Helping a Puzzled Clock

One day, Sancar was investigating the genetic sequence of plant and bacterial photolyase genes and found strikingly similar sequences in human genes. At first, thinking it was a mistake, he cloned the human genes and tested whether they repaired DNA. They didn’t, and Sancar realized he was face to face with a group of proteins that had evolved from photolyases called cryptochromes, which were known to exist in plants. No one knew what these mammalian cryptochromes looked like or how they interacted in the body.

Although her desk stood in a distant corner of the lab against a white cinder block wall, Partch was always visible by her bright-red dyed hair that she tended to pull into a messy bun on the top of her head. She spent her time trying to figure out what cryptochromes did, which involved studying protein interactions in cells. She was in the lab so much that her jeans were stained with bleach and blue dye from coloring proteins.

On one typical day, Partch left her pipettes and protein dyes on the bench and decided to tackle cryptochromes with “sheer brute force,” she says. She had an idea: compare cryptochromes and photolyase from different species to see which areas of the cryptochromes were different from their photolyase ancestors, but exactly the same within the cryptochromes themselves. Although photolyases and cryptochromes are from the same family, they have different functions. The former repairs DNA, while the latter keeps time. Partch thought that although cryptochromes differ among species, if some regions were the same and didn’t appear in photolyase, they must be critical to the clock’s function.

Graphic: Yasemin Saplakoglu

By the time Partch stepped foot into the circadian rhythm field, she could go online on the PubMed database to find the identity and location of each amino acid, or protein building block, that made up the different cryptochromes. The string of amino acids that makes up a protein is called its protein sequence. She printed out the cryptochrome and photolyase protein sequences of species ranging from humans to insects and taped these printouts on a wall.

Partch peered through her thin-rimmed glasses as she spent hours highlighting similar areas of different proteins. Scotch-taped printouts of the protein sequences gathered dirt as they crinkled near her feet.

Partch highlighted every amino acid that matched up among mammalian cryptochromes. Using her highlighted pieces of paper and a base model of the structure of the most similar proteins at the time—the photolyases and plant cryptochromes, whose structures had been deciphered—she created a 3D mockup of a mammalian cryptochrome.

With its nooks and folds, the model looked like a rainbow-dyed oatmeal cookie. In a spattering of orange, yellow, green, blue, and pink, Partch highlighted five regions that remained the same across mammalian cryptochromes.

At the time, she had no bioinformatics experience, which would have allowed her to use computer programs to test her hypothesis about the structure of CRY proteins. So she shut the thick, black, leather-bound thesis notebook in which she’d drawn her colorful model. Partch then left Sancar’s lab to pursue postdoctoral research at the University of Texas Southwestern, where she studied the clock and similar proteins. In 2011 she began to set up her own lab at UC Santa Cruz, knowing that she wanted to understand what those five regions of the mammalian cryptochromes do.

The animal cryptochrome

While she was starting her lab, other researchers figured out what the blue, pink, and green regions did.

Five years ago, a lab at the University of Washington in Seattle first published the structure of a mammalian cryptochrome. Ning Zheng, a structural biologist, had been looking for something else altogether: a ligase, a protein that glues DNA strands together. The blue and pink regions of the mammalian cryptochrome turned out to be where the ligase attached to break down CRY. “We were more interested in the ligase without realizing that the cryptochrome was in fact more interesting,” Zheng says.

Zheng remembers how Partch sent him an excited email after he published his result. When he saw the diagram from her graduate thesis where she’d identified the blue region of cryptochrome as one of its more important components, Zheng was equally amazed. “She really had a lot of insight on the structure before it was even published,” he says.

Soon after, Eva Wolf, a crystallographer at the Institute of Molecular Biology in Munich, Germany, figured out how CRY communicates with a protein called PER, which helps CRY inactivate the clock complex. PER binds to a hotdog-like helix of CRY, corresponding to the pink area on Partch’s diagram. PER also gloms onto to the green area that Partch had identified on the backside of CRY and the blue area where the ligase binds. “So PER kind of gives CRY a big bear hug,” Partch says—a hug that keeps CRY from degrading at the wrong time.

All that remained was to find the final two regions that Partch had predicted could be important, the ones colored yellow and orange in her thesis. In 2015 Partch and her collaborator Andrew Liu, a circadian rhythm researcher at the University of Memphis, used a detailed imaging technique called nuclear magnetic resonance to find exactly where CRY binds to inhibit the molecular clock complex to prepare for slower hours of the day.

Then, this year, Partch’s lab solved the last piece of the puzzle she had outlined as a graduate student by using small x-ray scattering—a different imaging method that not only reveals the structure of a molecule, but also how it moves. This movement is hard to capture with other techniques, says Partch, but it is necessary to understand how a dynamic complex like the molecular clock actually changes.

Scientists had known that twice a day, the cryptochromes bind to the protein complex to slow down the clock. Partch found out how. A loop extends out of the molecular clock protein complex and fits into a pocket—formerly known to her as the yellow and orange area—on the cryptochrome. Almost a decade after Partch predicted that five colored regions of CRY would be important, she now finally understands why and how these proteins interact to help the body keep time.

The binding pocket is ancient, Partch notes. Photolyase, CRY’s oldest relative, also has a pocket that binds a molecule to help it harvest light. “The pocket has been there since the origins of this family,” she says. “It has just gotten bigger, so that now it can hold a protein.” While her decade-old diagram correctly predicted that the region of CRY containing its pocket is important, she had underestimated how large the pocket would turn out to be.

Liu thinks Partch’s new paper improves the understanding of how these four proteins interact together to make the molecular clock tick. “The pocket provides an opportunity for fine-tuning,” he says. For instance, perhaps the pocket could serve as a dock for a drug that would allow precise control of the clock. “The idea is that you can make the clock work a little bit better,” Liu adds.

Other researchers, such as Zheng, plan on tailoring their own research based on these results. “I will have to make my models less static,” he says.

Stifling a CRY

Scientists in other fields are turning their attention to these studies as well, Liu says, because they provide a way to link clock function with physiology and disease.

Disruption of the body’s internal clock can happen with aging, taking on night shifts, traveling across time zones, and in physiological conditions such as obesity and cancer. Partch was the first to show that a unique protein found in some cancer cells was a time thief and could entirely stop the clock. This disruption is dangerous because the clock is interconnected with bodily processes. And it can wreak havoc; studies have linked a number of diseases to disturbed clocks.

Upsetting the clock can disrupt sleep, mood, the brain’s reward center, and learning, leading to sleeping disorders and depression. In the blood, it can increase the amount of fats, leading to high cholesterol. It can increase the amount of cytokines, small proteins secreted in the immune system, and contribute to autoimmune disorders. It can cause the body to store more fat, leading to obesity. It can even tamper with DNA repair proteins, which can lead to tumors. “The clock is not just something that sits in my office on my wall,” Liu says.

Meanwhile, Partch is racing against time. She is looking for a small molecule that could bind to a confused cryptochrome and keep it off the clock complex, allowing the molecular clock to return to its dawn-like state. Such a molecule might serve as a drug that travelers could take to synchronize their body clocks to the time zone to which they’re traveling, thus preventing the ill effects of jet lag.

Other labs are also working on developing possible drugs to control the clock, but the U.S. Patent Office has ruled that no other lab can patent such small molecules for one year.

“It is a model that is supported by a lot of evidence,” says Eva Wolf of Munich. “One could probably put a drug into that pocket and disrupt the main interaction between cryptochrome and [the other proteins].”

Reflecting on her years of research on CRY, Partch pulls out her thesis to a page showing her decade-old mammalian cryptochrome mock-up model—decorated with its blue, pink, yellow, orange, and green swatches. At the time, she noted the conserved areas of the protein. Though she didn’t know their function, she wrote that they were “striking.” “Yes, striking indeed,” she now says. These regions stayed constant across millions of years. Sure enough, they turned out to play major roles in the ticking of our internal clocks.

If Partch’s hunch about the pocket is right, there may come a time when we pack a time-zone-switch pill along with our passport, ibuprofen, and phone chargers—an era when we may have to remember to swallow the correct time.

© 2017 Yasemin Saplakoglu / UC Santa Cruz Science Communication Program