosphorylation status, LiWang suggests. LiWang's lab unveiled an NMR structure of the C-terminal half of KaiA earlier this year.

A FLURRY of KaiA crystal structures soon followed, including one of full-length KaiA from LiWang and Texas A&M biochemistry and biophysics professor James C. Sacchettini [J. Biol. Chem., 279, 20511 (2004)]. In addition, grad students Robert G. Garces and Ning Wu and medical biophysics professor Emil F. Pai of the University of Toronto revealed the structure of KaiB, which bears little resemblance to any structurally characterized protein [EMBO J., 23, 1688 (2004)]. But perhaps the most anticipated structure was that of KaiC. The crystal structure of the central clock protein has just been published by Vanderbilt associate professor of biochemistry Martin Egli, postdoc Rekha Pattanayek, structural biologist Jimin Wang of Yale University, Johnson, and coworkers [Mol. Cell, published online July 22, http://www.molecule.org/content/article/
abstract?uid=PIIS1097276504004356]. As Johnson's earlier electron microscopy and ultracentrifugation studies had predicted, KaiC forms a doughnut-like hexamer with a partially sealed central pore and a nipped-in waist.

EGLI NOTES that some of the residues known to be important to KaiA-KaiC interactions are located at the protein's waist, whereas others are found on its domed top. Johnson tells C&EN that the structure also has allowed the team to identify three key phosphorylation sites on KaiC. Texas A&M's Golden says that what's needed to complete the picture are structures of complexes between the clock proteins. In a step in that direction, LiWang and Vakonakis recently published the NMR structure of a KaiC-derived peptide bound to the C-terminal portion of KaiA [Proc. Natl. Acad. Sci. USA, published online July 15, http://dx.doi.org/10.1073/pnas0403037101].

The peptide-protein interface is stabilized by both hydrophobic and electrostatic interactions and includes many of the amino acid side chains already known to be key to keeping time, LiWang notes. He points out that binding of the KaiC peptide to KaiA's C-terminal domain causes subtle changes in the conformation of KaiA's N-terminal domain. Other proteins that are yet to be identified could regulate the circadian system by binding KaiA's N terminus and perturbing the conformation of its C terminus, making it easier or harder for KaiA to bind KaiC, he proposes.

Unfortunately, the portion of KaiC seen in LiWang's complex is too floppy to be visible in Egli and Johnson's KaiC crystal structure. Egli and Johnson--and likely many others--are now pursuing the crystal structure of KaiA-KaiC, as well as the KaiA-KaiB-KaiC complex.

"The fact that so many groups have amassed so much structural data in such a short period of time is remarkable," Golden says. She hopes recent success in structurally characterizing cyanobacterial clocks will challenge the circadian rhythm community to go after the structures of clocks in other organisms.