A member of the G-coupled-protein-receptor (GPCR) family has been visualized like no GPCR has ever been imaged before. Previous attempts at visualizing GPCRs have tended to neglect GPCR dynamics, settling for static before-and-after views (as in before and after activation), or broad impressions derived from observations of the most flexible parts of GPCRs. A new study, however, has managed to capture motions that occur at the heart of a GPCR—that is, motions that occur in a GPCR’s intracellular regions in response to extracellular stimuli.

The new structural insights, which may help drug designers improve the targeting of GPCRs, come from a team of scientists based at The Scripps Research Institute (TSRI). These scientists used nuclear magnetic resonance (NMR) spectroscopy to capture many different conformations of a GPCR protein called the A2A adenosine receptor (A2AAR).

A2AAR, which regulates blood flow and inflammation and mediates the effects of caffeine, is just one of more than 800 GPCRs that have been discovered in the human body. Nonetheless, the TSRI team, which was led by Nobel laureate Kurt Wüthrich, Ph.D., emphasizes that its A2AAR findings may be relevant for many other GPCRs. In fact, structural details from this study could apply to more than 600 “class A” GPCRs in our bodies.

Details of the TSRI team’s work appeared December 28 in the journal Cell, in an article entitled “Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor.” The article describes how TSRI scientists used NMR spectroscopy to observe how wild-type-like A2AAR behaves in solution. This work allowed the scientists to characterize A2AAR’s signaling-related structural dynamics.

“All six tryptophan indole and eight glycine backbone 15N–1H NMR signals in A2AAR were individually assigned,” wrote the article’s authors. “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 research built on previous studies where scientists used an imaging technique called 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 used NMR spectroscopy, which creates strong magnetic fields to locate the positions of probes in a sample. The NMR work, which was spearheaded by TSRI's Matthew Eddy, Ph.D., first author of the new study, captured A2AAR in many different conformations, shedding light on how it changes shape on the surface of human cells in response to drug treatments.

“This basic knowledge is potentially helpful for improving drug design,” said Dr. Wüthrich, the senior author of the current study and winner of the Nobel Prize in Chemistry in 2002 for pioneering work in NMR to study the structures of biological molecules.

Importantly, NMR let the TSRI team visualize changes in the internal architecture of A2AAR. This took them beyond previous solution NMR studies, which focused on the technically less demanding observation of NMR-observable probes attached to flexible parts of GPCRs, mostly located at or near the surface of the receptor. The approach in the new study 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,” noted 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.

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