Orthosteric Ligand Binding Site Research Articles

Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1β2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3β2 ≅ α3β4, indicating that β2/β4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9′, 13′, and 16′, whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (–) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3

Nicotinic acetylcholine (ACh) receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [215, 3, 159, 225, 259]. All nicotinic receptors are pentamers in which each of the five subunits contains 4 TM domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [120]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [159]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). Nicotinic ACh receptors contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the 4 TM domains [264, 87]; see also [106]). The high resolution crystal structure of the molluscan ACh binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[35]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94Â resolution [55], has revealed the orthosteric binding site in detail (reviewed in [215, 120, 39, 198]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [96, 93, 159]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [159]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [159] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [263]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [159]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [158, 9, 118]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [143]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses- see also [46]).

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Kinetic analysis of fluorescent ligand binding to cell surface receptors: Insights into conformational changes and allosterism in living cells.

Equilibrium binding assays are one of the mainstays of current drug discovery efforts to evaluate the interaction of drugs with receptors in membranes and intact cells. However, in recent years, there has been increased focus on the kinetics of the drug-receptor interaction to gain insight into the lifetime of drug-receptor complexes and the rate of association of a ligand with its receptor. Furthermore, drugs that act on topically distinct sites (allosteric) from those occupied by the endogenous ligand (orthosteric site) can induce conformational changes in the orthosteric binding site leading to changes in the association and/or dissociation rate constants of orthosteric ligands. Conformational changes in the orthosteric ligand binding site can also be induced through interaction with neighbouring accessory proteins and receptor homodimerisation and heterodimerisation. In this review, we provide an overview of the use of fluorescent ligand technologies to interrogate ligand-receptor kinetics in living cells and the novel insights that they can provide into the conformational changes induced by drugs acting on a variety of cell surface receptors including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that thechannel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation.

The A3 adenosine receptor (A3AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure-activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo[4,5-c]quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A3AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A3AR signaling preferentially toward activation of Gαi3 and GαoA isoproteins, which are coexpressed with the A3AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A3AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A3AR PAMs, which includes leads for future pharmaceutical development.

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2.

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed fluorescence resonance energy transfer (FRET) sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM (egative allosteric modulator) increases the occupancy of one of the intermediate states while a positive allosteric modulator increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

Allosteric Modulators of Metabotropic Glutamate Receptors as Novel Therapeutics for Neuropsychiatric Disease.

Metabotropic glutamate (mGlu) receptors, a family of G-protein-coupled receptors, have been identified as novel therapeutic targets based on extensive research supporting their diverse contributions to cell signaling and physiology throughout the nervous system and important roles in regulating complex behaviors, such as cognition, reward, and movement. Thus, targeting mGlu receptors may be a promising strategy for the treatment of several brain disorders. Ongoing advances in the discovery of subtype-selective allosteric modulators for mGlu receptors has provided an unprecedented opportunity for highly specific modulation of signaling by individual mGlu receptor subtypes in the brain by targeting sites distinct from orthosteric or endogenous ligand binding sites on mGlu receptors. These pharmacological agents provide the unparalleled opportunity to selectively regulate neuronal excitability, synaptic transmission, and subsequent behavioral output pertinent to many brain disorders. Here, we review preclinical and clinical evidence supporting the utility of mGlu receptor allosteric modulators as novel therapeutic approaches to treat neuropsychiatric diseases, such as schizophrenia, substance use disorders, and stress-related disorders.Significance StatementAllosteric modulation of metabotropic glutamate (mGlu) receptors represents a promising therapeutic strategy to normalize dysregulated cellular physiology associated with neuropsychiatric disease. This review summarizes preclinical and clinical studies using mGlu receptor allosteric modulators as experimental tools and potential therapeutic approaches for the treatment of neuropsychiatric diseases, including schizophrenia, stress, and substance use disorders.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Human Three-Finger Protein Lypd6 Is a Negative Modulator of the Cholinergic System in the Brain.

Lypd6 is a GPI-tethered protein from the Ly-6/uPAR family expressed in the brain. Lypd6 enhances the Wnt/β-catenin signaling, although its action on nicotinic acetylcholine receptors (nAChRs) have been also proposed. To investigate a cholinergic activity of Lypd6, we studied a recombinant water-soluble variant of the human protein (ws-Lypd6) containing isolated “three-finger” LU-domain. Experiments at different nAChR subtypes expressed in Xenopus oocytes revealed the negative allosteric modulatory activity of ws-Lypd6. Ws-Lypd6 inhibited ACh-evoked currents at α3β4- and α7-nAChRs with IC50 of ∼35 and 10 μM, respectively, and the maximal amplitude of inhibition of 30–50%. EC50 of ACh at α3β4-nAChRs (∼30 μM) was not changed in the presence of 35 μM ws-Lypd6, while the maximal amplitude of ACh-evoked current was reduced by ∼20%. Ws-Lypd6 did not elicit currents through nAChRs in the absence of ACh. Application of 1 μM ws-Lypd6 significantly inhibited (up to ∼28%) choline-evoked current at α7-nAChRs in rat hippocampal slices. Similar to snake neurotoxin α-bungarotoxin, ws-Lypd6 suppressed the long-term potentiation (LTP) in mouse hippocampal slices. Colocalization of endogenous GPI-tethered Lypd6 with α3β4- and α7-nAChRs was detected in primary cortical and hippocampal neurons. Ws-Lypd6 interaction with the extracellular domain of α7-nAChR was modeled using the ensemble protein-protein docking protocol. The interaction of all three Lypd6 loops (“fingers”) with the entrance to the orthosteric ligand-binding site and the loop C of the primary receptor subunit was predicted. The results obtained allow us to consider Lypd6 as the endogenous negative modulator involved in the regulation of the cholinergic system in the brain.

Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2021.3

Nicotinic acetylcholine (ACh) receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [215, 3, 159, 225, 259]. All nicotinic receptors are pentamers in which each of the five subunits contains 4 TM domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [120]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [159]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). Nicotinic ACh receptors contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the 4 TM domains [264, 87]; see also [106]). The high resolution crystal structure of the molluscan ACh binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[35]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94Â resolution [55], has revealed the orthosteric binding site in detail (reviewed in [215, 120, 39, 198]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [96, 93, 159]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [159]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [159] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [263]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [159]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [158, 9, 118]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [143]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses- see also [46]).

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.

Cooperativity between the orthosteric and allosteric ligand binding sites of RORγt

Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.

Loss of a water-mediated network results in reduced agonist affinity in a β2-adrenergic receptor clinical variant

The β2-adrenergic receptor (β2AR) is a member of the G protein-coupled receptor (GPCR) family that is an important drug target for asthma and COPD. Clinical studies coupled with biochemical data have identified a critical receptor variant, Thr164Ile, to have a reduced response to agonist-based therapy, although the molecular mechanism underlying this seemingly “non-deleterious” substitution is not clear. Here, we couple molecular dynamics simulations with network analysis and free-energy calculations to identify the molecular determinants underlying the differential drug response. We are able to identify hydration sites in the transmembrane domain that are essential to maintain the integrity of the binding site but are absent in the variant. The loss of these hydration sites in the variant correlates with perturbations in the intra-protein interaction network and rearrangements in the orthosteric ligand binding site. In conjunction, we observe an altered binding and reduced free energy of a series of agonists, in line with experimental trends. Our work identifies a functional allosteric pathway connected by specific hydration sites in β2AR that has not been reported before and provides insight into water-mediated networks in GPCRs in general. Overall, the work is one of the first step towards developing variant-specific potent and selective agonists.

Allosteric interactions via the orthosteric ligand binding sites in a constitutive G-protein-coupled receptor homodimer

I interpret some recent data to indicate that co-operative effects take place between the (identical) orthosteric binding sites in a G-protein-coupled receptor dimer. In the current study, the reasonability of this concept was tested by creating a mathematical model. The model is composed of a symmetrical constitutive receptor dimer in which the protomers are able to affect each other allosterically, and it includes binding, receptor activation and signal amplification steps. The model was utilized for analyses of previous data as well as simulations of predicted behaviour. The model demonstrates the behaviour stated in the hypotheses, i.e. even an apparently neutral receptor ligand can allosterically affect agonist binding or receptor activation by binding to the normal orthosteric ligand binding site. Therewith the speculated allosteric action originating from the orthosteric binding site of the dimeric receptor is a realistic possibility. The results of the simulations and curve fitting constitute a reasonable starting point for further studies, and the model can be utilized to design meaningful experiments to investigate these questions.

Effect of Cholesterol on the Structural Dynamics of Metabotropic Glutamate Receptor (mGluR1): A Molecular Dynamics Study

Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors (GPCRs) which regulate neurotransmission and are prevalently expressed in the central nervous system. The mGluR subfamily has eight members divided into three subgroups (I (mGluR1,5), II (mGluR2,3) and III (mGluR4,6,7,8)) based on their sequence homology, agonist selectivity and signaling function. The group I mGluRs (mGluR1 and mGluR5) are useful for treating diseases such as cancer, pain, schizophrenia, Alzheimer's disease, anxiety, and autism. Unlike most GPCRs which have the orthosteric binding pocket located in the transmembrane helices, the extracellular VFT domain of the mGluRs serves as the orthosteric ligand binding site while the 7-transmembrane (7TM) domain has allosteric binding sites. Using all-atom equilibrium MD simulations, we have studied the effect of different amounts of membrane cholesterol on the local structural dynamics of the mGlur1 7TM region in the inactive state. In addition, we have also studied the effect of thermostabilized apocytochrome b562RIL (BRIL) on the 7TM region. We observed that BRIL and cholesterol had contrasting effects on the 7TM domain of mGluR1. While the presence of BRIL was found to maintain the inactive conformation of the protein, increasing the amount of membrane cholesterol was found to trigger a conformational transition towards the active state. A salt bridge (E783-K834) present in the inactive state was found to break when membrane cholesterol was increased to 25%. We also observed similar protein fluctuations in the absence of BRIL and with the increased membrane cholesterol. This study thus sheds light on conformational changes observed in mGluR1 and the effect of cholesterol within the 7TM region, providing a framework for determining the activation process of mGluRs.

Enhancing the Signaling of GPCRs via Orthosteric Ions.

G protein-coupled receptors play essential roles in cellular processes such as neuronal signaling, vision, olfaction, tasting, and metabolism. As GPCRs are the most important drug targets, understanding their interactions with ligands is of utmost importance for discovering related new medicines. In many GPCRs, an allosteric sodium ion next to the highly conserved residue D2.50 has been proposed to stabilize the inactive receptor state by mediating interactions between transmembrane helices. Here, we probed the existence of internal and functionally important sodium ions in the dopamine D2 receptor, using molecular dynamics simulations. Besides a new sodium ion at the allosteric ligand binding site, we discovered an additional sodium ion, located close to the orthosteric ligand binding site. Through cell-based activation assays, the signaling of D2 receptor with site-specific mutations was tested against a series of chemically modified agonists. We concluded an important structural role of this newly discovered orthosteric sodium ion in modulating the receptor signaling: It enables the coordination of a polar residue in the ligand binding site with an appropriately designed agonist molecule. An identical interaction was also observed in a recently released high-resolution crystal structure of mu-opioid receptor, which was reresolved in this work. Probably because of similar interactions, various metal ions have been found to increase the signaling of many other GPCRs. This unique principle and strategy could be used to optimize the drug activity of GPCR. Our findings open a new mechanistic opportunity of GPCR signaling and help design the next generation of drugs targeting GPCRs.

Structural insights into melatonin receptors.

The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT1 and MT2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT1 and MT2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects.