Design and preparation of the class B G protein‐coupled receptors GLP‐1R and GCGR for ¹⁹F‐NMR studies in solution

Abstract

The human glucagon‐like peptide‐1 receptor (GLP‐1R) and the glucagon receptor (GCGR) are class B G protein‐coupled receptors (GPCRs) that are activated by interactions with, respectively, the glucagon‐like peptide‐1 (GLP‐1) and glucagon (GCG). These polypeptide hormones are involved in the regulation of lipid and cholic acid metabolism, and thus play an important role in the pathogenesis of glucose metabolism and diabetes mellitus, which attracts keen interest of these GPCRs as drug targets. GLP‐1R and GCGR have therefore been extensively investigated by X‐ray crystallography and cryo‐electron microscopy (cryo‐EM), so that their structures are well known. Here, we present the groundwork for using nuclear magnetic resonance (NMR) spectroscopy in solution to complement the molecular architectures with information on intramolecular dynamics and on the thermodynamics and kinetics of interactions with physiological ligands and extrinsic drug candidates. This includes the generation of novel, near‐wild‐type constructs of GLP‐1R and GCGR, optimization of the solution conditions for NMR studies in detergent micelles and in nanodiscs, post‐translational chemical introduction of fluorine‐19 NMR probes, and sequence‐specific assignments of the 19F‐labels attached to indigenous cysteines. Addition of the negative allosteric modulator (NAM) NNC0640 was critically important for obtaining the long‐time stability needed for our NMR experiments, and we report on novel insights into the allosteric effects arising from binding of NNC0640 to the transmembrane domain of GLP‐1R (GLP‐1R[TMD]).

Through-Space Scalar 19F–19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins

“…Site-Specific Incorporation of CF 3 -Tyr and CF 3 -Phe. The amino acids CF 3 -Tyr and CF 3 -Phe were incorporated into the protein PpiB by replacing Phe27 and Phe98 with the respective noncanonical amino acids, producing the samples PpiB CF 3 -Tyr (27,98) and PpiB CF 3 -Phe (27,98), respectively. As Phe4 is close to both residues in position 27 and 98 37 and to provide more space for the CF 3 groups, two more samples were prepared that contained the mutation Phe4Ala in addition to the CF 3 residues in positions 27 and 98.…”

“…As Phe4 is close to both residues in position 27 and 98 37 and to provide more space for the CF 3 groups, two more samples were prepared that contained the mutation Phe4Ala in addition to the CF 3 residues in positions 27 and 98. In the following, we refer to these samples as PpiB F4A/CF 3 -Tyr (27,98) and PpiB F4A/CF 3 -Phe (27,98), respectively. Finally, a version of PpiB was produced with three CF 3 -Tyr residues, yielding PpiB CF 3 -Tyr (4,27,98).…”

“…In the following, we refer to these samples as PpiB F4A/CF 3 -Tyr (27,98) and PpiB F4A/CF 3 -Phe (27,98), respectively. Finally, a version of PpiB was produced with three CF 3 -Tyr residues, yielding PpiB CF 3 -Tyr (4,27,98). The mutant proteins were produced with the help of a previously published aminoacyl-tRNA synthetase, tfm-Phe2.…”

“…To a large extent, this must be attributed to the large chemical shift anisotropy associated with 19 F spins which, at high magnetic field strength, causes substantial line broadening in high-molecular weight systems such as proteins. Although the CSA relaxation can be alleviated by positioning the fluorine spins in highly flexible chemical groups, such as a CF 3 group at the end of a flexible tether, and this approach has been very successful in monitoring different conformations of integral membrane proteins induced by different ligands, − it does not lend itself to the observation of through-space scalar 19 F– 19 F couplings.…”

Small, but powerful and attractive: 19F in biomolecular NMR

“…In most cases, 19 F labels are conjugated to naturally present cysteines near the cytoplasmic face of the receptor (Frei et al, 2020) or on strategically introduced Cys residues at positions sensitive to signal transduction. Using 19 F NMR, it has been possible to identify inactive intermediates and partial-or full-agonist-driven activated states for the adenosine A2A receptor (Huang et al, 2021;Wang et al, 2021a). Such studies inform about immensely important physiological processes, and, given that large numbers of drugs are being designed for pharmacological interventions in GPCR-mediated signaling, fluorine NMR in this context has enormous potential.…”