Researchers have used new techniques and one of the brightest X-ray sources on the planet to map the 3-D structure of an important cellular gatekeeper in a more natural state than possible before.
The new approach, published recently in the journal Science, is a major advance in exploring G protein-coupled receptors (GPCRs)—a vast, hard-to-study family of proteins that play a key role in human health. GPCRs are targeted by an estimated 40 percent of modern medicines.
“For the first time we have a room-temperature, high-resolution structure of one of the most difficult-to-study but medically important families of membrane proteins,” said Vadim Cherezov, a structural biologist at The Scripps Research Institute (TSRI) who led the research. “And we have validated this new method so that it can be confidently used for solving new structures.”
In the study, the scientists examined the human serotonin receptor, which plays a role in learning, mood and sleep and is the target of drugs that combat obesity, depression and migraines. The scientists prepared crystallized samples of the receptor in a fatty gel that mimics its environment in the cell.
Working at the Linac Coherent Light Source (LCLS) X-ray laser at the Department of Energy’s (DOE’s) SLAC National Accelerator Laboratory, the scientists then used a newly designed injection system, engineered by a team from Arizona State University, to stream the gel into the path of the X-ray pulses, which hit the crystals and produced patterns used to reconstruct a high-resolution, 3-D model of the receptor.
The method eliminates one of the biggest hurdles in the study of GPCRs. It is notoriously difficult to grow sufficiently large crystals of these proteins needed for conventional X-ray studies at synchrotrons. Because LCLS is a billion times brighter than synchrotrons and produces ultrafast snapshots, it enables researchers to use tiny crystals and collect data in the instant before any damage sets in.
“This is one of the niches that LCLS is perfect for,” said SLAC Staff Scientist Sébastien Boutet, a co-author of the report. “With really challenging proteins like this you often need years to develop crystals that are large enough to study at synchrotron X-ray facilities.”
Wei Liu, a TSRI staff scientist who was first author of the study, said, “It’s a big advantage that you don’t have to harvest individual crystals—you can just load the whole gel-like sample with embedded microcrystals in the injector and start collecting data. It’s also significant that the crystals don’t have to be cryo-cooled in liquid nitrogen to protect them from radiation damage. Instead of looking at the samples at minus 173 degrees Celsius, we can look at them at room temperature—much closer to the temperature of their natural environment, which is body temperature.”
While a team led by Scripps Research Institute scientists had previously determined the human serotonin structure with conventional methods, that effort required the receptor to be frozen. It also took much longer.
Even after samples of a GPCR are crystallized and imaged, with conventional methods it can take several months to optimize the crystal size and collect enough synchrotron X-ray data to produce structural information, Cherezov noted. This new method can potentially condense that timeline to a matter of days.
‘Just the Beginning’
Disorders linked to GPCRs include hypertension, asthma, schizophrenia and Parkinson's disease. Because of their vital role in regulating cells' signaling and response mechanisms and their importance to human health, advances in receptor-related research garnered the 2012 Nobel Prize in Chemistry.
So far, scientists have been able to map the structures of fewer than two dozen of the estimated 800 GPCRs in humans. The more accurate the structure, the better scientists can use it to create effective drug treatments without side effects.
“I view these recent experiments as just the beginning,” Cherezov said. “Now it is time to start making a serious impact on the field of structural biology of G protein-coupled receptors and other challenging membrane proteins and complexes. The pace of structural studies in this field is breathtaking, and there is still a lot unknown.”
In addition to Cherezov, Liu and Boutet, the study, “Serial Femtosecond Crystallography of G Protein–Coupled Receptors,” was authored by Daniel Wacker, Gye Won Han, Vsevolod Katritch, Chong Wang and Raymond C. Stevens of TSRI; Cornelius Gati, Anton Barty, Kenneth R. Beyerlein and Thomas A. White of Deutsches Elektronen-Synchrotron; Daniel James, Dingjie Wang, Garrett Nelson, Uwe Weierstall, Nadia A. Zatsepin, Shibom Basu, Raimund Fromme, Christopher Kupitz, Kimberley N. Rendek, Ingo Grotjohann, Petra Fromme and John C. H. Spence of Arizona State University; Dianfan Li, Syed T. A. Shah and Martin Caffrey of Trinity College, Dublin; Marc Messerschmidt, Garth J. Williams and Jason E. Koglin of SLAC; M. Marvin Seibert of SLAC and Uppsala University; Richard A. Kirian of Deutsches Elektronen-Synchrotron and Arizona State University; and Henry N. Chapman of Deutsches Elektronen-Synchrotron, University of Hamburg and Center for Ultrafast Imaging. See http://www.sciencemag.org/content/342/6165/1521.full
The research was supported by the National Institutes of Health Common Fund in Structural Biology (grants P50 GM073197, P50 GM073210, R01 GM095583), the National Institute of General Medical Sciences (PSI:Biology grants U54 GM094618, U54 GM094599) and the National Science Foundation (award 1231306), with additional support from the Helmholz Association, the German Federal Ministry of Education and Research, and Science Foundation Ireland (07/IN.1/B1836, 12/IA/1255).
The research team for the new study included (left to right): Daniel Wacker (TSRI), Uwe Weierstall (Arizona State University), Dianfan Li (Trinity College Dublin), Vadim Cherezov (TSRI), Ingo Grotjohann (ASU), Petra Fromme (ASU), Chris Kupitz (ASU), Wei Liu (TSRI), Shibom Basu (ASU), Nadia Zatsepin (ASU), Raimund Fromme (ASU), Cornelius Gati (Center for Free-Electron Laser Science), Martin Caffrey (TCD), and Daniel James (ASU). (Photo courtesy Vadim Cherezov, TSRI)
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