GPCR structural characterization by NMR spectroscopy in solution

Abstract

In the human proteome, 826 G-protein-coupled receptors (GPCRs) interact with extracellular stimuli to initiate cascades of intracellular signaling. Determining conformational dynamics and intermolecular interactions are key to understand GPCR function as a basis for drug design. X-ray crystallography and cryo-electron microscopy (cryo-EM) contribute molecular architectures of GPCRs and GPCR-signaling complexes. NMR spectroscopy is complementary by providing information on the dynamics of GPCR structures at physiological temperature. In this review, several NMR approaches in use to probe GPCR dynamics and intermolecular interactions are discussed. The topics include uniform stable-isotope labeling, amino acid residue-selective stable-isotope labeling, site-specific labeling by genetic engineering, the introduction of ¹⁹F-NMR probes, and the use of paramagnetic nitroxide spin labels. The unique information provided by NMR spectroscopy contributes to our understanding of GPCR biology and thus adds to the foundations for rational drug design.

Keywords: G protein-coupled receptor dynamics; GPCR biology; drug development; fluorine-19 NMR; stable-isotope labeling.

Figure 1

GPCR molecular structure and strategies for NMR characterization (A) Schematic view of a GPCR molecular structure and its interactions with orthosteric and allosteric small molecule ligands (potentially druggable binding sites) and intracellular partner proteins. The black triangle, pentagon and hexagon represent different orthosteric ligands, which all target the orthosteric binding site on the extracellular surface. The purple polygon represents allosteric ligands, which may target a variety of binding sites in GPCR structures. Three NMR spectroscopy approaches to study conformational dynamics and intermolecular interactions of GPCRs are indicated on the right, i.e., uniform stable-isotope labeling (blue, B–D), residue-selective stable-isotope labeling (red, E–F) and 19F-NMR probes (green, G–J). In the GPCR structure, thin blue lines represent extracellular and intracellular loops, thick blue lines are the TMs, red spots and a green circle indicate sites for selective labelling with 13C or 15N, and with a 19F-label, respectively. (B–D) 2D [15N, 1H]-transverse relaxation optimized spectroscopy (TROSY) of A2AAR in complex with the antagonist ZM241385. (E) 2D [15N, 1H]-TROSY spectrum of [2,3,3-2H, 15N]-leu-labeled β2AR. (F) 2D [13C, 1H]-heteronuclear multiple quantum correlation (HMQC) spectrum of ε-N[13CH3]2-lysines in the μ-opioid receptor (μOR). (G–I) 1D 19F-NMR spectra of A2AAR[A289CTET] bound to an inverse agonist, in the apo-form, and bound to an agonist. Spectral components contained in the observed signal envelope are shown on the right of this panel. (J) 2D [19F, 19F]-EXSY spectrum of the A2AAR–agonist complex in I, recorded at 280 K with a mixing time of 100 ms. B to D are adapted from Figure1 in reference [20]. E is adapted from Figure1 in reference [23]. F is adapted from Figure1 in reference [28]. G to J are adapted from Figure1 in reference [29].

Figure 2

Chemical structures of ¹⁹F-NMR probes used for studies of GPCRs (A) 2,2,2-trifluoroethanethiol (TET). (B) 2-bromo-N-(4-(trifluoromethyl) phenyl) acetamide (BTFMA). (C) 3-bromo-1,1,1-trifluoroacetone (BTFA). (D) 3-trifluoromethyl-L-phenylalanine (mtfF).

Figure 3

Schematic view of cannabinoid receptor 1 (CB1) with ¹⁹F-NMR probe and its ¹⁹F-NMR spectra (A) Schematic view of CB1 with a genetically engineered 19F-NMR probe, 3-(trifluoromethyl) phenylalanine (mtfF) at the sequence position 3376.29. The brown sphere represents the position of the mtfF. (B,D) 1D 19F-NMR spectra of CB1 [M3376.29mtfF] in complexes with the antagonist AM6538 and the agonist 2-AG, respectively. (C,E) Spectra of B,D, respectively, with indication of the component signals (blue and red) identified by Lorentzian deconvolution. The spectra B to E were previously included in Figure2 of reference [47].