A team led by structural biologists at The Scripps Research Institute (TSRI) has taken a big step toward understanding the intricate molecular mechanism of a metabolic enzyme produced in most forms of life on Earth.
The finding, published in the January 9 issue of Science, concerns nicotinamide nucleotide transhydrogenase (TH), an ancient evolutionary enzyme found throughout the animal kingdom as well as in plants and many simpler species. The enzyme is part of a process key to maintaining healthy cells and has also recently been linked to diseases such as diabetes and cancer.
“Despite its importance, TH has been one of the least-studied of mitochondrial enzymes,” said TSRI Associate Professor C. David Stout. “Our new study helps clear up some mysteries—suggesting how the enzyme structure might harness protons and indicating that its two sides are able to alternate functions, always staying in balance.”
Powering the Cell
In humans and other higher organisms, TH enzymes work within mitochondria, the tiny, double-hulled oxygen reactors that help power most cellular processes.
As a mitochondrion burns oxygen, it pumps protons (hydrogen atoms denuded of their electrons) out of its inner compartment (“matrix”), creating an excess of these charged particles just outside its inner membrane. TH enzymes, which are fixed at one end within this membrane, allow a one-by-one flow of protons back through the membrane within the matrix. This process—which is similar to that which makes ATP, the cell’s universal source of energy—has also been linked to the production of a compound called NADPH, which is crucial for defusing oxygen free radicals to maintain cell health.
Stout’s laboratory and others have previously described portions of the TH enzyme that protrude from the membrane into the mitochondrial matrix. But a precise understanding of TH’s mechanism has been elusive. In its entirety, the enzyme has an exceptionally loose structure that makes it hard to evaluate using X-ray crystallography, the standard tool for determining the structures of large proteins at atomic-level resolution.
“Key details we’ve been lacking include the structure of TH’s transmembrane portion, and the way in which the parts assemble into the whole enzyme,” said Josephine H. Leung, a graduate student in the Stout laboratory who was lead author of the study.
New Clues to a Dynamic Structure
In the new study, thanks to technology developed by Professor Vadim Cherezov, now of University of Southern California, Leung and her colleagues were able for the first time to form crystals (neatly lined-up groupings) of the TH transmembrane portion and use X-ray crystallography to determine its structure—to an atomic-level resolution of 2.8 angstroms (280 trillionths of a meter).
The team also was able to grow crystals of the whole TH enzyme. These yielded a much lower-resolution structural image, but the researchers were able to enhance the resolution to 6.9 angstroms by plugging in data from crystallography of individual TH portions. In a further study, Professor Bridget Carragher and colleagues at the TSRI-based National Resource for Automated Molecular Microscopy (NRAMM) imaged individual copies of the enzyme to 18 angstroms using electron microscopy. Stout emphasized that such seamless collaborations at TSRI made this work possible: “Only an environment as at Scripps would enable the study of transhydrogenase.”
The electron microscopy data confirmed that TH naturally exists as a “dimer”—two identical copies bound together—and provided major clues to how TH manages to work in this conformation.
Directly above TH’s transmembrane structure, just inside the mitochondrial matrix, is the “domain III” structure that binds NADPH’s precursor molecule, NADP+, during conversion to NADPH. Structural biologists haven’t understood how two such structures could work side by side in the TH dimer and not interfere with each other’s activity. The new structural data suggest that these side-by-side structures are highly flexible and always have different orientations.
“Our most striking finding was that the two domain III structures are not symmetric—one of them faces up while the other faces down,” said Leung.
In particular, one of structures is oriented apparently to catalyze the production of NADPH, while the other is turned towards the membrane, perhaps to facilitate transit of a proton. The new structural model suggests that with each proton transit, the two domain III structures flip and switch their functions. “We suspect that the passage of the proton is what somehow causes this flipping of the domain III structures,” said Leung.
But much work remains to be done to determine TH’s precise structure and mechanism. For example, the new structural data provide evidence of a likely proton channel in the TH transmembrane region, but show only a closed conformation of that structure. “We suspect that this channel can have another, open conformation that lets the proton pass through, so that’s one of the details we want to study further,” said Leung.
“There are many experiments to follow,” Stout said.
Other co-authors of the study, “Division of labor in transhydrogenase by alternating proton translocation and hydride transfer,” were Robert B. Gennis, professor of biochemistry and biophysics at the University of Illinois at Urbana-Champaign, and a research associate in his laboratory, Lici A. Schurig-Briccio, who produced whole TH proteins for analysis and characterized the activity of TH when mutated at key structural sites; Jeffrey A. Speir of NRAAM; former NRAAM member Arne Moeller, now at Aarhus University; and Mutsuo Yamaguchi, staff scientist in the Stout laboratory at TSRI.
Support for the study was provided by the National Institutes of Health (5R01GM061545) and by the National Institute of General Medical Sciences (1R01GM103838, GM095600, GM073197 and P41GM103310).
About the Scripps Research Institute
The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists—including three Nobel laureates—work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see www.scripps.edu
Madeline McCurry Schmidt | newswise
Nanoparticle Exposure Can Awaken Dormant Viruses in the Lungs
16.01.2017 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt
Cholera bacteria infect more effectively with a simple twist of shape
13.01.2017 | Princeton University
Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.
As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...
At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).
Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...
Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.
The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...
UMD, NOAA collaboration demonstrates suitability of in-orbit datasets for weather satellite calibration
"Traffic and weather, together on the hour!" blasts your local radio station, while your smartphone knows the weather halfway across the world. A network of...
Fiber-reinforced plastics (FRP) are frequently used in the aeronautic and automobile industry. However, the repair of workpieces made of these composite materials is often less profitable than exchanging the part. In order to increase the lifetime of FRP parts and to make them more eco-efficient, the Laser Zentrum Hannover e.V. (LZH) and the Apodius GmbH want to combine a new measuring device for fiber layer orientation with an innovative laser-based repair process.
Defects in FRP pieces may be production or operation-related. Whether or not repair is cost-effective depends on the geometry of the defective area, the tools...
10.01.2017 | Event News
09.01.2017 | Event News
05.01.2017 | Event News
16.01.2017 | Trade Fair News
16.01.2017 | Architecture and Construction
13.01.2017 | Life Sciences