DALLAS - April 15, 2020 - Researchers at UT Southwestern Medical Center have uncovered the detailed shape of a key protein involved in muscle contraction. The report, published today in Neuron, may lead to improved understanding of muscle-weakening genetic conditions called congenital myasthenic syndromes (CMS).
The protein sits on the surface of nerve cells that connect to muscles and is integral to triggering the muscle cell to contract just milliseconds after instructions are sent through the spinal cord. Called a nicotinic receptor, it has been a challenge to study because it sits in the cell's membrane.
"The nicotinic receptor at the neuromuscular junction has been a target of interest for over a century. It was the first ion channel to be purified, the first to have its genes cloned, and the first to be imaged by an electron microscope," says Ryan Hibbs, Ph.D., associate professor of Neuroscience and Biophysics at UTSW and a corresponding author of the study.
Many groups had tried to determine the receptor's structure using an earlier technology called X-ray crystallography as well as first generation cryogenic electron microscopes (cryo-EM) but they were only able to obtain low-resolution images, he adds.
Normally the nicotinic receptor is activated by a molecule called acetylcholine. However, the nicotinic receptor is also the target of various venoms that cause muscle paralysis.
So the team used this to their advantage to isolate enough of the receptor protein to study its shape and structure. They mixed the toxin from snake venom with fish tissue known to contain high amounts of the receptor protein.
The team then flash froze the receptor bound to the toxin and used the rapidly evolving technique of cryo-electron microscopy to uncover the shape of the structure. Before recent developments in cryo-EM, the only method by which to solve protein structures like the nicotinic receptor was X-ray crystallography, which involved slowly growing crystals of proteins. But proteins that sit in membranes usually do not crystallize well.
"This never could have been done using X-ray crystallography - hundreds of researchers had attempted it. The new microscopes allowed us to get to a very high resolution near the atomic level. At that resolution, we can precisely make out the positions of most of more than 2,000 amino acids that make up the receptor protein," Hibbs says. "The results were stunning. They revealed, at a very fine level of detail, the 3D architecture of the receptor, with two toxin molecules bound to it exactly where we know the much smaller acetylcholine binds."
The results showed how the toxin blocks acetylcholine by competing for the same binding site, paralyzing the receptor in the closed configuration and preventing the flow of electrochemical messages. The structure also reveals why the toxin binds so tightly and selectively to the receptor and how the venom paralyzes prey, including humans, who are unfortunate enough to get bitten by the snake.
"We learned a tremendous amount from the new 3D structure beyond how the toxin works to poison the receptor," Hibbs says. Lead author Md. Mahfuzur Rahman, Ph.D., a postdoctoral researcher in the Hibbs lab, adds that the nicotinic acetylcholine neuromuscular receptor is a therapeutic target for several genetic diseases that cause muscle weakness, which the team also investigated.
"Here we mapped the mutations related to CMS to three principal regions on the receptor structure. This new structural information sheds light on how mutations in those areas result in muscle-weakening syndromes," Rahman says.
UTSW co-authors include Jinfeng Teng and Colleen Noviello. Co-authors from the University of Colorado, Boulder, include co-corresponding author Michael H.B. Stowell, Brady Worrell, and Myeongseon Lee. Arthur Karlin of Columbia University also participated.
The research received support from the Cancer Prevention and Research Institute of Texas (grant No. RP170644), the National Institutes of Health (grant Nos. GM12957, DA037492, DA42072, NS095899, AG061829) and the MCDB Neurodegenerative Disease Fund. The authors declare no competing interests.
Hibbs is an Effie Marie Cain Scholar in Medical Research.
About UT Southwestern Medical Center
UT Southwestern, one of the premier academic medical centers in the nation, integrates pioneering biomedical research with exceptional clinical care and education. The institution's faculty has received six Nobel Prizes, and includes 22 members of the National Academy of Sciences, 17 members of the National Academy of Medicine, and 14 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 2,500 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UT Southwestern physicians provide care in about 80 specialties to more than 105,000 hospitalized patients, nearly 370,000 emergency room cases, and oversee approximately 3 million outpatient visits a year.