New research sheds light on how circadian rhythms work

Using innovative cryoelectron microscopy techniques, researchers have identified the structure of the circadian rhythm photosensor and its target in the fruit fly (Drosophila melanogaster), one of the main organisms used to study circadian rhythms. The research, “Cryptochromatic Timeless Structure Reveals Circadian Clock Timing Mechanisms,” published April 26 in Nature.

The research focused on fruit fly cryptochromes, key components of the circadian clocks of plants and animals, including humans. In flies and other insects, cryptochromes, activated by blue light, serve as the primary light sensors for establishing circadian rhythms. The target of the cryptochrome photosensor, known as “Timeless” (TIM), is a large and complex protein that could not be previously imaged, and therefore its interactions with cryptochrome are not well understood.

Circadian rhythms work through what are basically genetic feedback loops. The researchers found that the TIM protein, along with its partner, the Period (PER) protein, act together to inhibit the genes that are responsible for their own production. With appropriate delays between gene expression and repression events, an oscillation in protein levels is established.

This oscillation represents “the ticking of the clock and seems to be quite unique to the circadian rhythm,” said lead author Brian Crane, the George W. and Grace L. Todd Professor and chair of chemistry and chemical biology in the College of Arts. and Sciences.

The blue light, Crane said, changes the chemistry and structure of the cryptochrome flavin cofactor, allowing the protein to bind to the TIM protein and inhibit TIM’s ability to repress gene expression and thereby restore the oscillation.

Much of the hard work in the study went into figuring out how to make the cryptochrome-TIM complex so that it could be studied, because TIM is such a large and unwieldy protein, Crane said. To achieve their results, first author Changfan Lin, MS ’17, Ph.D. ’21, modified the cryptochrome protein to improve the stability of the cryptochrome-TIM complex and used innovative techniques to purify the samples, making them suitable for high-resolution imaging.

“These new methods allowed us to obtain detailed images of protein structures and gain valuable insight into their function,” said Lin, a postdoctoral fellow in the Friedrich Ataxia Research Alliance at the California Institute of Technology. “This research not only deepens our understanding of circadian rhythm regulation, but also opens up new possibilities for the development of therapies that target related processes.”

Co-author Shi Feng, a doctoral student in the field of biophysics, did much of the cryoelectron microscopy work. Cristina C. DeOliveira, a doctoral student in the field of biochemistry and molecular and cell biology, was also a co-author.

An unexpected result from the study sheds light on how DNA damage is repaired in a cell. Cryptochromes are closely related to a family of enzymes involved in DNA damage repair called photolyases. Crane said the research “explains why these families of proteins are closely related to each other, even though they are doing quite different things: they are making use of the same molecular recognition in different contexts.”

The study also offers an explanation for the genetic variation in flies that allows them to adapt to higher latitudes, where days are shorter in winter and it is colder. These flies have more than one certain genetic variant involving a change in the TIM protein, and it was not clear why the variation might help them. The researchers found that because of how cryptochrome binds to TIM, the variation reduces the affinity of TIM for cryptochrome. The interaction between proteins is modulated and the ability of light to restore oscillation is changed, thus disrupting the circadian clock and extending the fly’s dormant period, helping it survive the winter.

“Some of the interactions we see here in the fruit fly can be mapped to human proteins,” Crane said. “This study may help us understand key interactions between the components that regulate sleep behavior in people, such as how critical delays in the basic timing mechanism are integrated into the system.”

Another exciting finding, Lin said, was the discovery of an important structural area in TIM, called a “groove,” which helps explain how TIM enters the cell nucleus. Previous studies had identified some factors involved in this process, but the exact mechanism remained unclear. “Our research provided a clearer understanding of this phenomenon,” Lin said.