Cryo-EM imaging of STING protein reveals novel binding pocket

Near-atomic resolution imaging of a key immune protein commonly called STING has revealed a previously unrecognized binding site that appears to be essential for launching immune attacks, UT Southwestern scientists report in a new study. The findings, published in Naturecould lead to new ways to manipulate STING to trigger stronger immune responses or reverse its action in autoimmune diseases.

“For the first time, this work provides an accurate picture of the activated state of STING, essential for understanding its role in normal immunity as well as autoimmune diseases,” said study author Xuewu. Zhang, Ph.D., professor of pharmacology and biophysics at UT Southwest. Dr. Zhang co-directed the study with Xiaochen Bai, Ph.D., associate professor of biophysics and cell biology at UT Southwestern, and their postdoctoral fellows Defen Lu and Guijun Shang. Dr. Zhang and Dr. Bai are members of the Harold C. Simmons Comprehensive Cancer Center.

STING, short for “interferon gene stimulator”, is a central component of the innate immune system, which serves as the body’s first line of defense against viruses, bacteria and cancers. After a sensor known as cGAS detects foreign DNA in cells, it generates a messenger molecule known as cyclic GMP-AMP (cGAMP) that activates STING. In turn, STING initiates several signaling pathways that stimulate the production of inflammatory molecules and chemical signals that prompt cells to clean up detritus to eliminate invaders.

Together with UT Southwestern researcher Zhijian “James” Chen, Ph.D., a professor of molecular biology and at the Center for Host Defense Genetics, Zhang Lab and Bai Lab previously reported the first images of STING taken with cryogenic electron microscopy (cryo-EM), a technique that freezes proteins in place to accurately assess their structure, in UTSW’s cryo-electron microscopy facility.

Although this work has elucidated some of the fundamental mechanisms that control STING activity, exactly how this protein transitions into an active form is unclear. To answer this question, Zhang and Bai labs mixed the purified STING protein with cGAMP and used cryo-EM to image the resulting product. However, the researchers saw few activated STING molecules, and those that were present were unstable.

Hoping to increase the amount of activated STING available for imaging, scientists have added an experimental drug known as Compound 53 (C53) which is currently being tested as a STING activator for cancer therapy. C53 was thought to bind to the same site as cGAMP on STING.

The combination of cGAMP and C53 produced significantly more activated STING molecules. But when the researchers looked for C53 in the cryo-EM images, they found it in a completely different location from cGAMP, at the opposite end of the molecule.

“This newly discovered binding site for STING activation came as a complete surprise,” Dr. Bai explained. “We call it a ‘cryptic pocket’ because it appears to form in response to the presence of C53. No evidence for this site exists when C53 is absent.”

The fact that STING appears to require both cGAMP and C53 to activate strongly and stably suggests that an unknown C53-like molecule may exist in cells to fulfill the same role, Dr. Zhang said. Future research will focus on finding this molecule and better understanding its function.

One day, the researchers add, drugs that attach to or block this newly discovered binding site could be used to boost or weaken immunity to fight infectious or autoimmune diseases.

Jie Li and Yong Lu of UT Southwestern also contributed to this study.

Drs. Both Zhang and Bai are Virginia Murchison Linthicum Fellows in Medical Research. Dr. Chen is the George L. MacGregor Distinguished Chair in Biomedical Sciences and a research fellow at the Howard Hughes Medical Institute.

This work was supported in part by grants from the National Institutes of Health (R35GM130289 and R01GM143158), the Welch Foundation (I-1702 and I-1944), and the Cancer Prevention and Research Institute of Texas (RP160082)

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