Abstract
GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.
Highlights
Our understanding of the molecular underpinnings of GPCR function has been greatly advanced over the past two decades through a combination of X-ray crystal structures, computational simulations, and spectroscopic studies of protein dynamics
For GPCRs, changes in sidechain dynamics may play an energetic role in binding to ligands and G proteins, we have focused on allosteric mechanisms connecting these two functionally important binding sites
The spectra we obtained for the labeled Ile d1 methyl groups of perdeuterated A2AR are comparable or superior to previously published NMR spectra on other wild-type GPCRs, the limited dispersion and signal-to-noise contributed to significant error in the relaxation values derived from the data (Figure 6A)
Summary
Our understanding of the molecular underpinnings of GPCR function has been greatly advanced over the past two decades through a combination of X-ray crystal structures, computational simulations, and spectroscopic studies of protein dynamics. Biochemistry Biophysics and Structural Biology eLife digest Almost every aspect of the human body – from our senses to our moods – depends, in one way or another, on a large family of proteins called G-protein-coupled receptors. These receptor proteins, known as GPCRs for short, detect signals from outside the cell and trigger activity within the cell. Since GPCRs regulate many processes in the body that are involved in disease, it is perhaps unsurprising that over a third of all approved drugs target these receptors
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