Investigators provide first atomic-level images of the CLOCK complex that drives circadian rhythm

Dr. Joseph Takahashi
Dr. Hong Zhang, Dr. Carla Green, Dr. Joseph Takahashi, and Yogarany Chelliah.

DALLAS – June 4, 2012 –  UT Southwestern Medical Center researchers have taken a major step toward understanding the cellular clock, mapping for the first time the atomic-level architecture of a key component of the timekeeper that governs the body’s daily rhythms.

The daily, or circadian, cycles guided by the body’s clocks affect our ability to get a good night’s sleep, how fast we recover from jet lag, and even the best time to give cancer treatments, said Dr. Joseph Takahashi, senior author of the Science study published online and a pioneer in the study of circadian rhythms. The work was completed in collaboration with the laboratory of Dr. Hong Zhang, an associate professor of biochemistry.

Understanding the structure of the cellular clock could lead to better treatments for insomnia, diabetes, and even cancer.

“The clock is found in virtually every cell of the body, and is important for controlling many different metabolic functions,” said Dr. Takahashi, chairman of neuroscience and a Howard Hughes Medical Institute (HHMI) investigator at UT Southwestern.

Mapping the 3-D structure of the key component in the cellular clock – called the CLOCK:BMAL1 transcriptional activator complex – will have a great impact on the study of circadian rhythms and in other areas like toxicology and the growth of nerve cells, in which proteins in the same family play central roles, he said.

“Ultimately, we have to go to the atomic level to really understand how these proteins work” Dr. Takahashi said.

“Ever since Dr. Takahashi identified the Clock gene using positional cloning in 1997, it has been a goal of many labs to determine the 3-D structure of CLOCK. It is satisfying that the structure was determined at UT Southwestern,” Dr. Zhang said.

The two laboratories have spent years working on the X-ray crystallography of the CLOCK:BMAL1 complex, a process marked by a series of breakthroughs. One breakthrough was the decision to focus on the area of the complex in which the CLOCK and BMAL1 proteins interact rather than on the full-length proteins, which contained areas of instability, Dr. Zhang said.

It was also necessary to switch from using bacterial cells (the common approach) to utilizing insect cells to make the protein. Another breakthrough came in the spring of 2011 when Yogarany Chelliah, an HHMI research specialist at UT Southwestern, was able to crystallize the proteins for the first time. That breakthrough was followed by Dr. Nian Huang, a postdoctoral researcher of biochemistry and lead author of the study, and graduate student Clinton Taylor modifying the purification protocols and optimizing the crystallization conditions to allow successful X-ray diffraction, the wave-scattering procedure that resulted in the 3-D images, Dr. Zhang explained.

Atomic-level images of the CLOCK:BMAL1 transcriptional activator complex
The CLOCK:BMAL1 transcriptional
activator complex. Image courtesy of Dr. Joseph Takahashi.

The researchers found that the CLOCK protein is tightly wrapped around the BMAL1 protein in an unusually asymmetrical fashion. They identified three distinct areas for interactions between CLOCK and BMAL1 as well as regions for interactions with other molecules that might affect the cellular clock by changing the sleep-wake cycle or other body processes that depend on circadian rhythm, he said.

Dr. Takahashi’s research on the subject goes back almost 20 years. That’s when he began a behavioral study of mice looking for those animals in which their biological clocks seemed out of sync. After screening hundreds of mice, his laboratory in 1994 identified one mutant mouse whose daily cycle was four hours longer than normal. He named that mouse the Clock mutant.

Dr. Takahashi then used that mouse to identify the world’s first circadian rhythm gene in a mammal. Researchers in his laboratory cloned the Clock gene in 1997. In 1998, they discovered that the CLOCK protein worked in concert with the BMAL1 protein in a study done in collaboration with Dr. Charles Weitz at Harvard Medical School.

Two years ago, Dr. Takahashi’s team – in collaboration with Dr. Joseph T. Bass at Northwestern University Feinberg School of Medicine in Chicago – reported in Nature that disruptions in the Clock and Bmal1 genes in mice can alter the release of insulin by the pancreas, which results in diabetes.

“We started on this path a long time ago, and it actually began with a mouse, which then allowed us to find the Clock gene, and then from this gene we now see the proteins from their crystal structure,” Dr. Takahashi said. “For that to all happen after such a long quest is particularly satisfying.”

Other UT Southwestern researchers involved in the study include Dr. Yongli Shan, postdoctoral researcher in neuroscience; Dr. Seung-Hee Yoo, instructor of neuroscience; and Dr. Carla Green, professor of neuroscience. Dr. Carrie Partch – a former postdoctoral researcher in biochemistry, neuroscience, and the HHMI – is now an assistant professor of chemistry and biochemistry at UC Santa Cruz.

This work was supported by the HHMI, the American Heart Association, and the National Institutes of Health.

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Media Contact: Deborah Wormser
214-648-3404
deborah.wormser@utsouthwestern.edu

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