Crystal structure reveals the binding mode and selectivity of a photoswitchable ligand for the adenosine A2A receptor.

Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.

In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A2A Receptor Antagonist/Inverse Agonist.

KW-6356 is a novel adenosine A2A (A2A) receptor antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing "OFF" episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined cocrystal structures of A2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A2A receptor (the -log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 minute-1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas istradefylline exhibits surmountable antagonism. Crystallography of KW-6356- and istradefylline-bound A2A receptor have indicated that interactions with His2506.52 and Trp2466.48 are essential for the inverse agonism, whereas the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important differences in vivo and help predict better clinical performance. SIGNIFICANCE STATEMENT: KW-6356 is a potent and selective adenosine A2A receptor antagonist/inverse agonist and exhibits insurmountable antagonism, whereas istradefylline, a first-generation adenosine A2A receptor antagonist, exhibits surmountable antagonism. Structural studies of adenosine A2A receptor in complex with KW-6356 and istradefylline explain the characteristic differences in the pharmacological properties of KW-6356 and istradefylline.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Structural insight into an anti-BRIL Fab as a G-protein-coupled receptor crystallization chaperone.

Structure determination of G-protein-coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti-BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL-fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high-resolution crystal structure of the BRIL-SRP2070Fab complex. The structure of the BRIL-SRP2070Fab complex was determined at 2.1 Šresolution. This high-resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL-SRP2070Fab co-crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL-fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure-based drug design of membrane-protein drug targets.

Structural insights into the agonists binding and receptor selectivity of human histamine H4 receptor.

Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H4 receptor (H4R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H4R-Gq complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe3447.39, which, in turn, form the "aromatic slot". The results provide insights into the molecular underpinnings of the agonism of H4R and subtype selectivity of histamine receptors, and show that the H4R structures may be valuable in rational drug design of drugs targeting the H4R.

Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.

BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.

Molecular basis for anti-insomnia drug design from structure of lemborexant-bound orexin 2 receptor.

Orexin receptors are a family of G protein-coupled receptors that consist of two subtypes: orexin-1 receptors (OX1Rs) and OX2Rs. They are expressed throughout the central nervous system and are involved in regulating the sleep-wake cycle. The development of antagonists to orexin receptors has become important in drug discovery because modulation of these receptors can lead to novel treatments for diseases related to the regulation of sleep and wakefulness, such as insomnia. In this study, we determined that the structure of OX2R bound to lemborexant, a dual orexin receptor antagonist (DORA), at 2.89 Å resolution. Comparisons of kinetic and dynamic properties of DORAs based on structures and simulations suggest that the enthalpy of molecular binding to receptors and the entropy derived from intramolecular structure can be separately controlled. These results complement existing structural information and allow us to discuss the usefulness of pharmacophore models and target selectivity to OXRs.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium.

Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl(-) and HCO3(-)across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-ß-D-maltoside. His-tagged PcAEP was purified as a single band on SDS-PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins.

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.

BACKGROUND: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely ß2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. RESULTS: The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. CONCLUSION: Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein.

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.

Endogenous agonist-bound S1PR3 structure reveals determinants of G protein-subtype bias.

Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics.

The discovery of a new antibody for BRIL-fused GPCR structure determination.

G-protein-coupled receptors (GPCRs)-the largest family of cell-surface membrane proteins-mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

Structure of an antagonist-bound ghrelin receptor reveals possible ghrelin recognition mode.

Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin's activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

The Crystal Structure of Angiotensin II Type 2 Receptor with Endogenous Peptide Hormone.

Angiotensin II (AngII) is a peptide hormone that plays a key role in regulating blood pressure, and its interactions with the G protein-coupled receptors, AngII type-1 receptor (AT1R) and AngII type-2 receptor (AT2R), are central to its mechanism of action. We solved the crystal structure of human AT2R bound to AngII and its specific antibody at 3.2-Å resolution. AngII (full agonist) and [Sar1, Ile8]-AngII (partial agonist) interact with AT2R in a similar fashion, except at the bottom of the AT2R ligand-binding pocket. In particular, the residues including Met1283.36, which constitute the deep end of the cavity, play important roles in angiotensin receptor (ATR) activation upon AngII binding. These differences that occur upon endogenous ligand binding may contribute to a structural change in AT2R, leading to normalization of the non-canonical coordination of helix 8. Our results will inform the design of more effective ligands for ATRs.

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae.

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-beta-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.

Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

Structures of the 5-HT2A receptor in complex with the antipsychotics risperidone and zotepine.

Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.