XCL1: Two Folds Are Better Than One

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About twenty years ago, when Brian Volkman was still a postdoctoral student, he was approached by a colleague who was studying HIV and looking to characterize a protein he had encountered in his studies on the immune system. The protein, now referred to as XCL1, was identified as having an important role in the immune system’s response. Essentially, Volkman was tasked with determining how the protein folds in three dimensions, like a piece of paper folds to create an origami shape. Using nuclear magnetic resonance (NMR), he started to uncover the structure of XCL1, which would in turn tell him and his colleague a lot about its function. When he got his first batch of data back, he was at a loss; it was uninterpretable and seemed to suggest that the protein was unfolded, or had no consistent structure.

It was only after running many more tests under many different temperatures and salt concentrations that Volkman came to a startling conclusion: the XCL1 protein had two folds.

We now know that one fold of XCL1 tells white blood cells where to go in the body in order to fight off invaders. The other fold also directs an immune response, through a different mechanism, but can also directly fight off invasive cells. Because of these alternative forms, one protein can have a much greater impact on the body’s overall immune response.

But back then, not much was known about the protein’s form or function. As Volkman says, anyone who has taken a biochemistry class as an undergraduate will tell you that a protein folds in a single specific way that is most thermodynamically favorable. The structure of this protein then determines its specific function in the body. This principle is one of the core tenets of biochemistry, and Volkman’s work was challenging everything his field believed in.

“It took years to reach the point where I felt confident enough to know what was happening and write the paper. Even now, almost twenty years later, there are people, experts in biochemistry, who if you asked them, ‘Can a protein do this?’ They would say, ‘No, no, we know that’s not the case,’” Volkman said. “That’s the hardest part—looking at some data, some evidence, and realizing that it’s the opposite of what you learned in your biochemistry class and wondering what’s right: what you learned in the textbook or what you’re looking at on the page in front of you.”

After Volkman’s work was finally published, the term “metamorphic protein” was coined. Because of this, he considers XCL1 to be one of the first metamorphic proteins.

But his work with XCL1 didn’t end there. Volkman received several National Institutes of Health grants to continue his research. “I like to think that Tony Fauci has been supporting this over the years,” Volkman said, laughing. With this level of funding, he continued to study XCL1 until he met an interested graduate student, Caci Dishman.

Acacia “Caci” Dishamn met Volkman early in her MD/PhD program at the Medical College of Wisconsin, where he was her advisor. She was specifically excited about his research in metamorphic proteins because it went against what she had been taught for her entire undergraduate career. Dishman said early on in her conversations with Volkman she could see that this would be “paradigm-defying work.” 

She compiled preliminary data that other postdocs and graduate students had collected about the evolutionary ancestry of XCL1 and combined it with her own work, eventually being published as the first author on a recent paper about how XCL1 came to be. Volkman says that Dishman’s drive to push the project forward was the reason they found something genuinely new that they wanted to share with a broader audience.

Dishman’s work had two main findings. First of all, it was discovered that XCL1’s simultaneous evolution of two folds wasn’t an accident. When many scientists look at metamorphic proteins, they come to the conclusion that one fold must be an evolutionary artifact, like an appendix in humans. The prevailing belief was that XCL1 and other similar proteins only had two folds because they were unfinished with their evolutionary journey and someday would return to only having one fold. However, when Dishman and Volkman modelled the ancestry of the protein, they found that XCL1 started out with one fold. Over time, it evolved to have two folds, but primarily occupied the old fold in a ratio of about nine to one. As more time passed, the ratio became one to nine, now primarily occupying the new fold. If the protein was evolving to only occupy the new fold, as was the conventional belief, the next step would see only the new fold.

That wasn’t the case. The protein is now present in a ratio of one to one, found equally in both forms, and is able to spontaneously and randomly switch between folds under conditions within the human body. This protein wasn’t the result of an ill-timed snapshot or an evolutionary mistake; rather, it was evolution’s creative improvement to the immune response.

But how does a protein evolve from having only one fold to having two folds? Dishman’s next goal was to make changes in the amino acid sequence of a chemokine protein like XCL1, until it expressed two folds. At first, she made small changes, trying to isolate the specific amino acids that were important. After making about fifteen individual changes, one or two at a time, it didn’t seem like her approach was going to work. It was only when she had the idea to employ three sets of changes at once, eleven in total, that the protein finally became metamorphic.

Dishman says that was both the most challenging part of the project and the most rewarding. “When I finally figured out that set of mutations that caused XCL1 to become metamorphic, I was so excited,” she said. “I had collected data for an experiment overnight, and I went in the next morning, and the protein I had made was metamorphic and I was just so amped.” She added “People in the lab were starting to be like ‘Caci, I don’t know if you’re going to do this. You’ve been trying for a while. You might want to stop.’ But finally I got it.”

Why is all of this important? First of all, now that we know that XCL1 isn’t an accident, biologists across the world can search for more metamorphic proteins and learn about their functions. One important application is the creation of targeted therapies for diseases: if a metamorphic protein can cause a genetic disease, finding and understanding its structure is the next step in developing a treatment. Essentially, if one fold of a protein causes a disease, a powerful therapy would be to determine how to switch to only the other fold of that protein.

Why is this important? Now that they have an “instruction manual,” Dishman and Volkman are attempting to create metamorphic proteins with specific functions for biological sensors, self-assembling materials, and components of molecular machines. The level of control researchers have with metamorphic proteins is much greater than that with a regular protein—an “active” fold could be engineered only to occur in certain conditions. This would allow researchers to turn a protein “on” by changing the environment. The applications of metamorphic proteins are far-reaching, and will likely have a significant impact on the fields of biochemistry and bioengineering as more developments are made.

If Volkman and Dishman had believed in convention over their own data or listened to critics, such advancements would not be possible. It is only through their willingness to defy commonly held beliefs and their unwavering perseverance that we can even imagine these treatments and technologies, let alone develop them in years to come.


Dishman, A. F., Tyler, R. C., Fox, J. C., Kleist, A. B., Prehoda, K. E., Babu, M. M., … & Volkman, B. F. (2021). Evolution of fold switching in a metamorphic protein. Science, 371(6524), 86-90.

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