In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the “toggle switch tryptophan”. Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

Crystal and cryo-EM structures of the glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) bound with their peptide ligands have been obtained with full-length constructs, indicating that the extracellular domain (ECD) is indispensable for specific ligand binding. This article complements these data with studies of ligand recognition of the two receptors in solution. Paramagnetic NMR relaxation enhancement measurements using dual labeling with fluorine-19 probes on the receptor and nitroxide spin labels on the peptide ligands provided new insights. The glucagon-like peptide-1 (GLP-1) was found to interact with GLP-1R by selective binding to the extracellular surface. The ligand selectivity toward the extracellular surface of the receptor was preserved in the transmembrane domain (TMD) devoid of the ECD. The dual labeling approach further provided evidence of cross-reactivity of GLP-1R and GCGR with glucagon and GLP-1, respectively, which is of interest in the context of medical treatments using combinations of the two polypeptides.

Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii . Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6 th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.

Dear Editor, GPR52 is a class-A orphan GPCR, which has been reported as a target for treating Huntington’s disease, cognitive impairment, schizophrenia, and neuromuscular disorders. Previous structure determinations showed that GPR52 adopts an active conformation when bound to G-protein in the absence of any ligand.1 A self-activation model was proposed, attributed to a built-in agonist motif embedded in the extracellular loop 2 (ECL2) near the orthosteric ligand binding pocket; this would also explain the high basal activity of GPR52. The synthetic ligand c17 is bound to a GPR52 side pocket, as shown in the co-crystal structure.1 Recently, some new molecules with a similar chemical structure to c17 have entered pre-clinical trials for GPR52-related disorders.2 Structural and conformational dynamics studies of GPCRs are crucial for the screening of new ligands and understanding the molecular mechanisms of previously untapped drug targets. Here, we show a new cryo-electron microscopy (cryo-EM) structure of GPR52 bound with c17 at the extracellular side and G-protein at the intracellular side. 19F-NMR studies3 of the complex by observation of the trifluoromethyl group in c17 (Figure 1A) revealed local polymorphisms in GPR52. 19F-NMR data also indicate localized differences in the extracellular ligand binding pocket between the GPR52–c17 complex and the ternary complex with the G-protein. To investigate the dynamics of the ligand binding pocket of GPR52, the near-wild type receptor and some variants were expressed in Sf9 insect cells (Figure S1). The –CF3 group of c17 (Figure 1A) serves here as a natural 19FNMR probe for studies of the complex with GPR52. For NMR measurements, the GPR52 complexes were reconstituted in lauryl maltose neopentyl glycol/cholesteryl hemisuccinate tris salt mixed micelles. 1D 19F-NMR spectra of the ligand c17 in the free state as well as bound to GPR52* (see Table S1 for the

Neurokinin 3 receptor (NK3R) is a tachykinin receptor essential for the hypothalamic-pituitary-gonadal axis. The endogenous peptide agonist neurokinin B (NKB) preferentially activates NK3R, while substance P (SP) binds preferentially to NK1R. In addition, the SP analogue senktide more potently activates NK3R than NKB and SP. However, the mechanisms of preferential binding of peptide and NK3R activation remain elusive. Herein, we determined the cryogenic electron microscopy (cryo-EM) structures of the NK3R-Gq complex bound to NKB, SP and senktide. The three NK3R-Gq/peptide complexes utilize a class of noncanonical receptor activation mechanisms. Combining the structural analysis and functional assay illustrated that the consensus C-termini of the three peptide agonists share a conserved binding mode to NK3R, while the divergent N-termini of the peptides confer the preferential binding of the agonist to NK3R. In addition, the specific interactions between the N-terminus of senktide and the N-terminus and extracellular loops (ECL2 and ECL3) of NK3R lead to the improved activation displayed by senktide compared to SP and NKB. These findings pave the way to understand tachykinin receptor subtype selectivity and provide ideas to rationally develop drugs targeting NK3R.

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure–function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N–1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid–water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.

G protein-coupled receptors (GPCRs) are a large membrane protein family found in higher organisms, including the human body. GPCRs mediate cellular responses to diverse extracellular stimuli and thus control key physiological functions, which makes them important targets for drug design. Signaling by GPCRs is related to the structure and dynamics of these proteins, which are modulated by extrinsic ligands as well as by intracellular binding partners such as G proteins and arrestins. Here, we review some basics of using nuclear magnetic resonance (NMR) spectroscopy in solution for the characterization of GPCR conformations and intermolecular interactions that relate to transmembrane signaling.

Comparisons of G protein-coupled receptor (GPCR) complexes with agonists and antagonists based on X-ray crystallography and cryo-electron microscopy structure determinations show differences in the width of the orthosteric ligand binding groove over the range from 0.3 to 2.9 Å. Here, we show that there are transient structure fluctuations with amplitudes up to at least 6 Å. The experiments were performed with the neurokinin 1 receptor (NK1R), a GPCR of class A that is involved in inflammation, pain, and cancer. We used 19F-NMR observation of aprepitant, which is an approved drug that targets NK1R for the treatment of chemotherapy-induced nausea and vomiting. Aprepitant includes a bis-trifluoromethyl-phenyl ring attached with a single bond to the core of the molecule; 19F-NMR revealed 180° flipping motions of this ring about this bond. In the picture emerging from the 19F-NMR data, the GPCR transmembrane helices undergo large-scale floating motions in the lipid bilayer. The functional implication is of extensive promiscuity of initial ligand binding, primarily determined by size and shape of the ligand, with subsequent selection by unique interactions between atom groups of the ligand and the GPCR within the binding groove. This second step ensures the wide range of different efficacies documented for GPCR-targeting drugs. The NK1R data also provide a rationale for the observation that diffracting GPCR crystals are obtained for complexes with only very few of the ligands from libraries of approved drugs and lead compounds that bind to the receptors.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

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]).

Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca2+ -regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca2+ -regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.80 Å resolution. The structures of obelin-v and obelin bound with native CTZ revealed almost no difference; only the minor rearrangement in hydrogen-bond pattern and slightly increased distances between key active site residues and some atoms of 2-hydroperoxycoelenterazine-v were found. The fluorescence quantum yield (ΦFL ) of obelin bound with coelenteramide-v (0.24) turned out to be even higher than that of obelin with native coelenteramide (0.19). Since both obelins are in effect the enzyme-substrate complexes containing the 2-hydroperoxy adduct of CTZ-v or CTZ, we reasonably assume the chemical reaction mechanisms and the yields of the reaction products (ΦR ) to be similar for both obelins. Based on these findings we suggest that low bioluminescence activity of obelin-v is caused by the low efficiency of generating an electronic excited state (ΦS ). In turn, the low ΦS value as compared to that of native CTZ might be the result of small changes in the substrate microenvironment in the obelin-v active site.

The dynamics of GPCRs (G protein-coupled receptors) coupling for cognate G proteins play a critical role in signal transduction. Herein, we reported a site-specifically labelled small-sized fluorescent pair 7-HC/FlAsH ((7-hydroxycoumarin-4-yl)-ethylglycine/fluorescein arsenical hairpin) for fluorescence lifetime based FRET (fluorescence resonance energy transfer) to reveal conformational differences of Gαi1 (inhibitory G proteins) and Gαs (stimulatory G proteins) upon β2AR (β2-adrenergic receptor) coupling. It offers a new generally applicable method to probe protein dynamic interactions or conformational changes.

Neurokinin 1 receptor (NK1R) has key regulating functions in the central and peripheral nervous systems, and NK1R antagonists such as aprepitant have been approved for treating chemotherapy-induced nausea and vomiting. However, the lack of data on NK1R structure and biochemistry has limited further drug development targeting this receptor. Here, we combine NMR spectroscopy and X-ray crystallography to provide dynamic and static characterisation of the binding mode of aprepitant in complexes with human NK1R variants. 19F-NMR showed a slow off-rate in the binding site, where aprepitant occupies multiple substates that exchange with frequencies in the millisecond range. The environment of the bound ligand is affected by the amino acid in position 2.50, which plays a key role in ligand binding and receptor signaling in class A GPCRs. Crystal structures now reveal how receptor signaling relates to the conformation of the conserved NP7.50xxY motif in transmembrane helix VII.

Large membrane proteins such as G protein‐coupled receptors (GPCRs) are difficult for NMR study due to severe signal overlaps and unfavorable relaxation properties. We used a trimethylsilyl (TMS) group as a reporter group for 1H NMR study of conformational changes in proteins, utilizing high‐intensity 1H NMR signals near 0 p.p.m. The β2‐adrenergic receptor was labeled with TMS groups at two cysteines located at the cytoplasmic ends of helices VI and VII. Binding of various ligands led to changes in 1H NMR signals, which manifested that helix VI is sensitive to G protein‐specific activation, whereas helix VII is sensitive to β‐arrestin‐specific activation. Thus, the TMS group is a useful reporter group in NMR for studying conformational changes in membrane proteins such as GPCRs.

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A(2A)AR, which is an alpha-helical protein, and the beta-barrel protein OmpX from E. coli.

The amino acid 4-fluoro-l-phenylalanine (4F-Phe) was introduced at the positions of Phe6 and Phe22 in the 29-residue polypeptide hormone glucagon by expressing glucagon in E. coli in the presence of an excess of 4F-Phe. Glucagon regulates blood glucose homeostasis by interaction with the glucagon receptor (GCGR), a class B GPCR. By referencing to the 4F-Phe chemical shifts at varying D2O concentrations, the solvent exposure of the two Phe sites along the glucagon sequence was determined, showing that 4F-Phe6 was fully solvent exposed and 4F-Phe22 was only partially exposed. The incorporation of fluorine atoms in polypeptide hormones paves the way for novel studies of their interactions with membrane-spanning receptors, specifically by differentiating between effects on the solvent accessibility, the line shapes, and the chemical shifts from interactions with lipids, detergents and proteins. Studies of interactions of GCGR with ligands in solution is at this point of keen interest, given that recent crystallographic studies revealed that an apparent small molecule antagonist actually binds as an allosteric effector at a distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al., in Nature 533:274–277, 2016).

Crystal structures of the human GLP-1 receptor in complex with two negative allosteric modulators reveal a common binding pocket, and, together with mutagenesis and modelling studies, further our understanding of the receptor activation mechanism.Author: Please check the wording of the following statement, which will appear online only.

Fluorine-19 NMR markers are attractive reporter groups for use in studies of complex biomacromolecular systems, in particular also for studies of function-related conformational equilibria and rate processes in membrane proteins. Advantages of 19F-NMR probes include high sensitivity of the 19F chemical shifts to variations in the non-covalent environment. Nonetheless, in studies of G protein-coupled receptors (GPCR) we encountered situations where 19F chemical shifts were not responsive to conformational changes that had been implicated by other methods. This prompted us to examine possible effects of aromatic ring current fields on the chemical shifts of 19F-NMR probes used in GPCRs. Analysis of previously reported 19F-NMR data on the β2-adrenergic receptor and mammalian rhodopsin showed that all 19F-labeling sites which manifested conformational changes are located near aromatic residues. Although ring current effects are small when compared to other known non-covalent effects on 19F chemical shifts, there is thus an indication that their contributions are significant when studying activation processes in GPCRs, since the observed activation-related 19F-NMR chemical shifts are comparable in size to the calculated ring current shifts. Considering the impact of ring current shifts may thus be helpful in identifying promising indigenous or engineered labeling sites for future 19F-NMR studies of GPCR activation, and novel information may be obtained on the nature of conformational rearrangements near the 19F-labels. It will then also be interesting to see if the presently indicated role of ring current shifts in membrane protein studies with 19F-NMR markers can be substantiated by a more extensive data base resulting from future studies.