Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor

Abstract

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 A adenosine receptor (AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone N-H NMR signals in AAR were individually assigned. These NMR probes provided insight into the role of Asp52 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 246, and delineated the structural response to variable efficacy of bound drugs across AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp52.

Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor

“…For W201 and W290, which are located at the intracellular ends of helices V and VII, respectively, signals that would correspond to the indole 15 N– 1 H signals observed for the antagonist complexes were not observed in complexes with agonists, indicating the presence of exchange broadening. The response of the endogenous indole 15 N– 1 H signals 6 was highly similar for A 2A AR and the variant proteins.…”

“…Protein produced from this construct exhibited nearly identical ligand-binding activity to that of native A 2A AR expressed in mammalian or insect cells 6 both in isolated Pichia membranes and in mixed detergent micelles. These data were obtained without using ligand affinity purification, 6 which can significantly reduce the final protein yield. 25 …”

“…Specifically, in 2D [ 15 N, 1 H]-TROSY correlation spectra of the human A 2A adenosine receptor (A 2A AR), a class A GPCR, the tryptophan indole 15 N– 1 H lines are well separated from the other 15 N– 1 H signals in the spectra, thus enabling their unambiguous site-specific assignment and observation of their response to variable efficacy of drugs bound to the extracellular orthosteric site. 6 …”

X-ray structures of human ETBreceptor provide mechanistic insight into receptor activation and partial activation

“…6). W6.48 is also highly conserved among the class A GPCRs (71%) 42 , and the association between W6.48 and D2.50 plays a critical role in the GPCR activation process, as shown in the previous nuclear magnetic resonance (NMR) study of the adenosine A2A receptor 43 . Given the importance of W3.36 and D2.50 in the activation of GPCRs, our proposed model of the partial receptor activation by IRL1620 is consistent with the previous functional analyses of GPCRs.…”

Structure and dynamics of dynorphin peptide and its receptor

“…Most GPCR structures were obtained from membrane proteins expressed in eukaryotic insect cells where the flexible N-and C-termini, as well as the intracellular loops (mostly ICL3), were deleted or replaced by exogenous protein domains promoting thermostability and crystallization, such as T4 lysozyme or the thermostabilized apocytochrome b562 RIL (BRIL) (Chun et al, 2012;Lv et al, 2016). Other expression systems have been used such as the methylotrophic yeast P. pastoris (Talmont, Sidobre, Demange, Milon, & Emorine, 1996), which allows stable isotope labelling, including perdeuteration (Massou et al, 1999), mostly for NMR experiments (Eddy et al, 2018). E. coli is also an interesting host for isotope-labelled GPCR biosynthesis which can be achieved by receptor expression as inclusion bodies followed by in vitro refolding during the protein purification (Baneres et al, 2003;Baneres, Popot, & Mouillac, 2011;Casiraghi et al, 2016).…”