A Scripps Research Institute (TSRI) team reports that it has found structural features in a key protein used in drug design that may lead to new ways to target diseases. The A2a adenosine receptor (A2aAR) belongs to the G-protein-coupled receptor (GPCR) family, which is the target of roughly 40% of all approved pharmaceuticals. 

The new, more detailed image of A2aAR's signaling mechanism reveals critical parts of its inner system, including an amino acid that acts like a “toggle switch” to control signaling across the cell membrane, according to Nobel laureate Kurt Wüthrich, Ph.D., the Cecil H. and Ida M. Green Professor of Structural Biology at TSRI and senior author of the study (“Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2a Adenosine Receptor”), which appears in Cell.

“Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2a adenosine receptor (A2aAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N–1H NMR signals in A2aAR were individually assigned,” write the investigators.

“These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2aAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.”

“This basic knowledge is potentially helpful for improving drug design,” says Dr. Wüthrich.

A2aAR, which regulates blood flow and inflammation and mediates the effects of caffeine, is also a validated target for treating Parkinson's disease and a relatively new target for targeting cancers. “GPCRs do just about everything you can imagine,” notes Dr. Wüthrich. “But for a long time, drug design was being done without knowing how GPCRs looked.” 

For the new study, the researchers aimed to better understand the relationship between A2aAR function and dynamic changes in its structure to help inform drug design. 

The research built on previous studies where scientists used  X-ray crystallography to determine A2aAR's three-dimensional structure. The images showed that A2aAR looks like a chain that crisscrosses the cell membrane and has an opening on the side facing out of the cell. The region of the GPCR structure that sticks out of the membrane interacts with drugs and other molecules to signal to partner proteins inside the cell. 

Although crystal structures provided a key outline of the receptor's shape in inactive and active-like states, they could not show motion and changes in structure when A2aAR meets new binding partners, such as pharmaceutical candidates. In short, the researchers in the new study needed to investigate why A2aAR works the way it does.

To solve this problem, the researchers relied on nuclear magnetic resonance (NMR), which creates strong magnetic fields to locate the positions of probes in a sample. Dr. Wüthrich won the Nobel Prize in Chemistry in 2002 for pioneering work in NMR to study the structures of biological molecules. 

The Scripps researchers used NMR to observe A2aAR in many different conformations, shedding light on how it changes shape on the surface of human cells in response to drug treatments, and to visualize changes in the internal architecture of A2aAR. The approach enabled researchers to follow the effects of drug binding at the extracellular surface on changes in protein structure and dynamics at the intracellular surface—the structural basis of signal transfer—across the heart of the GPCR.

Two details in A2aAR's structure gave researchers insight into how future drugs could manipulate the receptor. One key finding was that replacing one particular amino acid in the receptor's center destroyed the receptor's ability to send signals into the cell.

“With this finding, we can say 'A-ha! It is this change in structure that kills the signaling activity.” Maybe we can make a change in a drug to overcome this limit,” says Dr. Wüthrich. 

The researchers also revealed the activity of a toggle switch in A2aAR. Previous studies suggested that one of the tryptophan amino acids in A2aAR flips up and down in concert with A2aAR's activity. With NMR, the scientists directly observed this unique tryptophan as it changed orientations in response to different drugs. Chemists could potentially modify drugs to manipulate this switch and control A2aAR signaling.

The researchers emphasize that this new study is potentially relevant for much of the large family of GPCRs. In fact, structural details from this study could apply to more than 600 “class A” GPCRs in our bodies, notes Dr. Wüthrich.

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