Proteins shaping up
The premier scientific journal Nature recently published two studies describing the first structures solved in UT Southwestern Medical Center’s new $22.5 million cryo-electron microscopy facility.
Drs. Youxing Jiang and Xiaochen Bai are corresponding authors on both studies, which were among several conducted during the facility’s first year of operation. The most recent study, published in December, described the structure of the TRPM4 cation channel – providing the scientific community’s first glimpse into the molecular architecture of the TRPM family. Dr. Jiang is a Professor of Physiology and Biophysics, and Dr. Bai is an Assistant Professor of Biophysics and Cell Biology.
Biophysics Chair Dr. Michael Rosen says, “It’s just amazing what they have accomplished in the last year. It shows you what can happen when you combine very deep expertise in structural biology and general biochemistry – making proteins – with somebody who has equally deep expertise in cryo-EM.”
“Xiaochen Bai is our resident expert for single-particle cryo-EM.”
The melastatin-related transient receptor potential (TRPM) channels comprise the largest subfamily of TRP channels and have diverse functions in various physiological processes, including temperature sensing. The researchers determined the structures of the mouse TRPM4 channel in two conformations – unbound from and bound to the universal energy provider ATP – to a resolution of 3.1 and 2.9 Å, respectively, using single-particle cryo-EM. Lead authors were former and current postdoctoral researchers Dr. Jiangtao Guo and Dr. Ji She.
“These structures represent the first among TRPM family members and reveal a unique three-tiered architecture,” Dr. Jiang says.
The TRPML1 ion channel viewed from different angles. Images courtesy of Y. Jiang and X. Bai Labs, UT Southwestern
“Along with electrophysiological analysis, we defined the location and physiological role of ATP binding in TRPM4 and revealed the structural basis of the distinct selectivity property of TRPM4. These structures pave the way for our future studies to understand the complex regulation of the TRPM4 channel,” he adds.
As a member of the TRPM family, TRPM4 functions as a calcium-activated, phosphatidylinositol bisphosphate (PIP2) modulated, nonselective cation channel. TRPM4 is ubiquitously expressed in the pancreas, heart, prostate, renal tubule, and many other tissues and organs, Dr. Jiang says. The C-terminal domain’s structure revealed a coiled-coil helix and a long preceding stretcher helix, named for its architectural resemblance to the stretchers of an umbrella, the researchers said.
Only two months before the TRPM4 study – in October 2017 – the pair published a 3-D atomic structure of the TRPML1 (transient receptor potential mucolipin 1) ion channel found in mammals and implicated in a rare, inherited human neurodegenerative disease called mucolipidosis type IV, directly caused by loss of function mutations in genes governing the TRPML1 channels.
The disease – one of about 50 lysosomal storage diseases (LSDs) identified in humans – is marked by delayed development of mental and motor skills and vision impairment, according to the National Institutes of Health.
The TRPML1 channel, which regulates the flow of calcium ions, is found in every mammal. The channel sits in the membrane of organelles inside cells called lysosomes, which contain enzymes that aid in cellular recycling by breaking down large molecules. Proteins embedded in the membrane, and therefore resistant to the crystallization required for X-ray crystallography, are some of the previously elusive structures now attainable with cryo-EM.
“Due to its link to that class of lysosomal storage diseases, TRPML1 has been a potential target for small-molecule therapeutics, and several potential agonists [channel openers] have been developed,” says Dr. Jiang, a W.W. Caruth, Jr. Scholar in Biomedical Research and a Howard Hughes Medical Institute (HHMI) Investigator.
A distinction shared by both studies is the successful use of a relatively new sample preparation technique that embeds the protein of interest in a nanodisc structure made from lipids and other biological materials. The sample was created by lead author Dr. Qingfeng Chen, a postdoctoral researcher in the Jiang laboratory.
“For a long time, detergent has been used to extract proteins from membranes for study. People have suggested that detergent might change the protein structure from its native state,” says Dr. Bai, a Virginia Murchison Linthicum Scholar in Research. “Membrane proteins, such as those we studied, are usually wrapped in lipids. Nanodiscs are used to provide a native environment for the protein sample.”
“Xiaochen Bai is our resident expert for single-particle cryo-EM. He came to UT Southwestern from the MRC Laboratory of Molecular Biology in Cambridge, U.K., where his next-door laboratory director was Dr. Richard Henderson, who shared the Nobel Prize in December for developing key technologies that resulted in the ‘resolution revolution’ in cryo-electron microscopy technology,” says Dr. Daniela Nicastro, Director of the cryo-EM facility and an Associate Professor of Cell Biology and Biophysics. Dr. Nicastro and Dr. Bai are also Cancer Prevention and Research Institute of Texas (CPRIT) Scholars.
“Xiaochen benefits from collaborating with other structural biologists at UT Southwestern, like Dr. Youxing Jiang, who is an expert X-ray crystallographer. In that field, you need outstanding biochemists to purify large amounts of clean protein samples,” she explains.
Dr. Rosen agrees.
“Xiaochen was able to walk in and work with a number of different groups and bam, bam, bam, everybody’s getting structures done. I think we’re just going to be rolling out tons of structures over the next few years,” says Dr. Rosen, an HHMI Investigator, Professor in both Biophysics and the Cecil H. and Ida Green Comprehensive Center for Molecular, Computational, and Systems Biology, and holder of Mar Nell and F. Andrew Bell Distinguished Chair in Biochemistry.