Most interferons – proteins that send signals throughout the immune system – are beneficial when it comes to helping the body fight a virus. But interferon lambda 4 (IFNλ4) does the opposite; it makes an immune response worse. Studies have shown that people with the gene for IFNλ4 (up to 70% of people in some parts of the world), are less effective at fighting hepatitis C and can be more susceptible to COVID-19 and other respiratory viruses.
Understanding the IFNλ4 conundrum has been challenging since its discovery ten years ago. In particular, researchers have struggled to produce or purify high levels of the IFNλ4 protein.
Now, researchers at the University of Chicago Pritzker School of Molecular Engineering (UChicago PME) have developed a new method of producing and isolating IFNλ4. That enabled them to determine the structure of the protein for the first time, and it revealed an unusual, floppy region of the protein. Their studies reveal that this unstructured region contributes to why IFNλ4 has been hard to make in cells. Now, future studies can probe whether modifying that region could minimize the negative impact of IFNλ4 on the immune response.
“This protein is one of the strongest genetic predictors of viral susceptibility and it impacts a number of diseases,” said UChicago PME Assistant Professor Juan Mendoza, the senior author of a new work featured by the Editor in Nature Communications. “We can now start to actually understand why that is.”
Mendoza, who is also a Howard Hughes Medical Institute Freeman Hrabowski Scholar, wanted to know what made IFNλ4 different from closely related interferons which help protect against viral infections. But he knew that other labs had struggled to produce the protein.
Over the past several years, William Grubbe, PhD'24, a former PME graduate student of Mendoza and Juan de Pablo, developed a new method to express the protein in cells by combining the gene for IFNλ4 with the gene for its receptor. This helped to stabilize IFNλ4 as it was being produced and to protect it as it travels from the inside to the outside of the cell.
“It was incredibly exciting to isolate a stable version of IFNλ4 for the first time and enable further characterization of its function,” said Grubbe, lead author of the new paper.
Together with the lab of Prof. Minglei Zhao in the Department of Biochemistry and Molecular Biology, the team then used cryogenic electron microscopy (cryoEM) to create detailed three-dimensional images of both IFNλ4 bound to its receptors as well as a related interferon, IFNλ3, bound to the same receptors.
IFNλ4 binds to the receptors even more tightly than IFNλ3 and attaches at a different angle, potentially explaining how it has different cellular consequences. Moreover, Mendoza and Grubbe spotted a region of IFNλ4 that stood out: a floppy, positively charged region in the middle of the protein. When the team replaced this disordered region with a more structured region from IFNλ3, the protein was able to be produced even in the absence of its receptor.
The scientists hypothesize that the highly charged, unstructured region makes IFNλ4 fold incorrectly, accumulate in cells, and cause cell stress. This explains why it has been so difficult to produce in cells in the lab, but also why it might lead to a stymied immune response to viruses.
“IFNλ4 might make immune cells stressed and less effective rather than triggering an effective immune response like other interferons,” said Mendoza.
More work is needed to understand whether this region is indeed responsible for the negative health consequences of IFNλ4 when it comes to hepatitis and respiratory viruses. If it is, Mendoza says therapeutics could be explored that stabilize the structure of the protein or prevent it from causing problems in immune cells.
Citation: “Structural studies of the IFNλ4 receptor complex using cryoEM enabled by protein engineering,” Grubbe et al, Nature Communications, January 18, 2025. DOI: 10.1038/s41467-025-56119-y
Funding: This work was supported by funding from the Howard Hughes Medical Institute (HHMI), the National Institutes of Health (NIH) (1R35GM147179-01, 5R35GM143052-03), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (#RS-2023- 737 00268767).