Differential pathogenetic mechanisms of mutations in helix 2 and helix 6 of rhodopsin.

Variants in rhodopsin (RHO) have been linked to autosomal dominant congenital stationary night blindness (adCSNB), which affects the ability to see in dim light, and the pathogenetic mechanism is still not well understood. In this study we report two novel RHO variants found in adCSNB families, p.W265R and p.A269V, that map in the sixth transmembrane domain of RHO protein. We applied in silico molecular simulation and in vitro biochemical and molecular studies to characterize the two new variants and compare the molecular determinants to two previously characterized adCSNB variants, p.G90D and p.T94I, that map in the second transmembrane domain of the RHO protein. We demonstrate that W265R and A269V cause constitutive activation of RHO with light-independent G protein coupling and impaired binding to arrestin. Differently, G90D and T94I are characterized by slow kinetics of RHO activation and deactivation. This study provides new evidence on the differential contribution of transmembrane α-helixes two and six to the interaction with intracellular transducers of RHO and mutations in these helixes result in a similar phenotype in patients but with distinct molecular effects.

Development of an odorant sensor with a cell-free synthesized olfactory receptor and a graphene field-effect transistor.

Animals sense odorants using olfactory receptors. Many trials have been conducted to develop artificial odorant sensors using olfactory receptors. However, the development has been hindered by the difficulty in obtaining olfactory receptors. In this study, we expressed an olfactory receptor, cOR52, using a wheat germ cell-free synthesis system. The functionality of the expressed cOR52 was confirmed by ligand concentration-dependent interactions with the mini-G protein. The expressed cOR52 was immobilized on a graphene field-effect transistor. The cOR52-modified graphene field-effect transistor exhibited a ligand-specific response between 100 nM and 100 µM. This approach seems to be applicable for other olfactory receptors. Therefore, it will be possible to develop an odorant sensor equipped with various olfactory receptors by this method.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein.

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the ßγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the ß subunit of the G protein was observed.

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands.

Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein.

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A2A receptor (A2AR) coupled to mini-Gs. Although this is extremely encouraging, A2AR is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.

Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein.

G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR-G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging targets for structural studies due to their conformationally dynamic and inherently transient nature. We recently developed an engineered G protein, mini-Gs, which addressed these problems and allowed the formation of a stable GPCR-G protein complex. Mini-Gs facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its G protein-bound conformation at 3.4 Å resolution. Here, we describe a step by step protocol for the expression and purification of A2AR, and crystallisation of the A2AR-mini-Gs complex.

Expression and Purification of Mini G Proteins from Escherichia coli.

Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation.

G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and ß-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gßγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised ß1-adrenergic receptor (ß1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.