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Sample records for 7tm receptor structures

  1. Galaxy7TM: flexible GPCR-ligand docking by structure refinement.

    PubMed

    Lee, Gyu Rie; Seok, Chaok

    2016-07-01

    G-protein-coupled receptors (GPCRs) play important physiological roles related to signal transduction and form a major group of drug targets. Prediction of GPCR-ligand complex structures has therefore important implications to drug discovery. With previously available servers, it was only possible to first predict GPCR structures by homology modeling and then perform ligand docking on the model structures. However, model structures generated without explicit consideration of specific ligands of interest can be inaccurate because GPCR structures can be affected by ligand binding. The Galaxy7TM server, freely accessible at http://galaxy.seoklab.org/7TM, improves an input GPCR structure by simultaneous ligand docking and flexible structure refinement using GALAXY methods. The server shows better performance in both ligand docking and GPCR structure refinement than commonly used programs AutoDock Vina and Rosetta MPrelax, respectively. PMID:27131365

  2. Galaxy7TM: flexible GPCR–ligand docking by structure refinement

    PubMed Central

    Lee, Gyu Rie; Seok, Chaok

    2016-01-01

    G-protein-coupled receptors (GPCRs) play important physiological roles related to signal transduction and form a major group of drug targets. Prediction of GPCR–ligand complex structures has therefore important implications to drug discovery. With previously available servers, it was only possible to first predict GPCR structures by homology modeling and then perform ligand docking on the model structures. However, model structures generated without explicit consideration of specific ligands of interest can be inaccurate because GPCR structures can be affected by ligand binding. The Galaxy7TM server, freely accessible at http://galaxy.seoklab.org/7TM, improves an input GPCR structure by simultaneous ligand docking and flexible structure refinement using GALAXY methods. The server shows better performance in both ligand docking and GPCR structure refinement than commonly used programs AutoDock Vina and Rosetta MPrelax, respectively. PMID:27131365

  3. Solution Structure and Sugar-Binding Mechanism of Mouse Latrophilin-1 RBL: a 7TM Receptor-Attached Lectin-Like Domain

    PubMed Central

    Vakonakis, Ioannis; Langenhan, Tobias; Prömel, Simone; Russ, Andreas; Campbell, Iain D.

    2008-01-01

    Summary Latrophilin-1 (Lat-1), a target receptor for α-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique α/β fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors. PMID:18547526

  4. Functionally biased signalling properties of 7TM receptors – opportunities for drug development for the ghrelin receptor

    PubMed Central

    Sivertsen, B; Holliday, N; Madsen, A N; Holst, B

    2013-01-01

    The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several different signalling pathways including Gαq, Gαi/o, Gα12/13 and arrestin recruitment. These multiple signalling pathways allow for functionally biased signalling, where one signalling pathway may be favoured over another either by selective ligands or through mutations in the receptor. In the present review, we have described how ligands and mutations in the 7TM receptor may bias the receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G–protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have been demonstrated to signal more strongly through the Gαq-coupled pathway than the Gα12/13-coupled pathway. Similarly a ligand that promotes Gαq coupling over Gαi coupling has been described and it has been suggested that several different active conformations of the receptor may exist dependent on the properties of the agonist. Importantly, ligands with such biased signalling properties may allow the development of drugs that selectively modulate only the therapeutically relevant physiological functions, thereby decreasing the risk of side effects. LINKED ARTICLES This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 PMID:24032557

  5. Delineating biased ligand efficacy at 7TM receptors from an experimental perspective.

    PubMed

    Galandrin, Ségolène; Onfroy, Lauriane; Poirot, Mathias Charles; Sénard, Jean-Michel; Galés, Céline

    2016-08-01

    During the last 10 years, the concept of "biased agonism" also called "functional selectivity" swamped the pharmacology of 7 transmembrane receptors and paved the way for developing signaling pathway-selective drugs with increased efficacy and less adverse effects. Initially thought to select the activation of only a subset of the signaling pathways by the reference agonist, bias ligands revealed higher complexity as they have been shown to stabilize variable receptor conformations that associate with distinct signaling events from the reference. Today, one major challenge relies on the in vitro determination of the bias and classification of these ligands, as a prerequisite for future in vivo and clinical translation. In this review, current experimental considerations for the bias evaluation related to choice of the cellular model, of the signaling pathway as well as of the assays are presented and discussed. PMID:27107932

  6. Opioid-induced redistribution of 6TM and 7TM μ opioid receptors: A hypothesized mechanistic facilitator model of opioid-induced hyperalgesia.

    PubMed

    Wang, Wei; Wang, Yan; Zhang, Wei; Jin, Xiaoju; Liu, Yusheng; Xu, Shiqin; Lei, Liming; Shen, Xiaofeng; Guo, Xirong; Xia, Xiaoqiong; Wang, Fuzhou

    2016-08-01

    Opioids are still the most popular form of pain treatment, but many unavoidable side effects make opioids a big challenge in effective pain management. Opioid-induced hyperalgesia (OIH), a paradoxical phenomenon, portrays an increased sensitivity to harmful stimuli caused by opioid exposure. Changes in the neural modulation are considered a major contributor to the development of OIH. Activation of opioid receptors (ORs) and corresponding downstream molecules are the vital composition of functional performance of opioids. Increasing interests were proposed of the interaction between ORs and other neural transmitter systems such as glutamatergic, GABAergic and adrenergic ones to the genesis of OIH. G protein coupled μ-opioid receptor (MOR) was studied comprehensively on its role in the development of OIH. In addition to the relationship between MOR and other neurotransmitter receptors, a new intracellular MOR that has six transmembrane (6TM) domains was identified, and found to perform a pro-nociceptive task in contrast to the counterpart 7TM isoform. A mechanistic model of OIH in which both 6TM and 7TM MORs undergoing membrane redistribution upon opioid exposure is proposed which eventually facilitates the neurons more sensitive to nociceptive stimulation than that of the preceding opioid exposure. PMID:27116700

  7. 7TM X-ray structures for class C GPCRs as new drug-discovery tools. 1. mGluR5.

    PubMed

    Topiol, Sid; Sabio, Michael

    2016-01-15

    We illustrate, with a focus on mGluR5, how the recently published, first X-ray structures of mGluR 7TM domains, specifically those of mGluR1 and mGluR5 complexed with negative allosteric modulators (NAMs), will begin to influence ligand- (e.g., drug- or sweetener-) discovery efforts involving class C GPCRs. With an extensive docking study allowing full ligand flexibility and full side chain flexibility of all residues in the ligand-binding cavity, we have predicted and analyzed the binding modes of a variety of structurally diverse mGluR5 NAM ligands, showing how the X-ray structures serve to effectively rationalize each ligand's binding characteristics. We demonstrated that the features that are inherent in our earlier overlay model are preserved in the protein structure-based docking models. We identified structurally diverse compounds, which potentially act as mGluR NAMs, and revealed binding-site differences by performing high-throughput docking using a database of approximately six million structures of commercially available compounds and the mGluR1 and mGluR5 X-ray structures. By comparing the 7TM domains of the mGluR5 and mGluR1 X-rays structures, we identified selectivity factors within group I of the mGluRs. Similarly, using homology models that we built for mGluR2 and mGluR4, we have identified the factors leading to the selectivity between group I and groups II and III for ligands occupying the deepest portion of the mGluR5 binding cavity. Finally, we have proposed a structure-based explanation of the pharmacological switching within a set of positive allosteric modulators (PAMs) and their corresponding, very close NAM analogs. PMID:26706173

  8. Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R).

    PubMed

    Yang, Dehua; de Graaf, Chris; Yang, Linlin; Song, Gaojie; Dai, Antao; Cai, Xiaoqing; Feng, Yang; Reedtz-Runge, Steffen; Hanson, Michael A; Yang, Huaiyu; Jiang, Hualiang; Stevens, Raymond C; Wang, Ming-Wei

    2016-06-17

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants. PMID:27059958

  9. Structural conservation among the rhodopsin-like and other G protein-coupled receptors

    PubMed Central

    Kinoshita, Mikitaka; Okada, Tetsuji

    2015-01-01

    Intramolecular remote coupling within the polypeptide backbones of membrane proteins is difficult to analyze owing to the limited structural information available at the atomic level. Nonetheless, recent progress in the crystallographic study of G protein-coupled receptors (GPCRs) has provided an unprecedented opportunity for understanding the sophisticated architecture of heptahelical transmembrane (7TM) bundles. These 7TM bundles can respond to a wide range of extracellular stimuli while retaining the common function of binding trimeric G proteins. Here we have systematically analyzed select sets of inactive-like 7TM bundles to highlight the structural conservation of the receptors, in terms of intramolecular Cα-Cα distances. Distances with the highest scores were found to be dominated by the intrahelical distances of helix III, regardless of the choice of bundles in the set, indicating that the intracellular half of this helix is highly conserved. Unexpectedly, the distances between the cytoplasmic side of helix I and the extracellular region of helix VI provided the largest contribution to the high score populations among the interhelical pairs in most of the selected sets, including class B, C and frizzled receptors. These findings are expected to be valuable in further studies of GPCRs with unknown structure and of other protein families. PMID:25775952

  10. Sugar-induced endocytosis of plant 7TM-RGS proteins

    PubMed Central

    Phan, Nguyen; Urano, Daisuke; Srba, Miroslav; Fischer, Lukas; Jones, Alan M.

    2013-01-01

    Plant cells use sugars mainly as a source or store of energy and carbon skeletons for anabolic reactions and for osmotic regulation. The perception of sugars and their responses are rather complex including the heterotrimeric G protein pathway and a seven-transmembrane RGS molecule. Previously, we found that endocytosis of the 7TM-RGS leads to sustained activation of the G protein pathway in the genetic model Arabidopsis. Here we show that other plants possess similar endocytosis systems of the 7TM-RGS proteins. A phosphorylation site essential for the endocytosis is well conserved in land plant 7TM-RGS proteins. In addition, conifer and tobacco 7TM-RGS proteins are internalized in response to sugar. These results indicate a universal mechanism to activate G signaling by endocytosis in plant cells that have 7TM-RGS proteins. PMID:23154506

  11. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  12. Conformational states of the full-length glucagon receptor

    PubMed Central

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-01-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

  13. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  14. Biased and G Protein-Independent Signaling of Chemokine Receptors

    PubMed Central

    Steen, Anne; Larsen, Olav; Thiele, Stefanie; Rosenkilde, Mette M.

    2014-01-01

    Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand–receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of “classic” redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors – in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution

  15. Crystal structures of the human adiponectin receptors.

    PubMed

    Tanabe, Hiroaki; Fujii, Yoshifumi; Okada-Iwabu, Miki; Iwabu, Masato; Nakamura, Yoshihiro; Hosaka, Toshiaki; Motoyama, Kanna; Ikeda, Mariko; Wakiyama, Motoaki; Terada, Takaho; Ohsawa, Noboru; Hato, Masakatsu; Ogasawara, Satoshi; Hino, Tomoya; Murata, Takeshi; Iwata, So; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yamauchi, Toshimasa; Kadowaki, Takashi; Yokoyama, Shigeyuki

    2015-04-16

    Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5' AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes. PMID:25855295

  16. Structure of toll-like receptors.

    PubMed

    Gay, Nicholas J; Gangloff, Monique

    2008-01-01

    The ten human Toll-like receptors are able to respond to an extremely diverse range of microbial products ranging from di- and tri-acylated lipids to nucleic acids. An understanding of the molecular structure adopted by the receptor extracellular, transmembrane, and cytoplasmic domains and the way in which these structures interact with ligands and downstream signaling adapters can explain how recognition and signal transduction are achieved at a molecular level. In this article we discuss how the leucine-rich repeats of the receptor ectodomain have evolved to bind a wide variety of biological molecules. We also discuss how ligand binding induces dimerization of two receptor chains and initiates a series of protein conformational changes that lead to a signaling event in the cytoplasm of the immune system cell. Thus, the signaling process of the TLRs can be viewed as a unidirectional molecular switch. PMID:18071660

  17. Structure biology of selective autophagy receptors

    PubMed Central

    Kim, Byeong-Won; Kwon, Do Hoon; Song, Hyun Kyu

    2016-01-01

    Autophagy is a process tightly regulated by various autophagy-related proteins. It is generally classified into non-selective and selective autophagy. Whereas non-selective autophagy is triggered when the cell is under starvation, selective autophagy is involved in eliminating dysfunctional organelles, misfolded and/or ubiquitylated proteins, and intracellular pathogens. These components are recognized by autophagy receptors and delivered to phagophores. Several selective autophagy receptors have been identified and characterized. They usually have some common domains, such as motif, a specific cargo interacting (ubiquitin-dependent or ubiquitin-independent) domain. Recently, structural data of these autophagy receptors has been described, which provides an insight of their function in the selective autophagic process. In this review, we summarize the most up-to-date findings about the structure-function of autophagy receptors that regulates selective autophagy. [BMB Reports 2016; 49(2): 73-80] PMID:26698872

  18. Effect of surface coating of KYb2F7:Tm(3+) on optical properties and biomedical applications.

    PubMed

    Pedraza, Francisco J; Avalos, Julio C; Yust, Brian G; Tsin, Andrew; Sardar, Dhiraj K

    2016-09-23

    This project aims to provide an insight on the effects of biocompatible polymers on the optical properties and the nanoparticle-cell interaction of KYb2F7:Tm(3+) nanocrystals that exhibit strong near infrared (NIR) fluorescence. KYb2F7:Tm(3+) nanocrystals were synthesized with a diameter of 20-30 nm and surface modified with poly(ethylene glycol), Pluronic(®) F-127, and poly(N-vinylpyrrolidone), due to the associated advantages. Some of these include biocompatibility and biodistribution in the instance of agglomeration and hydrophobicity as well as the addition of a targeting agent and drug loading by further functionalization. Despite the decrease in fluorescence intensity induced by the surface modification, thulium's emission fingerprint was easily detected. Moreover, surface modified KYb2F7:Tm(3+) nanocrystals failed to induce a toxic response on endothelial cells following a 24 h uptake period up to concentrations of 100 μg ml(-1). In vitro toxicity and confocal imaging have demonstrated the versatility of these NIR fluorescence nanocrystals in biomedical imaging, drug delivery, and photodynamic therapy. PMID:27518385

  19. Effect of surface coating of KYb2F7:Tm3+ on optical properties and biomedical applications

    NASA Astrophysics Data System (ADS)

    Pedraza, Francisco J.; Avalos, Julio C.; Yust, Brian G.; Tsin, Andrew; Sardar, Dhiraj K.

    2016-09-01

    This project aims to provide an insight on the effects of biocompatible polymers on the optical properties and the nanoparticle-cell interaction of KYb2F7:Tm3+ nanocrystals that exhibit strong near infrared (NIR) fluorescence. KYb2F7:Tm3+ nanocrystals were synthesized with a diameter of 20–30 nm and surface modified with poly(ethylene glycol), Pluronic® F-127, and poly(N-vinylpyrrolidone), due to the associated advantages. Some of these include biocompatibility and biodistribution in the instance of agglomeration and hydrophobicity as well as the addition of a targeting agent and drug loading by further functionalization. Despite the decrease in fluorescence intensity induced by the surface modification, thulium’s emission fingerprint was easily detected. Moreover, surface modified KYb2F7:Tm3+ nanocrystals failed to induce a toxic response on endothelial cells following a 24 h uptake period up to concentrations of 100 μg ml‑1. In vitro toxicity and confocal imaging have demonstrated the versatility of these NIR fluorescence nanocrystals in biomedical imaging, drug delivery, and photodynamic therapy.

  20. Crystal structure of human interferon-γ receptor 2 reveals the structural basis for receptor specificity.

    PubMed

    Mikulecký, Pavel; Zahradník, Jirí; Kolenko, Petr; Černý, Jiří; Charnavets, Tatsiana; Kolářová, Lucie; Nečasová, Iva; Pham, Phuong Ngoc; Schneider, Bohdan

    2016-09-01

    Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π-cation motif of stacked residues KWRWRH, a NAG-W-NAG sandwich (where NAG stands for N-acetyl-D-glucosamine) and finally a helix formed by residues 78-85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2. PMID:27599734

  1. Structural and Molecular Modeling Features of P2X Receptors

    PubMed Central

    Alves, Luiz Anastacio; da Silva, João Herminio Martins; Ferreira, Dinarte Neto Moreira; Fidalgo-Neto, Antonio Augusto; Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; Caffarena, Ernesto Raúl; de Freitas, Mônica Santos

    2014-01-01

    Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors. PMID:24637936

  2. Extra-helical binding site of a glucagon receptor antagonist.

    PubMed

    Jazayeri, Ali; Doré, Andrew S; Lamb, Daniel; Krishnamurthy, Harini; Southall, Stacey M; Baig, Asma H; Bortolato, Andrea; Koglin, Markus; Robertson, Nathan J; Errey, James C; Andrews, Stephen P; Teobald, Iryna; Brown, Alastair J H; Cooke, Robert M; Weir, Malcolm; Marshall, Fiona H

    2016-05-12

    Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors. PMID:27111510

  3. In silico characterization of Plasmodium falciparum purinergic receptor: a novel chemotherapeutic target.

    PubMed

    Gupta, Sonal; Singh, Deepak; Singh, Shailja

    2015-12-01

    Serpentine receptors with G-protein coupled receptor like seven transmembrane (7 TM) topology are identified in Plasmodium. A class of 7 TM receptors known as purinergic receptors binds to purines such as ADP, ATP and UTP and mediates important physiological functions including regulation of calcium signaling. Here we performed in silico analysis of Plasmodium falciparum serpentine receptors and found that one of the P. falciparum serpentine receptors, PfSR12 possess nucleotide binding consensus P-loop sequence in addition to seven transmembrane domains. The presence of conserved seven transmembrane domains and a consensus nucleotide binding sequence (P-loop) suggest that PfSR12 is a putative purinergic receptor. On further analysis using docking programmes we found four active binding residues Asn149, Lys150, Asn151 and Gly152 in P-loop of PfSR12, interact with ATP. This work gives insights into the interactions between putative purinergic receptor PfSR12 and its ligand ATP which can be explored in structure based drug designing against malaria. PMID:26702303

  4. Structural requirements of bitter taste receptor activation

    PubMed Central

    Brockhoff, Anne; Behrens, Maik; Niv, Masha Y.; Meyerhof, Wolfgang

    2010-01-01

    An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception. PMID:20534469

  5. A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum

    PubMed Central

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Helbo, Signe; Desmet, Filip; Berghmans, Herald; Van Doorslaer, Sabine; Hoogewijs, David; Schoofs, Liliane; Braeckman, Bart P.; Moens, Luc; Fago, Angela; Dewilde, Sylvia

    2015-01-01

    We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor. PMID:25666609

  6. Crystal structure of a heterotetrameric NMDA receptor ion channel

    PubMed Central

    Karakas, Erkan; Furukawa, Hiro

    2014-01-01

    N -methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Here we show the crystal structure of the intact heterotetrameric GluN1/GluN2B NMDA receptor ion channel at 4 Å. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the two-fold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors. PMID:24876489

  7. [Glutamate Metabotropic Receptors: Structure, Localisation, Functions].

    PubMed

    Perfilova, V N; Tyurenkov, I N

    2016-01-01

    The data on the structure, location and functions of the metabotropic glutamate receptor is shown. The family consists of 8 mGluRs subtypes and is divided into three groups: I group--mGluRs1/mGluRs5, II group--mGluRs2/mGluRs3, III group--mGluRs4/mGluRs6/mGluRs7/mGluRs8. They are associated with G-protein; signaling in the cells is carried out by IP3 or adenylate cyclase signaling pathways, in the result of which, mGluRs modify glial and neuronal excitability. Receptors are localized in the CNS and periphery in non-neuronal tissues: bone, heart, kidney, pancreas pod and platelets, the gastrointestinal tract, immune system. Their participation in the mechanisms of neurodegenerative diseases, mental and cognitive disorders, autoimmune processes, etc. is displayed. Agonists, antagonists, allosteric modulators of mGluRs are considered as potential medicines for treatment of mental diseases, including depression, fragile X syndrome, anxiety, obsessive-compulsive disorders, Parkinson's disease, etc. PMID:27530046

  8. Melanocortin 1 Receptor: Structure, Function, and Regulation

    PubMed Central

    Wolf Horrell, Erin M.; Boulanger, Mary C.; D’Orazio, John A.

    2016-01-01

    The melanocortin 1 receptor (MC1R) is a melanocytic Gs protein coupled receptor that regulates skin pigmentation, UV responses, and melanoma risk. It is a highly polymorphic gene, and loss of function correlates with a fair, UV-sensitive, and melanoma-prone phenotype due to defective epidermal melanization and sub-optimal DNA repair. MC1R signaling, achieved through adenylyl cyclase activation and generation of the second messenger cAMP, is hormonally controlled by the positive agonist melanocortin, the negative agonist agouti signaling protein, and the neutral antagonist β-defensin 3. Activation of cAMP signaling up-regulates melanin production and deposition in the epidermis which functions to limit UV penetration into the skin and enhances nucleotide excision repair (NER), the genomic stability pathway responsible for clearing UV photolesions from DNA to avoid mutagenesis. Herein we review MC1R structure and function and summarize our laboratory’s findings on the molecular mechanisms by which MC1R signaling impacts NER. PMID:27303435

  9. Structural basis for molecular recognition at serotonin receptors.

    PubMed

    Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

    2013-05-01

    Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs. PMID:23519210

  10. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.

    PubMed

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W; Frimurer, Thomas M

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were observed, despite their distinct chemical nature and inverse pharmacological profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer. PMID:27491650

  11. Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies

    PubMed Central

    2014-01-01

    Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general. PMID:25428871

  12. Crystallization and Structure Determination of Superantigens and Immune Receptor Complexes.

    PubMed

    Rödström, Karin E J; Lindkvist-Petersson, Karin

    2016-01-01

    Structure determination of superantigens and the complexes they form with immune receptors have over the years provided insight in their modes of action. This technique requires growing large and highly ordered crystals of the superantigen or receptor-superantigen complex, followed by exposure to X-ray radiation and data collection. Here, we describe methods for crystallizing superantigens and superantigen-receptor complexes using the vapor diffusion technique, how the crystals may be optimized, and lastly data collection and structure determination. PMID:26676036

  13. ROR nuclear receptors: structures, related diseases, and drug discovery

    PubMed Central

    Zhang, Yan; Luo, Xiao-yu; Wu, Dong-hai; Xu, Yong

    2015-01-01

    Nuclear receptors (NRs) are ligand-regulated transcription factors that regulate metabolism, development and immunity. The NR superfamily is one of the major classes of drug targets for human diseases. Retinoic acid receptor-related orphan receptor (ROR) α, β and γ belong to the NR superfamily, and these receptors are still considered as 'orphan' receptors because the identification of their endogenous ligands has been controversial. Recent studies have demonstrated that these receptors are regulated by synthetic ligands, thus emerge as important drug targets for the treatment of multiple sclerosis, rheumatoid arthritis, psoriasis, etc. Studying the structural basis and ligand development of RORs will pave the way for a better understanding of the roles of these receptors in human diseases. Here, we review the structural basis, disease relevance, strategies for ligand identification, and current status of development of therapeutic ligands for RORs. PMID:25500868

  14. A globin domain in a neuronal transmembrane receptor of Caenorhabditis elegans and Ascaris suum: molecular modeling and functional properties.

    PubMed

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Helbo, Signe; Desmet, Filip; Berghmans, Herald; Van Doorslaer, Sabine; Hoogewijs, David; Schoofs, Liliane; Braeckman, Bart P; Moens, Luc; Fago, Angela; Dewilde, Sylvia

    2015-04-17

    We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor. PMID:25666609

  15. Structure of a mammalian ryanodine receptor

    PubMed Central

    Zalk, Ran; Clarke, Oliver B.; des Georges, Amédée; Grassucci, Robert A.; Reiken, Steven; Mancia, Filippo; Hendrickson, Wayne A.; Frank, Joachim; Marks, Andrew R.

    2014-01-01

    Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. PMID:25470061

  16. A Structural Perspective on the Regulation of the EGF Receptor

    PubMed Central

    Kovacs, Erika; Zorn, Julie Anne; Huang, Yongjian; Barros, Tiago; Kuriyan, John

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. EGFR is unique in that its ligand-induced dimerization is established solely by contacts between regions of the receptor that are occluded within the monomeric, unliganded state. Activation of EGFR depends on the formation of an asymmetric dimer of the intracellular module of two receptor molecules, a configuration observed in crystal structures of the EGFR kinase domain in the active state. Coupling between the extracellular and intracellular modules is achieved by a switch between alternative geometries of the transmembrane and juxtamembrane segments within the receptor dimer. As the structure of the full-length receptor is yet to be determined, here we review recent structural studies on isolated modules of EGFR and molecular dynamics simulations that have provided much of our current understanding of its signaling mechanism, including how its regulation is compromised by oncogenic mutations. PMID:25621509

  17. Structural biology of the S1P1 receptor.

    PubMed

    Hanson, Michael A; Peach, Robert

    2014-01-01

    The sphingosine 1 phosphate receptor family has been studied widely since the initial discovery of its first member, endothelium differentiation gene 1. Since this initial discovery, the family has been renamed and the primary member of the family, the S1P1 receptor, has been targeted for a variety of disease indications and successfully drugged for the treatment of patients with relapsing multiple sclerosis. Recently, the three-dimensional structure of the S1P1 receptor has been determined by X-ray crystallography and the specifics of the sphingosine 1 phosphate ligand binding pocket mapped. Key structural features for the S1P1 receptor will be reviewed and the potential binding modes of additional pharmacologically active agents against the receptor will be analyzed in an effort to better understand the structural basis of important receptor-ligand interactions. PMID:24728592

  18. Functional role, structure, and evolution of the melanocortin-4 receptor.

    PubMed

    Schiöth, Helgi B; Lagerström, Malin C; Watanobe, Hajime; Jonsson, Logi; Vergoni, Anna Valeria; Ringholm, Aneta; Skarphedinsson, Jon O; Skuladottir, Gudrun V; Klovins, Janis; Fredriksson, Robert

    2003-06-01

    The melanocortin (MC)-4 receptor participates in regulating body weight homeostasis. We demonstrated early that acute blockage of the MC-4 receptor increases food intake and relieves anorexic conditions in rats. Our recent studies show that 4-week chronic blockage of the MC-4 receptor leads to robust increases in food intake and development of obesity, whereas stimulation of the receptor leads to anorexia. Interestingly, the food conversion ratio was clearly increased by MC-4 receptor blockage, whereas it was decreased in agonist-treated rats in a transient manner. Chronic infusion of an agonist caused a transient increase in oxygen consumption. Our studies also show that the MC-4 receptor plays a role in luteinizing hormone and prolactin surges in female rats. The MC-4 receptor has a role in mediating the effects of leptin on these surges. The phylogenetic relation of the MC-4 receptor to other GPCRs in the human genome was determined. The three-dimensional structure of the protein was studied by construction of a high-affinity zinc binding site between the helices, using two histidine residues facing each other. We also cloned the MC-4 receptor from evolutionary important species and showed by chromosomal mapping a conserved synteny between humans and zebrafish. The MC-4 receptor has been remarkably conserved in structure and pharmacology for more than 400 million years, implying that the receptor participated in vital physiological functions early in vertebrate evolution. PMID:12851300

  19. Predicting olfactory receptor neuron responses from odorant structure

    PubMed Central

    Schmuker, Michael; de Bruyne, Marien; Hähnel, Melanie; Schneider, Gisbert

    2007-01-01

    Background Olfactory receptors work at the interface between the chemical world of volatile molecules and the perception of scent in the brain. Their main purpose is to translate chemical space into information that can be processed by neural circuits. Assuming that these receptors have evolved to cope with this task, the analysis of their coding strategy promises to yield valuable insight in how to encode chemical information in an efficient way. Results We mimicked olfactory coding by modeling responses of primary olfactory neurons to small molecules using a large set of physicochemical molecular descriptors and artificial neural networks. We then tested these models by recording in vivo receptor neuron responses to a new set of odorants and successfully predicted the responses of five out of seven receptor neurons. Correlation coefficients ranged from 0.66 to 0.85, demonstrating the applicability of our approach for the analysis of olfactory receptor activation data. The molecular descriptors that are best-suited for response prediction vary for different receptor neurons, implying that each receptor neuron detects a different aspect of chemical space. Finally, we demonstrate that receptor responses themselves can be used as descriptors in a predictive model of neuron activation. Conclusion The chemical meaning of molecular descriptors helps understand structure-response relationships for olfactory receptors and their "receptive fields". Moreover, it is possible to predict receptor neuron activation from chemical structure using machine-learning techniques, although this is still complicated by a lack of training data. PMID:17880742

  20. Structure of the [delta]-opioid receptor bound to naltrindole

    SciTech Connect

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K.

    2012-07-11

    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  1. Emerging structural insights into the function of ionotropic glutamate receptors

    PubMed Central

    Karakas, Erkan; Regan, Michael C.; Furukawa, Hiro

    2015-01-01

    Summary Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission crucial for brain development and function including learning and memory formation. Recently a wealth of structural studies on iGluRs, including AMPA receptors (AMPARs), kainate receptors, and NMDA receptors (NMDARs) became available.. These studies showed structures of non-NMDARs including AMPAR and kainate receptor in various functional states, thereby providing the first visual sense of how non-NMDAR iGluRs may function in the context of homotetramers. Furthermore, they provided the first view of heterotetrameric NMDAR ion channels, which illuminated the similarities with and differences from non-NMDARs, thus raising a mechanistic distinction between the two groups of iGluRs. Here we review mechanistic insights into iGluR functions gained through structural studies of multiple groups. PMID:25941168

  2. Functional Insights from Glutamate Receptor Ion Channel Structures

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2014-01-01

    X-ray crystal structures for the soluble amino terminal and ligand binding domains of glutamate receptor ion channels, combined with a 3.6 Å resolution structure of the full length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of iGluR assembly and function. Increasingly sophisticated biochemical, computational and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists, and yield new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for development of subtype selective modulators. Many issues remain unsolved, including the role of the ATD in AMPA receptor signaling, and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how binding of glutamate opens the ion channel pore. PMID:22974439

  3. Structure of the human progesterone receptor gene.

    PubMed

    Misrahi, M; Venencie, P Y; Saugier-Veber, P; Sar, S; Dessen, P; Milgrom, E

    1993-11-16

    The complete organization of the human progesterone receptor (hPR) gene has been determined. It spans over 90 kbp and contains eight exons. The first exon encodes the N-terminal part of the receptor. The DNA binding domain is encoded by two exons, each exon corresponding to one zinc finger. The steroid binding domain is encoded by five exons. The nucleotide sequence of 1144 bp of the 5' flanking region has been determined. PMID:8241270

  4. Structural features for functional selectivity at serotonin receptors.

    PubMed

    Wacker, Daniel; Wang, Chong; Katritch, Vsevolod; Han, Gye Won; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Jiang, Yi; Chu, Meihua; Siu, Fai Yiu; Liu, Wei; Xu, H Eric; Cherezov, Vadim; Roth, Bryan L; Stevens, Raymond C

    2013-05-01

    Drugs active at G protein-coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-HT2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT2B receptor bound to ERG and compared it with the 5-HT1B/ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities. PMID:23519215

  5. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    SciTech Connect

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  6. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  7. Structural mechanism of glutamate receptor activation and desensitization.

    PubMed

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  8. Crystal Structure of a Lipid G Protein-Coupled Receptor

    SciTech Connect

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  9. Crystal structure of the human σ1 receptor.

    PubMed

    Schmidt, Hayden R; Zheng, Sanduo; Gurpinar, Esin; Koehl, Antoine; Manglik, Aashish; Kruse, Andrew C

    2016-04-28

    The human σ1 receptor is an enigmatic endoplasmic-reticulum-resident transmembrane protein implicated in a variety of disorders including depression, drug addiction, and neuropathic pain. Recently, an additional connection to amyotrophic lateral sclerosis has emerged from studies of human genetics and mouse models. Unlike many transmembrane receptors that belong to large, extensively studied families such as G-protein-coupled receptors or ligand-gated ion channels, the σ1 receptor is an evolutionary isolate with no discernible similarity to any other human protein. Despite its increasingly clear importance in human physiology and disease, the molecular architecture of the σ1 receptor and its regulation by drug-like compounds remain poorly defined. Here we report crystal structures of the human σ1 receptor in complex with two chemically divergent ligands, PD144418 and 4-IBP. The structures reveal a trimeric architecture with a single transmembrane domain in each protomer. The carboxy-terminal domain of the receptor shows an extensive flat, hydrophobic membrane-proximal surface, suggesting an intimate association with the cytosolic surface of the endoplasmic reticulum membrane in cells. This domain includes a cupin-like β-barrel with the ligand-binding site buried at its centre. This large, hydrophobic ligand-binding cavity shows remarkable plasticity in ligand recognition, binding the two ligands in similar positions despite dissimilar chemical structures. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important but poorly understood protein. PMID:27042935

  10. Structural design and molecular evolution of a cytokine receptor superfamily.

    PubMed Central

    Bazan, J F

    1990-01-01

    A family of cytokine receptors comprising molecules specific for a diverse group of hematopoietic factors and growth hormones has been principally defined by a striking homology of binding domains. This work proposes that the approximately 200-residue binding segment of the canonical cytokine receptor is composed of two discrete folding domains that share a significant sequence and structural resemblance. Analogous motifs are found in tandem approximately 100-amino acid domains in the extracellular segments of a receptor family formed by the interferon-alpha/beta and -gamma receptors and tissue factor, a membrane tether for a coagulation protease. Domains from the receptor supergroup reveal clear evolutionary links to fibronectin type III structures, approximately 90-amino acid modules that are typically found in cell surface molecules with adhesive functions. Predictive structural analysis of the shared receptor and fibronectin domains locates seven beta-strands in conserved regions of the chain; these strands are modeled to fold into antiparallel beta-sandwiches with a topology that is similar to immunoglobulin constant domains. These findings have strong implications for understanding the evolutionary emergence of an important class of regulatory molecules from primitive adhesive modules. In addition, the resulting double-barrel design of the receptors and the spatial clustering of conserved residues suggest a likely binding site for cytokine ligands. Images PMID:2169613

  11. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    SciTech Connect

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  12. Analysis of Human Dopamine D3 Receptor Quaternary Structure*

    PubMed Central

    Marsango, Sara; Caltabiano, Gianluigi; Pou, Chantevy; Varela Liste, María José; Milligan, Graeme

    2015-01-01

    The dopamine D3 receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D3 receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β1-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D3-D3 receptor interfaces within homo-oligomers and are consistent with the D3 receptor adopting a β1-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D3 protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D3 receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a ”rhombic” tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D3 receptor and possibly other GPCR quaternary structures. PMID:25931118

  13. The structural basis for receptor recognition of human interleukin-18

    PubMed Central

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-01-01

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity. PMID:25500532

  14. The structural basis for receptor recognition of human interleukin-18

    DOE PAGESBeta

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; et al

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  15. The structural basis for receptor recognition of human interleukin-18

    SciTech Connect

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.

  16. END-PLATE ACETYLCHOLINE RECEPTOR: STRUCTURE, MECHANISM, PHARMACOLOGY, AND DISEASE

    PubMed Central

    Sine, Steven M.

    2012-01-01

    The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction. PMID:22811427

  17. Glutamate Receptor Ion Channels: Structure, Regulation, and Function

    PubMed Central

    Wollmuth, Lonnie P.; McBain, Chris J.; Menniti, Frank S.; Vance, Katie M.; Ogden, Kevin K.; Hansen, Kasper B.; Yuan, Hongjie; Myers, Scott J.; Dingledine, Ray

    2010-01-01

    The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors. PMID:20716669

  18. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  19. NMDA receptor structures reveal subunit arrangement and pore architecture

    PubMed Central

    Lee, Chia-Hsueh; Lü, Wei; Michel, Jennifer Carlisle; Goehring, April; Du, Juan; Song, Xianqiang; Gouaux, Eric

    2014-01-01

    Summary N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present x-ray crystal structures of the GluN1/GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino terminal and ligand binding domains. The transmembrane domains harbor a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ~2-fold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors. PMID:25008524

  20. Inexpensive Method for Selecting Receptor Structures for Virtual Screening.

    PubMed

    Huang, Zunnan; Wong, Chung F

    2016-01-25

    This article introduces a screening performance index (SPI) to help select from a number of experimental structures one or a few that are more likely to identify more actives among its top hits from virtual screening of a compound library. It achieved this by docking only known actives to the experimental structures without considering a large number of decoys to reduce computational costs. The SPI is calculated by using the docking energies of the actives to all the receptor structures. We evaluated the performance of the SPI by applying it to study eight protein systems: fatty acid binding protein adipocyte FABP4, serine/threonine-protein kinase BRAF, beta-1 adrenergic receptor ADRB1, TGF-beta receptor type I TGFR1, adenosylhomocysteinase SAHH, thyroid hormone receptor beta-1 THB, phospholipase A2 group IIA PA2GA, and cytochrome P450 3a4 CP3A4. We found that the SPI agreed with the results from other popular performance metrics such as Boltzmann-Enhanced Discrimination Receiver Operator Characteristics (BEDROC), Robust Initial Enhancement (RIE), Area Under Accumulation Curve (AUAC), and Enrichment Factor (EF) but is less expensive to calculate. SPI also performed better than the best docking energy, the molecular volume of the bound ligand, and the resolution of crystal structure in selecting good receptor structures for virtual screening. The implications of these findings were further discussed in the context of ensemble docking, in situations when no experimental structure for the targeted protein was available, or under circumstances when quick choices of receptor structures need to be made before quantitative indexes such as the SPI and BEDROC can be calculated. PMID:26651874

  1. Structure and function of serotonin G protein-coupled receptors.

    PubMed

    McCorvy, John D; Roth, Bryan L

    2015-06-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein-coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  2. Structure and Function of Serotonin G protein Coupled Receptors

    PubMed Central

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  3. Structure and Pharmacologic Modulation of Inhibitory Glycine Receptors.

    PubMed

    Burgos, Carlos F; Yévenes, Gonzalo E; Aguayo, Luis G

    2016-09-01

    Glycine receptors (GlyR) are inhibitory Cys-loop ion channels that contribute to the control of excitability along the central nervous system (CNS). GlyR are found in the spinal cord and brain stem, and more recently they were reported in higher regions of the CNS such as the hippocampus and nucleus accumbens. GlyR are involved in motor coordination, respiratory rhythms, pain transmission, and sensory processing, and they are targets for relevant physiologic and pharmacologic modulators. Several studies with protein crystallography and cryoelectron microscopy have shed light on the residues and mechanisms associated with the activation, blockade, and regulation of pentameric Cys-loop ion channels at the atomic level. Initial studies conducted on the extracellular domain of acetylcholine receptors, ion channels from prokaryote homologs-Erwinia chrysanthemi ligand-gated ion channel (ELIC), Gloeobacter violaceus ligand-gated ion channel (GLIC)-and crystallized eukaryotic receptors made it possible to define the overall structure and topology of the Cys-loop receptors. For example, the determination of pentameric GlyR structures bound to glycine and strychnine have contributed to visualizing the structural changes implicated in the transition between the open and closed states of the Cys-loop receptors. In this review, we summarize how the new information obtained in functional, mutagenesis, and structural studies have contributed to a better understanding of the function and regulation of GlyR. PMID:27401877

  4. Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1

    PubMed Central

    Chrencik, Jill E.; Roth, Christopher B.; Terakado, Masahiko; Kurata, Haruto; Omi, Rie; Kihara, Yasuyuki; Warshaviak, Dora; Nakade, Shinji; Asmar-Rovira, Guillermo; Mileni, Mauro; Mizuno, Hirotaka; Griffith, Mark T.; Rodgers, Caroline; Han, Gye Won; Velasquez, Jeffrey; Chun, Jerold; Stevens, Raymond C.

    2015-01-01

    Summary Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with six cognate G protein-coupled receptors. Herein we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analysis. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease. PMID:26091040

  5. Novel lysophosphoplipid receptors: their structure and function

    PubMed Central

    Makide, Kumiko; Uwamizu, Akiharu; Shinjo, Yuji; Ishiguro, Jun; Okutani, Michiyo; Inoue, Asuka; Aoki, Junken

    2014-01-01

    It is now accepted that lysophospholipids (LysoGPs) have a wide variety of functions as lipid mediators that are exerted through G protein-coupled receptors (GPCRs) specific to each lysophospholipid. While the roles of some LysoGPs, such as lysophosphatidic acid and sphingosine 1-phosphate, have been thoroughly examined, little is known about the roles of several other LysoGPs, such as lysophosphatidylserine (LysoPS), lysophosphatidylthreonine, lysophosphatidylethanolamine, lysophosphatidylinositol (LPI), and lysophosphatidylglycerol. Recently, a GPCR was found for LPI (GPR55) and three GPCRs (GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3) were found for LysoPS. In this review, we focus on these newly identified GPCRs and summarize the actions of LysoPS and LPI as lipid mediators. PMID:24891334

  6. Novel lysophosphoplipid receptors: their structure and function.

    PubMed

    Makide, Kumiko; Uwamizu, Akiharu; Shinjo, Yuji; Ishiguro, Jun; Okutani, Michiyo; Inoue, Asuka; Aoki, Junken

    2014-10-01

    It is now accepted that lysophospholipids (LysoGPs) have a wide variety of functions as lipid mediators that are exerted through G protein-coupled receptors (GPCRs) specific to each lysophospholipid. While the roles of some LysoGPs, such as lysophosphatidic acid and sphingosine 1-phosphate, have been thoroughly examined, little is known about the roles of several other LysoGPs, such as lysophosphatidylserine (LysoPS), lysophosphatidylthreonine, lysophosphatidylethanolamine, lysophosphatidylinositol (LPI), and lysophosphatidylglycerol. Recently, a GPCR was found for LPI (GPR55) and three GPCRs (GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3) were found for LysoPS. In this review, we focus on these newly identified GPCRs and summarize the actions of LysoPS and LPI as lipid mediators. PMID:24891334

  7. LYRA, a webserver for lymphocyte receptor structural modeling

    PubMed Central

    Klausen, Michael Schantz; Anderson, Mads Valdemar; Jespersen, Martin Closter; Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    The accurate structural modeling of B- and T-cell receptors is fundamental to gain a detailed insight in the mechanisms underlying immunity and in developing new drugs and therapies. The LYRA (LYmphocyte Receptor Automated modeling) web server (http://www.cbs.dtu.dk/services/LYRA/) implements a complete and automated method for building of B- and T-cell receptor structural models starting from their amino acid sequence alone. The webserver is freely available and easy to use for non-specialists. Upon submission, LYRA automatically generates alignments using ad hoc profiles, predicts the structural class of each hypervariable loop, selects the best templates in an automatic fashion, and provides within minutes a complete 3D model that can be downloaded or inspected online. Experienced users can manually select or exclude template structures according to case specific information. LYRA is based on the canonical structure method, that in the last 30 years has been successfully used to generate antibody models of high accuracy, and in our benchmarks this approach proves to achieve similarly good results on TCR modeling, with a benchmarked average RMSD accuracy of 1.29 and 1.48 Å for B- and T-cell receptors, respectively. To the best of our knowledge, LYRA is the first automated server for the prediction of TCR structure. PMID:26007650

  8. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers

    PubMed Central

    Kojetin, Douglas J.; Matta-Camacho, Edna; Hughes, Travis S.; Srinivasan, Sathish; Nwachukwu, Jerome C.; Cavett, Valerie; Nowak, Jason; Chalmers, Michael J.; Marciano, David P.; Kamenecka, Theodore M.; Shulman, Andrew I.; Rance, Mark; Griffin, Patrick R.; Bruning, John B.; Nettles, Kendall W.

    2015-01-01

    A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses. PMID:26289479

  9. Structural basis of receptor sharing by interleukin 17 cytokines

    SciTech Connect

    Ely, Lauren K.; Fischer, Suzanne; Garcia, K. Christopher; Stanford-MED

    2010-02-19

    Interleukin 17 (IL-17)-producing helper T cells (T{sub H}-17 cells), together with their effector cytokines, including members of the IL-17 family, are emerging as key mediators of chronic inflammatory and autoimmune disorders. Here we present the crystal structure of a complex of IL-17 receptor A (IL-17RA) bound to IL-17F in a 1:2 stoichiometry. The mechanism of complex formation was unique for cytokines and involved the engagement of IL-17 by two fibronectin-type domains of IL-17RA in a groove between the IL-17 homodimer interface. Binding of the first receptor to the IL-17 cytokines modulated the affinity and specificity of the second receptor-binding event, thereby promoting heterodimeric versus homodimeric complex formation. IL-17RA used a common recognition strategy to bind to several members of the IL-17 family, which allows it to potentially act as a shared receptor in multiple different signaling complexes.

  10. Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

    PubMed

    Puthenkalam, Roshan; Hieckel, Marcel; Simeone, Xenia; Suwattanasophon, Chonticha; Feldbauer, Roman V; Ecker, Gerhard F; Ernst, Margot

    2016-01-01

    Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational

  11. Structure and organization of heteromeric AMPA-type glutamate receptors.

    PubMed

    Herguedas, Beatriz; García-Nafría, Javier; Cais, Ondrej; Fernández-Leiro, Rafael; Krieger, James; Ho, Hinze; Greger, Ingo H

    2016-04-29

    AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we report the first AMPAR heteromer structures, which deviate substantially from existing GluA2 homomer structures. Crystal structures of the GluA2/3 and GluA2/4 N-terminal domains reveal a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization is confirmed by cysteine cross-linking in full-length receptors, and it permitted us to determine the structure of an intact GluA2/3 receptor by cryogenic electron microscopy. Two models in the ligand-free state, at resolutions of 8.25 and 10.3 angstroms, exhibit substantial vertical compression and close associations between domain layers, reminiscent of N-methyl-D-aspartate receptors. Model 1 resembles a resting state and model 2 a desensitized state, thus providing snapshots of gating transitions in the nominal absence of ligand. Our data reveal organizational features of heteromeric AMPARs and provide a framework to decipher AMPAR architecture and signaling. PMID:26966189

  12. Structural and Functional Diversity of Estrogen Receptor Ligands

    PubMed Central

    Farooq, Amjad

    2015-01-01

    Estrogen receptors, comprised of ERα and ERβ isoforms in mammals, act as ligand-modulated transcription factors and orchestrate a plethora of cellular functions from sexual development and reproduction to metabolic homeostasis. Herein, I revisit the structural basis of the binding of ERα to DNA and estradiol in light of the recent discoveries and emerging trends in the field of nuclear receptors. A particular emphasis of this review is on the chemical and structural diversity of an ever-increasing repertoire of physiological, environmental and synthetic ligands of estrogen receptors that ultimately modulate their interactions with cognate DNA located within the promoters of estrogen-responsive genes. In particular, modulation of estrogen receptors by small molecule ligands represents an important therapeutic goal toward the treatment of a wide variety of human pathologies including breast cancer, cardiovascular disease, osteoporosis and obesity. Collectively, this article provides an overview of a wide array of small organic and inorganic molecules that can fine-tune the physiological function of estrogen receptors, thereby bearing a direct impact on human health and disease. PMID:25866274

  13. Structure and Assembly Mechanism for Heteromeric Kainate Receptors

    SciTech Connect

    Kumar, Janesh; Schuck, Peter; Mayer, Mark L.

    2012-10-25

    Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR57. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K{sub d} 11 nM, 32,000-fold lower than the K{sub d} for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.

  14. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    SciTech Connect

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  15. The perception of gibberellins: clues from receptor structure.

    PubMed

    Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

    2010-10-01

    The discovery of GID1, a soluble receptor for gibberellins (GAs), has revealed new insights into how GA is perceived. X-ray analysis has demonstrated similarities in the tertiary structure of GID1 to hormone sensitive lipase (HSL), and the GA-binding pocket of GID1 corresponds to the active site of HSL. X-ray analysis has also revealed the structural basis of the GA-GID1 interaction, and evolutionary aspects of GID1 have been discovered by comparison to GID1 from non-flowering plants. Recent studies have also demonstrated the complexity of GA signaling in Arabidopsis, which is mediated by three GID1 and five DELLA proteins. Finally, mechanistic and structural similarities for hormone signaling are compared for GA, auxin and abscisic acid, three hormones where the receptor protein structure was recently described. PMID:20851040

  16. Structural biology. Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine.

    PubMed

    Qin, Ling; Kufareva, Irina; Holden, Lauren G; Wang, Chong; Zheng, Yi; Zhao, Chunxia; Fenalti, Gustavo; Wu, Huixian; Han, Gye Won; Cherezov, Vadim; Abagyan, Ruben; Stevens, Raymond C; Handel, Tracy M

    2015-03-01

    Chemokines and their receptors control cell migration during development, immune system responses, and in numerous diseases, including inflammation and cancer. The structural basis of receptor:chemokine recognition has been a long-standing unanswered question due to the challenges of structure determination for membrane protein complexes. Here, we report the crystal structure of the chemokine receptor CXCR4 in complex with the viral chemokine antagonist vMIP-II at 3.1 angstrom resolution. The structure revealed a 1:1 stoichiometry and a more extensive binding interface than anticipated from the paradigmatic two-site model. The structure helped rationalize a large body of mutagenesis data and together with modeling provided insights into CXCR4 interactions with its endogenous ligand CXCL12, its ability to recognize diverse ligands, and the specificity of CC and CXC receptors for their respective chemokines. PMID:25612609

  17. Structure of the BoNT/A1--receptor complex.

    PubMed

    Benoit, Roger M; Frey, Daniel; Wieser, Mara M; Thieltges, Katherine M; Jaussi, Rolf; Capitani, Guido; Kammerer, Richard A

    2015-12-01

    Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C. Based on our findings, we discuss the potential implications for BoNT/A function. PMID:26260692

  18. Crystal Structures of the β2-Adrenergic Receptor

    NASA Astrophysics Data System (ADS)

    Weis, William I.; Rosenbaum, Daniel M.; Rasmussen, Søren G. F.; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Yao, Xiao-Jie; Day, Peter W.; Parnot, Charles; Fung, Juan J.; Ratnala, Venkata R. P.; Kobilka, Brian K.; Cherezov, Vadim; Hanson, Michael A.; Kuhn, Peter; Stevens, Raymond C.; Edwards, Patricia C.; Schertler, Gebhard F. X.; Burghammer, Manfred; Sanishvili, Ruslan; Fischetti, Robert F.; Masood, Asna; Rohrer, Daniel K.

    G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are responsible for the majority of signal transduction events involving hormones and neuro-transmitters across the cell membrane. GPCRs that bind to diffusible ligands have low natural abundance, are relatively unstable in detergents, and display basal G protein activation even in the absence of ligands. To overcome these problems two approaches were taken to obtain crystal structures of the β2-adrenergic receptor (β2AR), a well-characterized GPCR that binds cate-cholamine hormones. The receptor was bound to the partial inverse agonist carazolol and co-crystallized with a Fab made to a three-dimensional epitope formed by the third intracellular loop (ICL3), or by replacement of ICL3 with T4 lysozyme. Small crystals were obtained in lipid bicelles (β2AR-Fab) or lipidic cubic phase (β2AR-T4 lysozyme), and diffraction data were obtained using microfocus technology. The structures provide insights into the basal activity of the receptor, the structural features that enable binding of diffusible ligands, and the coupling between ligand binding and G-protein activation.

  19. Scavenger Receptor Structure and Function in Health and Disease

    PubMed Central

    Abdul Zani, Izma; Stephen, Sam L.; Mughal, Nadeem A.; Russell, David; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2015-01-01

    Scavenger receptors (SRs) are a ‘superfamily’ of membrane-bound receptors that were initially thought to bind and internalize modified low-density lipoprotein (LDL), though it is currently known to bind to a variety of ligands including endogenous proteins and pathogens. New family of SRs and their properties have been identified in recent years, and have now been classified into 10 eukaryote families, defined as Classes A-J. These receptors are classified according to their sequences, although in each class they are further classified based in the variations of the sequence. Their ability to bind a range of ligands is reflected on the biological functions such as clearance of modified lipoproteins and pathogens. SR members regulate pathophysiological states including atherosclerosis, pathogen infections, immune surveillance, and cancer. Here, we review our current understanding of SR structure and function implicated in health and disease. PMID:26010753

  20. Structural basis for selective activation of ABA receptors

    SciTech Connect

    Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F.

    2010-11-01

    Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

  1. The human prostacyclin receptor from structure function to disease.

    PubMed

    Martin, Kathleen A; Gleim, Scott; Elderon, Larkin; Fetalvero, Kristina; Hwa, John

    2009-01-01

    Thirty years have passed since Vane and colleagues first described a substance, prostanoid X, from microsomal fractions (later called prostacyclin) that relaxed rather than contracted mesenteric arteries. The critical role of prostacyclin in many pathophysiological conditions, such as atherothrombosis, has only recently become appreciated (through receptor knockout mice studies, selective cyclooxygenase-2 inhibition clinical trials, and the discovery of dysfunctional prostacyclin receptor genetic variants). Additionally, important roles in such diverse areas as pain and inflammation, and parturition are being uncovered. Prostacyclin-based therapies, currently used for pulmonary hypertension, are accordingly emerging as possible treatments for such diseases, fueling interests in structure function studies for the receptor and signal transduction pathways in native cells. The coming decade is likely to yield many further exciting advances. PMID:20374736

  2. [Molecular structure of luminal diuretic receptors].

    PubMed

    Gamba, G

    1995-01-01

    Since day to day sodium and water intake is more or less constant, the output by urinary sodium excretion is the key to maintain extracellular fluid volume within physiologic ranges. To achieve this goal, the kidneys ensure that most of the large quantities of filtered sodium are reabsorbed, a function that takes place in the proximal tubule, the loop of Henle and the distal tubule, and then the kidneys adjust the small amount of sodium that is excreted in urine in such a way that sodium balance is maintained. This adjustment occurs in the collecting duct. Three groups of diuretic-sensitive sodium transport mechanisms have been identified in the apical membranes of the distal nephron based on their different sensitivities to diuretics and requirements for chloride and potassium: 1) the sulfamoylbenzoic (or bumetanide)-sensitive Na+:K+:2CI- and Na+:CI- symporters in the thick ascending loop of Henle; 2) the benzothiadiazine (or thiazide)-sensitive Na+:CI- cotransporter in the distal tubule; and 3) the amiloride-sensitive Na+ channel in the collecting tubule. The inhibition of any one of these proteins by diuretics results in increased sodium urinary excretion. Recently, the use of molecular biology techniques, specially the functional expression cloning in Xenopus laevis oocytes, has led to the identification of cDNA's encoding members of the three groups of diuretic-sensitive transport proteins. The present paper reviews the primary structure and some aspects of the relationship between structure and function of these transporters as well as the new protein families emerging from these sequences. It also discusses the future implications of these discoveries on the physiology and pathophysiology of kidney disease and sodium retaining states. PMID:7569367

  3. Structural and functional interactions between six-transmembrane μ-opioid receptors and β2-adrenoreceptors modulate opioid signaling

    PubMed Central

    Samoshkin, Alexander; Convertino, Marino; Viet, Chi T.; Wieskopf, Jeffrey S.; Kambur, Oleg; Marcovitz, Jaclyn; Patel, Pinkal; Stone, Laura S.; Kalso, Eija; Mogil, Jeffrey S.; Schmidt, Brian L.; Maixner, William; Dokholyan, Nikolay V.; Diatchenko, Luda

    2015-01-01

    The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β2-adrenergic receptors (β2-ARs) through an interaction with the fifth and sixth helices of β2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy. PMID:26657998

  4. Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

    PubMed Central

    Mathäs, Marianne; Burk, Oliver; Gödtel-Armbrust, Ute; Herlyn, Holger; Wojnowski, Leszek; Windshügel, Björn

    2014-01-01

    The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution. PMID:24797902

  5. Estrogen receptor transcription and transactivation: Structure-function relationship in DNA- and ligand-binding domains of estrogen receptors

    PubMed Central

    Ruff, Marc; Gangloff, Monique; Marie Wurtz, Jean; Moras, Dino

    2000-01-01

    Estrogen receptors are members of the nuclear receptor steroid family that exhibit specific structural features, ligand-binding domain sequence identity and dimeric interactions, that single them out. The crystal structures of their DNA-binding domains give some insight into how nuclear receptors discriminate between DNA response elements. The various ligand-binding domain crystal structures of the two known estrogen receptor isotypes (α and β) allow one to interpret ligand specificity and reveal the interactions responsible for stabilizing the activation helix H12 in the agonist and antagonist positions. PMID:11250728

  6. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    SciTech Connect

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Sawaya, Michael R.; Xu, Qingping; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Yefanov, Oleksandr M.; White, Thomas A.; Wang, Chong; Ishchenko, Andrii; Tirupula, Kalyan C.; Desnoyer, Russell; Coe, Jesse; Conrad, Chelsie E.; Fromme, Petra; Stevens, Raymond C.; Katritch, Vsevolod; Karnik, Sadashiva S.; Cherezov, Vadim

    2015-05-07

    We report that angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. The AT1R-ZD7155 complex structure revealed key structural features ofAT1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.

  7. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    DOE PAGESBeta

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; et al

    2015-05-07

    We report that angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. Themore » AT1R-ZD7155 complex structure revealed key structural features ofAT1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.« less

  8. Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

    PubMed Central

    Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; Han, Gye Won; Liu, Wei; Zatsepin, Nadia A.; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Sawaya, Michael R.; Xu, Qingping; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Yefanov, Oleksandr M.; White, Thomas A.; Wang, Chong; Ishchenko, Andrii; Tirupula, Kalyan C.; Desnoyer, Russell; Coe, Jesse; Conrad, Chelsie E.; Fromme, Petra; Stevens, Raymond C.; Katritch, Vsevolod; Karnik, Sadashiva S.; Cherezov, Vadim

    2015-01-01

    SUMMARY Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. PMID:25913193

  9. μ Opioid receptor: novel antagonists and structural modeling

    NASA Astrophysics Data System (ADS)

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

    2016-02-01

    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

  10. μ Opioid receptor: novel antagonists and structural modeling

    PubMed Central

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

    2016-01-01

    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates. PMID:26888328

  11. Structural basis for collagen recognition by the immune receptor OSCAR

    PubMed Central

    Zhou, Long; Hinerman, Jennifer M.; Blaszczyk, Michal; Miller, Jeanette L. C.; Conrady, Deborah G.; Barrow, Alexander D.; Chirgadze, Dimitri Y.; Bihan, Dominique; Farndale, Richard W.

    2016-01-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  12. Structural Allostery and Binding of the Transferring Receptor Complex

    SciTech Connect

    Xu,G.; Liu, R.; Zak, O.; Aisen, P.; Chance, M.

    2005-01-01

    The structural allostery and binding interface for the human serum transferrin (Tf){center_dot}transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions. The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

  13. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  14. Structure of the heterodimeric ecdysone receptor DNA-binding complex

    PubMed Central

    Devarakonda, Srikripa; Harp, Joel M.; Kim, Youngchang; Ożyhar, Andrzej; Rastinejad, Fraydoon

    2003-01-01

    Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). The EcR–USP heterodimer preferentially mediates transcription through highly degenerate pseudo-palindromic response elements, resembling inverted repeats of 5′-AGGTCA-3′ separated by 1 bp (IR-1). The requirement for a heterodimeric arrangement of EcR–USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. We describe the 2.24 Å structure of the EcR–USP DNA-binding domain (DBD) heterodimer bound to an idealized IR-1 element. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein–protein contacts. As retinoid X receptor (RXR) is the mammalian homolog of USP, we also solved the 2.60 Å crystal structure of the EcR–RXR DBD heterodimer on IR-1 and found the dimerization and DNA-binding interfaces to be the same as in the EcR–USP complex. Sequence alignments indicate that the EcR–RXR heterodimer is an important model for understanding how the FXR–RXR heterodimer binds to IR-1 sites. PMID:14592980

  15. The venus kinase receptor (VKR) family: structure and evolution

    PubMed Central

    2013-01-01

    Background Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. Results Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. Conclusions This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology. PMID:23721482

  16. Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers

    PubMed Central

    Park, Sang Ho; Das, Bibhuti B.; Casagrande, Fabio; Tian, Ye; Nothnagel, Henry J.; Chu, Mignon; Kiefer, Hans; Maier, Klaus; De Angelis, Anna; Marassi, Francesca M.; Opella, Stanley J.

    2013-01-01

    CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth1-3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors4-7. Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are critical for G-protein interactions11 and activation of CXCR1 involves both N-terminal residues and extracellular loops2,12,13. Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. PMID:23086146

  17. Bioinformatic Analysis of Toll-Like Receptor Sequences and Structures.

    PubMed

    Monie, Tom P; Gay, Nicholas J; Gangloff, Monique

    2016-01-01

    Continual advancements in computing power and sophistication, coupled with rapid increases in protein sequence and structural information, have made bioinformatic tools an invaluable resource for the molecular and structural biologist. With the degree of sequence information continuing to expand at an almost exponential rate, it is essential that scientists today have a basic understanding of how to utilise, manipulate and analyse this information for the benefit of their own experiments. In the context of Toll-Interleukin I Receptor domain containing proteins, we describe here a series of the more common and user-friendly bioinformatic tools available as Internet-based resources. These will enable the identification and alignment of protein sequences; the identification of functional motifs; the characterisation of protein secondary structure; the identification of protein structural folds and distantly homologous proteins; and the validation of the structural geometry of modelled protein structures. PMID:26803620

  18. The D3 dopamine receptor: From structural interactions to function.

    PubMed

    Fiorentini, Chiara; Savoia, Paola; Bono, Federica; Tallarico, Paola; Missale, Cristina

    2015-09-01

    Novel structural and functional aspects of the dopamine (DA) D3 receptors (D3R) have been recently described. D3R expressed in dopaminergic neurons have been classically considered to play the role of autoreceptors inhibiting, as the D2R, DA release. However, evidence for D3R-mediated neurotrophic and neuroprotective effects on DA neurons suggests their involvement in preventing pathological alterations leading to neurodegeneration. On the other hand, given its localization and functional role at postsynaptic striatal levels, the D3R may also be involved in the pathogenesis of movement disorders and psychiatric diseases. Functional interactions of D3R with other receptor systems are crucial for the modulation of several physiological events. On this line, the discovery that the D3R can form heteromers with other receptors has opened the possibility of uncover novel molecular mechanisms of brain functions and dysfunctions. This paper summarizes the functional and physical interactions of D3R with other receptors both at pre-synaptic sites, where it is co-expressed with the D2R and nicotinic receptors, and at post-synaptic sites where it interacts with the DA D1 receptors (D1R). The biochemical and functional properties of the D1R-D3R heteromer will be especially discussed. Both D1R and D3R have been in fact implicated in several disorders, including schizophrenia and motor dysfunctions. Therefore, the D1R-D3R heteromer may represent a potential drug target for the treatment of these diseases. PMID:25532864

  19. A combined computational and structural model of the full-length human prolactin receptor

    PubMed Central

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-01-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg. PMID:27174498

  20. A combined computational and structural model of the full-length human prolactin receptor

    NASA Astrophysics Data System (ADS)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-05-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  1. Crystal Structure of the Human Laminin Receptor Precursor

    SciTech Connect

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.

    2008-01-01

    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  2. Structural homology between lymphocyte receptors for high endothelium and class III extracellular matrix receptor.

    PubMed Central

    Gallatin, W M; Wayner, E A; Hoffman, P A; St John, T; Butcher, E C; Carter, W G

    1989-01-01

    We have identified extensive structural homology between one type of heterotypic adhesion receptor (HAR) involved in lymphocyte interactions with high endothelium in lymphoid organs and a collagen-binding protein, termed class III extracellular matrix receptor (ECMRIII), expressed on most nucleated cell types. Both receptors have been described as heterogeneous 90-kDa transmembrane glycoproteins, referred to here as gp90. Monoclonal anti-HAR antibodies, Hermes-1 and Hutch-1, and monoclonal anti-ECMRIII antibodies, P1G12 and P3H9, were utilized to compare the two receptors. (i) All these monoclonal antibodies immunoprecipitated major gp90 components as well as uncharacterized additional higher molecular mass antigens of 120-200 kDa in human and macaque fibroblasts and peripheral blood mononuclear cells. (ii) Competitive binding analyses with the antibodies identified distinct epitopes present on gp90. (iii) Enzymatic and chemical digestions generated identical peptide fragments from all the antigens in human and macaque fibroblasts and peripheral blood mononuclear cells. (iv) Sequential immunoprecipitation with P1G12 followed by the other monoclonal antibodies indicated that all gp90 species reactive with Hermes-1 and Hutch-1 also expressed the P1G12 defined epitope. In reciprocal experiments, Hermes-1 and Hutch-1 immunoprecipitation did not completely remove all P1G12-reactive gp90 from cellular extracts. One inference from these data would be that gp90 is serologically heterogeneous, encompassing HARs as a major subset of this broadly expressed class of molecules. Images PMID:2471973

  3. Androgen receptor: structure, role in prostate cancer and drug discovery

    PubMed Central

    Tan, MH Eileen; Li, Jun; Xu, H Eric; Melcher, Karsten; Yong, Eu-leong

    2015-01-01

    Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2–3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein. PMID:24909511

  4. Structural insights into μ-opioid receptor activation

    PubMed Central

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A. J.; Laeremans, Toon; Feinberg, Evan N.; Sanborn, Adrian L.; Kato, Hideaki E.; Livingston, Kathryn E.; Thorsen, Thor S.; Kling, Ralf; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M.; Traynor, John R.; Weis, William I.; Steyaert, Jan; Dror, Ron O.; Kobilka, Brian K.

    2015-01-01

    Summary Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To understand the structural basis for μOR activation, we obtained a 2.1 Å X-ray crystal structure of the μOR bound to the morphinan agonist BU72 and stabilized by a G protein-mimetic camelid-antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2 adrenergic receptor (β2AR) and the M2 muscarinic receptor (M2R). Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three GPCRs. PMID:26245379

  5. Structural and Functional Attributes of the Interleukin-36 Receptor.

    PubMed

    Yi, Guanghui; Ybe, Joel A; Saha, Siddhartha S; Caviness, Gary; Raymond, Ernest; Ganesan, Rajkumar; Mbow, M Lamine; Kao, C Cheng

    2016-08-01

    Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP. PMID:27307043

  6. Differential Regulation of 6- and 7-Transmembrane Helix Variants of μ-Opioid Receptor in Response to Morphine Stimulation

    PubMed Central

    Convertino, Marino; Samoshkin, Alexander; Viet, Chi T.; Gauthier, Josee; Li Fraine, Steven P.; Sharif-Naeini, Reza; Schmidt, Brian L.; Maixner, William; Diatchenko, Luda; Dokholyan, Nikolay V.

    2015-01-01

    The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids. PMID:26554831

  7. Structure of human Aichi virus and implications for receptor binding.

    PubMed

    Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Yuan, Shuai; Yamashita, Teruo; Tuthill, Tobias J; Fry, Elizabeth E; Rao, Zihe; Stuart, David I

    2016-01-01

    Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries(1,2). The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life(3,4). There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification. PMID:27595320

  8. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  9. Structural and Functional Profiling of Environmental Ligands for Estrogen Receptors

    PubMed Central

    Delfosse, Vanessa; Grimaldi, Marina; Cavaillès, Vincent

    2014-01-01

    Background: Individuals are exposed daily to environmental pollutants that may act as endocrine-disrupting chemicals (EDCs), causing a range of developmental, reproductive, metabolic, or neoplastic diseases. With their mostly hydrophobic pocket that serves as a docking site for endogenous and exogenous ligands, nuclear receptors (NRs) can be primary targets of small molecule environmental contaminants. However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable. Objectives: We conducted a correlative analysis of structural and functional data to gain insight into the mechanisms by which 12 members of representative families of pollutants bind to and activate the estrogen receptors ERα and ERβ. Methods: We used a battery of biochemical, structural, biophysical, and cell-based approaches to characterize the interaction between ERs and their environmental ligands. Results: Our study revealed that the chemically diverse compounds bound to ERs via varied sets of protein–ligand interactions, reflecting their differential activities, binding affinities, and specificities. We observed xenoestrogens binding to both ERs—with affinities ranging from subnanomolar to micromolar values—and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ. Conclusions: The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment. Citation: Delfosse V, Grimaldi M, Cavaillès V, Balaguer P, Bourguet W. 2014. Structural and functional profiling of environmental ligands for estrogen

  10. Structure of the LDL receptor extracellular domain at endosomalpH

    SciTech Connect

    Rudenko, Gabby; Henry, Lisa; Henderson, Keith; Ichtchenko,Konstantin; Brown, Michael S.; Goldstein, Joseph L.; Deisenhofer, Johann

    2002-09-05

    The structure of the low-density lipoprotein receptor extracellular portion has been determined. The document proposes a mechanism for the release of lipoprotein in the endosome. Without this release, the mechanism of receptor recycling cannot function.

  11. Structural rearrangement of the intracellular domains during AMPA receptor activation.

    PubMed

    Zachariassen, Linda G; Katchan, Ljudmila; Jensen, Anna G; Pickering, Darryl S; Plested, Andrew J R; Kristensen, Anders S

    2016-07-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  12. Human antibody-Fc receptor interactions illuminated by crystal structures.

    PubMed

    Woof, Jenny M; Burton, Dennis R

    2004-02-01

    Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies. PMID:15040582

  13. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

    PubMed Central

    Hermans, E; Challiss, R A

    2001-01-01

    In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders. PMID:11672421

  14. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

    PubMed

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S; Balle, Thomas

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. PMID:26572235

  15. Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

    PubMed

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei; Laustsen, Anne Kjær; Koldsø, Heidi; Skeby, Katrine Kirkeby; Schiøtt, Birgit; Andersen, Gregers R; Vorup-Jensen, Thomas

    2016-08-12

    The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists. PMID:27339893

  16. Structure of the STRA6 receptor for retinol uptake.

    PubMed

    Chen, Yunting; Clarke, Oliver B; Kim, Jonathan; Stowe, Sean; Kim, Youn-Kyung; Assur, Zahra; Cavalier, Michael; Godoy-Ruiz, Raquel; von Alpen, Desiree C; Manzini, Chiara; Blaner, William S; Frank, Joachim; Quadro, Loredana; Weber, David J; Shapiro, Lawrence; Hendrickson, Wayne A; Mancia, Filippo

    2016-08-26

    Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer. PMID:27563101

  17. Defining the structural relationship between kainate receptor deactivation and desensitization

    PubMed Central

    Dawe, G. Brent; Musgaard, Maria; Andrews, Elizabeth D.; Daniels, Bryan A.; Aurousseau, Mark R.P.; Biggin, Philip C.; Bowie, Derek

    2016-01-01

    Desensitization is an important mechanism that curtails the activity of ligand-gated ion-channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by the physicochemical properties of the bound ligand. Here, we show the importance of an allosteric cation binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs, identifying cations as gatekeepers of KAR gating, a role unique among even closely-related LGICs. PMID:23955023

  18. Structural basis for gibberellin recognition by its receptor GID1.

    PubMed

    Shimada, Asako; Ueguchi-Tanaka, Miyako; Nakatsu, Toru; Nakajima, Masatoshi; Naoe, Youichi; Ohmiya, Hiroko; Kato, Hiroaki; Matsuoka, Makoto

    2008-11-27

    Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA(4) and GA(3) at 1.9 A resolution. The overall structure of both complexes shows an alpha/beta-hydrolase fold similar to that of HSLs except for an amino-terminal lid. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val-residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s-caused an increase in the binding affinity for GA(34), a 2beta-hydroxylated GA(4). These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution. PMID:19037316

  19. Structures and regulation of non-X orphan nuclear receptors: A retinoid hypothesis.

    PubMed

    Zhi, Xiaoyong; Zhou, X Edward; Melcher, Karsten; Xu, H Eric

    2016-03-01

    Nuclear receptors are defined as a family of ligand regulated transcription factors [1-6]. While this definition reflects that ligand binding is a key property of nuclear receptors, it is still a heated subject of debate if all the nuclear receptors (48 human members) can bind ligands (ligands referred here to both physiological and synthetic ligands). Recent studies in nuclear receptor structure biology and pharmacology have undoubtedly increased our knowledge of nuclear receptor functions and their regulation. As a result, they point to new avenues for the discovery and development of nuclear receptor regulators, including nuclear receptor ligands. Here we review the recent literature on orphan nuclear receptor structural analysis and ligand identification, particularly on the orphan nuclear receptors that do not heterodimerize with retinoid X receptors, which we term as non-X orphan receptors. We also propose a speculative "retinoid hypothesis" for a subset of non-X orphan nuclear receptors, which we hope to help shed light on orphan nuclear receptor biology and drug discovery. This article is part of a Special Issue entitled 'Orphan Nuclear Receptors'. PMID:26159912

  20. Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction.

    PubMed

    Watanabe, T; Shintani, A; Nakata, M; Shing, Y; Folkman, J; Igarashi, K; Sasada, R

    1994-04-01

    Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR. PMID:8144591

  1. Androgen Receptor Structure, Function and Biology: From Bench to Bedside

    PubMed Central

    Davey, Rachel A; Grossmann, Mathis

    2016-01-01

    The actions of androgens such as testosterone and dihydrotestosterone are mediated via the androgen receptor (AR), a ligand-dependent nuclear transcription factor and member of the steroid hormone nuclear receptor family. Given its widespread expression in many cells and tissues, the AR has a diverse range of biological actions including important roles in the development and maintenance of the reproductive, musculoskeletal, cardiovascular, immune, neural and haemopoietic systems. AR signalling may also be involved in the development of tumours in the prostate, bladder, liver, kidney and lung. Androgens can exert their actions via the AR in a DNA binding-dependent manner to regulate target gene transcription, or in a non-DNA binding-dependent manner to initiate rapid, cellular events such as the phosphorylation of 2nd messenger signalling cascades. More recently, ligand-independent actions of the AR have also been identified. Given the large volume of studies relating to androgens and the AR, this review is not intended as an extensive review of all studies investigating the AR, but rather as an overview of the structure, function, signalling pathways and biology of the AR as well as its important role in clinical medicine, with emphasis on recent developments in this field. PMID:27057074

  2. Structural basis of dynamic glycine receptor clustering by gephyrin

    PubMed Central

    Sola, Maria; Bavro, Vassiliy N; Timmins, Joanna; Franz, Thomas; Ricard-Blum, Sylvie; Schoehn, Guy; Ruigrok, Rob W H; Paarmann, Ingo; Saiyed, Taslimarif; O'Sullivan, Gregory A; Schmitt, Bertram; Betz, Heinrich; Weissenhorn, Winfried

    2004-01-01

    Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR β-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the β-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity. PMID:15201864

  3. Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin

    NASA Astrophysics Data System (ADS)

    Ward, Meaghan E.; Brown, Leonid S.; Ladizhansky, Vladimir

    2015-04-01

    Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic α-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination. In this Perspective article we describe our recent efforts directed at determining the structure and functional dynamics of Anabaena Sensory Rhodopsin, a heptahelical transmembrane (7TM) protein. We review some of the established and emerging methods which can be utilized for SSNMR-based structure determination, with a particular focus on those used for ASR, a bacterial protein which shares its 7TM architecture with G-protein coupled receptors.

  4. Angiotensin Receptors: Structure, Function, Signaling and Clinical Applications

    PubMed Central

    Singh, Khuraijam Dhanachandra; Karnik, Sadashiva S

    2016-01-01

    Angiotensinogen – a serpin family protein predominantly produced by the liver is systematically processed by proteases of the Renin Angiotensin system (RAS) generating hormone peptides. Specific cell surface receptors for at least three distinct angiotensin peptides produce distinct cellular signals that regulate system-wide physiological response to RAS. Two well characterized receptors are angiotensin type 1 receptor (AT1 receptor) and type 2 receptor (AT2 receptor). They respond to the octapeptide hormone angiotensin II. The oncogene product MAS is a putative receptor for Ang (1–7). While these are G-protein coupled receptors (GPCRs), the in vivo angiotensin IV binding sites may be type 2 transmembrane proteins. These four receptors together regulate cardiovascular, hemodynamic, neurological, renal, and endothelial functions; as well as cell proliferation, survival, matrix-cell interactions and inflammation. Angiotensin receptors are important therapeutic targets for several diseases. Thus, researchers and pharmaceutical companies are focusing on drugs targeting AT1 receptor than AT2 receptor, MAS and AngIV binding sites. AT1 receptor blockers are the cornerstone of current treatment for hypertension, heart failure, renal failure and many types of vascular diseases including atherosclerosis, aortic aneurism and Marfan syndrome. PMID:27512731

  5. Magnetically-refreshable receptor platform structures for reusable nano-biosensor chips

    NASA Astrophysics Data System (ADS)

    Yoo, Haneul; Lee, Dong Jun; Cho, Dong-guk; Park, Juhun; Nam, Ki Wan; Tak Cho, Young; Park, Jae Yeol; Chen, Xing; Hong, Seunghun

    2016-01-01

    We developed a magnetically-refreshable receptor platform structure which can be integrated with quite versatile nano-biosensor structures to build reusable nano-biosensor chips. This structure allows one to easily remove used receptor molecules from a biosensor surface and reuse the biosensor for repeated sensing operations. Using this structure, we demonstrated reusable immunofluorescence biosensors. Significantly, since our method allows one to place receptor molecules very close to a nano-biosensor surface, it can be utilized to build reusable carbon nanotube transistor-based biosensors which require receptor molecules within a Debye length from the sensor surface. Furthermore, we also show that a single sensor chip can be utilized to detect two different target molecules simply by replacing receptor molecules using our method. Since this method does not rely on any chemical reaction to refresh sensor chips, it can be utilized for versatile biosensor structures and virtually-general receptor molecular species.

  6. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  7. Structural basis of transcobalamin recognition by human CD320 receptor

    PubMed Central

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  8. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  9. Structural basis of transcobalamin recognition by human CD320 receptor

    NASA Astrophysics Data System (ADS)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  10. Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

    PubMed Central

    Poulter, L; Earnest, J P; Stroud, R M; Burlingame, A L

    1989-01-01

    Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide. Images PMID:2771948

  11. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems.

    PubMed

    Toll, Lawrence; Bruchas, Michael R; Calo', Girolamo; Cox, Brian M; Zaveri, Nurulain T

    2016-04-01

    The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor. PMID:26956246

  12. The Design and Structure of Outer Membrane Receptors from Peroxisomes, Mitochondria, and Chloroplasts.

    PubMed

    Panigrahi, Rashmi; Kubiszewski-Jakubiak, Szymon; Whelan, James; Vrielink, Alice

    2015-10-01

    The eukaryotic cell is defined by compartments that allow specialization of function. This compartmental structure generates a new concept in cell biology compared with the simpler prokaryotic cell structure, namely the specific targeting of proteins to intracellular compartments. Protein targeting is achieved by the action of specialized signals on proteins destined for organelles that are recognized by cognate receptors. An understanding of the specificity of targeting signal recognition leading to import requires an understanding of the receptor structures. Here, we focus on the structures of receptors of different import machineries located on the outer membrane of three organelles: peroxisomes, mitochondria, and chloroplasts. This review provides an overview of the structural features of outer membrane import receptors that recognize targeting signals. Finally, we briefly discuss combinatorial approaches that might aid in understanding the structural factors mediating receptor targeting signal recognition. PMID:26365798

  13. The structure and function of cutaneous sensory receptors.

    PubMed

    Munger, B L; Ide, C

    1988-03-01

    The present review of cutaneous sensory receptors begins with a consideration of free nerve endings (FNEs) that can be considered as sensory terminals evidencing the least structural specialization of the axon and associated cells. Using the criteria established by Kruger et al (1981), FNEs of both A delta and C fibers can be identified on the basis of ultrastructural characteristics that include an intimate relationship between axons and the associated epithelium, the lack of a complete Schwann cell investment, the accumulation of numerous vesicles and other cytoplasmic organelles, and for A delta terminals a 1:1 relationship between axon and investing Schwann cell. Using these criteria, the so-called genital end bulbs of the human glans penis are merely a skein of FNEs based on the ultrastructural study of Halata and Munger (1986). Hair follicles of most species studied to date (the exception being the rabbit and to some extent the guinea pig) are multiply innervated with lanceolate, Ruffini and FNEs. The lanceolate terminals are the rapidly adapting terminals that are numerous in guard hairs. Ruffini terminals of hairs resemble those of the periodontal ligament or joint capsules and both are remarkably similar to Golgi tendon organs in terms of ultrastructural characteristics. The key ultrastructural characteristic is the encircling of collagen bundles by axons and associated Schwann and connective tissue cells. Axons frequently enter the epidermis either to terminate as FNEs or become associated with Merkel cells in glabrous skin at the base of the papillary ridges or in clusters of Merkel cells in hairy skin in touch domes or Haarscheiben. Merkel cells have clusters of apparent secretory granules polarized toward the axon and the axon is typically a slowly adapting mechanoreceptor. The function of the granules is not known. Pacinian corpuscles are the largest of the corpuscular receptors of the dermis and are characterized by an elaborate inner core of stacks

  14. New insights into structural determinants for prostanoid thromboxane A2 receptor- and prostacyclin receptor-G protein coupling.

    PubMed

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Bhullar, Rajinder Pal; Hwa, John; Dakshinamurti, Shyamala; Chelikani, Prashen

    2013-01-01

    G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease. PMID:23109431

  15. New Insights into Structural Determinants for Prostanoid Thromboxane A2 Receptor- and Prostacyclin Receptor-G Protein Coupling

    PubMed Central

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Bhullar, Rajinder Pal; Hwa, John; Dakshinamurti, Shyamala

    2013-01-01

    G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA2) receptor (TP) and prostacyclin (PGI2) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease. PMID:23109431

  16. Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system.

    PubMed

    Deleuze, C; Runquist, M; Orcel, H; Rabié, A; Dayanithi, G; Alonso, G; Hussy, N

    2005-01-01

    Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments. PMID:16125853

  17. GPCRDB: an information system for G protein-coupled receptors.

    PubMed

    Isberg, Vignir; Vroling, Bas; van der Kant, Rob; Li, Kang; Vriend, Gert; Gloriam, David

    2014-01-01

    For the past 20 years, the GPCRDB (G protein-coupled receptors database; http://www.gpcr.org/7tm/) has been a 'one-stop shop' for G protein-coupled receptor (GPCR)-related data. The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data, such as multiple sequence alignments and homology models. The GPCRDB also provides visualization and analysis tools, plus a number of query systems. In the latest GPCRDB release, all multiple sequence alignments, and >65,000 homology models, have been significantly improved, thanks to a recent flurry of GPCR X-ray structure data. Tools were introduced to browse X-ray structures, compare binding sites, profile similar receptors and generate amino acid conservation statistics. Snake plots and helix box diagrams can now be custom coloured (e.g. by chemical properties or mutation data) and saved as figures. A series of sequence alignment visualization tools has been added, and sequence alignments can now be created for subsets of sequences and sequence positions, and alignment statistics can be produced for any of these subsets. PMID:24304901

  18. Structure-based receptor MIMICS targeted against bacterial superantigen toxins

    DOEpatents

    Gupta, Goutam; Hong-Geller, Elizabeth; Shiflett, Patrick R.; Lehnert, Nancy M.

    2009-08-18

    The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

  19. Melatonin receptors and their regulation: biochemical and structural mechanisms.

    PubMed

    Witt-Enderby, Paula A; Bennett, Jennifer; Jarzynka, Michael J; Firestine, Steven; Melan, Melissa A

    2003-04-01

    There is growing evidence demonstrating the complexity of melatonin's role in modulating a diverse number of physiological processes. This complexity could be attributed to the fact that melatonin receptors belong to two distinct classes of proteins, that is, the G-protein coupled receptor superfamily (MT(1), MT(2)) and the quinone reductase enzyme family (MT(3)) which makes them unique at the molecular level. Also, within the G-protein coupled receptor family of proteins, the MT(1) and MT(2) receptors can couple to multiple and distinct signal transduction cascades whose activation can lead to unique cellular responses. Also, throughout the 24-hour cycle, the receptors' sensitivity to specific cues fluctuates and this sensitivity can be modulated in a homologous fashion, that is, by melatonin itself, and in a heterologous manner, that is, by other cues including the photoperiod or estrogen. This sensitivity of response may reflect changes in melatonin receptor density that also occurs throughout the 24-hour light/dark cycle but out of phase with circulating melatonin levels. The mechanisms that underlie the changes in melatonin receptor density and function are still not well-understood, but data is beginning to show that transcriptional events and G-protein uncoupling may be involved. Even though this area of research is still in its infancy, great strides are being made everyday in elucidating the mechanisms that underlie melatonin receptor function and regulation. The focus of this review is to highlight some of these discoveries in an attempt to reveal the uniqueness of the melatonin receptor family while at the same time provide thought-provoking ideas to further advance this area of research. Thus, a brief overview of each of the mammalian melatonin receptor subtypes and the signal transduction cascades to which they couple will be discussed with a greater emphasis placed on the mechanisms underlying their regulation and the domains within the receptors

  20. Structure of the retinoid X receptor α-liver X receptor β (RXRα-LXRβ) heterodimer on DNA

    SciTech Connect

    Lou, Xiaohua; Toresson, Gudrun; Benod, Cindy; Suh, Ji Ho; Philips, Kevin J; Webb, Paul; Gustafsson, Jan-Ake

    2014-03-11

    Nuclear receptors (NRs) are conditional transcription factors with common multidomain organization that bind diverse DNA elements. How DNA sequences influence NR conformation is poorly understood. Here we report the crystal structure of the human retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on its cognate element, an AGGTCA direct repeat spaced by 4 nt. The complex has an extended X-shaped arrangement, with DNA- and ligand-binding domains crossed, in contrast to the parallel domain arrangement of other NRs that bind an AGGTCA direct repeat spaced by 1 nt. The LXRβ core binds DNA via canonical contacts and auxiliary DNA contacts that enhance affinity for the response element. Comparisons of RXRα–LXRβs in the crystal asymmetric unit and with previous NR structures reveal flexibility in NR organization and suggest a role for RXRα in adaptation of heterodimeric complexes to DNA.

  1. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2004-05-18

    Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

  2. Structural basis and functions of abscisic acid receptors PYLs

    PubMed Central

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

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

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    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.

  4. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    PubMed

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition. PMID:17077558

  5. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    PubMed

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  6. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  7. High potency olfactory receptor agonists discovered by virtual high-throughput screening: molecular probes for receptor structure and olfactory function

    PubMed Central

    Triballeau, Nicolas; Van Name, Eric; Laslier, Guillaume; Cai, Diana; Paillard, Guillaume; Sorensen, Peter W.; Hoffmann, Rémy; Bertrand, Hugues-Olivier; Ngai, John; Acher, Francine C.

    2008-01-01

    The detection and discrimination of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study we used a computational high-throughput screening strategy to discover novel high affinity agonists of an olfactory G protein-coupled receptor tuned to recognize amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor’s known natural ligands. Computational modeling revealed molecular interactions involved in ligand recognition by this receptor, and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo, and include a natural product that may be used to signal the presence of bacteria in the aquatic environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo. PMID:19081373

  8. The Structural Basis for Class II Cytokine Receptor Recognition by JAK1.

    PubMed

    Ferrao, Ryan; Wallweber, Heidi J A; Ho, Hoangdung; Tam, Christine; Franke, Yvonne; Quinn, John; Lupardus, Patrick J

    2016-06-01

    JAK1 is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases that are activated in response to cytokines and interferons. Here, we present two crystal structures of the human JAK1 FERM and SH2 domains bound to peptides derived from the class II cytokine receptors IFN-λ receptor 1 and IL-10 receptor 1 (IFNLR1 and IL10RA). These structures reveal an interaction site in the JAK1 FERM that accommodates the so-called "box1" membrane-proximal receptor peptide motif. Biophysical analysis of the JAK1-IFNLR1 interaction indicates that the receptor box1 is the primary driver of the JAK1 interaction, and identifies residues conserved among class II receptors as important for binding. In addition, we demonstrate that a second "box2" receptor motif further stabilizes the JAK1-IFNLR1 complex. Together, these data identify a conserved JAK binding site for receptor peptides and elucidate the mechanism by which class II cytokine receptors interact with JAK1. PMID:27133025

  9. Structure-Based, Rational Design of T Cell Receptors

    PubMed Central

    Zoete, V.; Irving, M.; Ferber, M.; Cuendet, M. A.; Michielin, O.

    2013-01-01

    Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157–165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, KD = ∼1 − 5 μM. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function, in line with the “half-life” model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method

  10. Xenobiotic-sensing nuclear receptors involved in drug metabolism: a structural perspective

    PubMed Central

    Wallace, Bret D.; Redinbo, Matthew R.

    2016-01-01

    Xenobiotic compounds undergo a critical range of biotransformations performed by the phase I, II, and III drug-metabolizing enzymes. The oxidation, conjugation, and transportation of potentially harmful xenobiotic and endobiotic compounds achieved by these catalytic systems are significantly regulated, at the gene expression level, by members of the nuclear receptor (NR) family of ligand-modulated transcription factors. Activation of NRs by a variety of endo- and exogenous chemicals are elemental to induction and repression of drug-metabolism pathways. The master xenobiotic sensing NRs, the promiscuous pregnane X receptor and less-promiscuous constitutive androstane receptor are crucial to initial ligand recognition, jump-starting the metabolic process. Other receptors, including farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4 alpha, peroxisome proliferator activated receptor, glucocorticoid receptor, liver X receptor, and RAR-related orphan receptor, are not directly linked to promiscuous xenobiotic binding, but clearly play important roles in the modulation of metabolic gene expression. Crystallographic studies of the ligand-binding domains of nine NRs involved in drug metabolism provide key insights into ligand-based and constitutive activity, coregulator recruitment, and gene regulation. Structures of other, noncanonical transcription factors also shed light on secondary, but important, pathways of control. Pharmacological targeting of some of these nuclear and atypical receptors has been instituted as a means to treat metabolic and developmental disorders and provides a future avenue to be explored for other members of the xenobiotic-sensing NRs. PMID:23210723

  11. The βc receptor family - Structural insights and their functional implications.

    PubMed

    Broughton, Sophie E; Nero, Tracy L; Dhagat, Urmi; Kan, Winnie L; Hercus, Timothy R; Tvorogov, Denis; Lopez, Angel F; Parker, Michael W

    2015-08-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure-function studies, giving new insights into cytokine-receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics. PMID:25982846

  12. International Union of Pharmacology. LXXXIX. Update on the Extended Family of Chemokine Receptors and Introducing a New Nomenclature for Atypical Chemokine Receptors

    PubMed Central

    Bachelerie, Francoise; Ben-Baruch, Adit; Burkhardt, Amanda M.; Combadiere, Christophe; Farber, Joshua M.; Graham, Gerard J.; Horuk, Richard; Sparre-Ulrich, Alexander Hovard; Locati, Massimo; Luster, Andrew D.; Mantovani, Alberto; Matsushima, Kouji; Nibbs, Robert; Nomiyama, Hisayuki; Power, Christine A.; Proudfoot, Amanda E. I.; Rosenkilde, Mette M.; Rot, Antal; Sozzani, Silvano; Thelen, Marcus; Yoshie, Osamu; Zlotnik, Albert

    2014-01-01

    Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145–176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human

  13. Progress toward advanced understanding of metabotropic glutamate receptors: structure, signaling and therapeutic indications

    PubMed Central

    Yin, Shen; Niswender, Colleen M.

    2014-01-01

    The metabotropic glutamate (mGlu) receptors are a group of Class C Seven Transmembrane Spanning/G Protein Coupled Receptors (7TMRs/GPCRs). These receptors are activated by glutamate, one of the standard amino acids and the major excitatory neurotransmitter. By activating G protein-dependent and non G protein-dependent signaling pathways, mGlus modulate glutamatergic transmission in both the periphery and throughout the central nervous system. Since the discovery of the first mGlu receptor, especially the last decade, a great deal of progress has been made in understanding the signaling, structure, pharmacological manipulation and therapeutic indications of the 8 mGlu members. PMID:24793301

  14. Structural and functional evolution of the P2Y12-like receptor group

    PubMed Central

    Hermsdorf, Thomas; Engemaier, Eva; Engel, Kathrin; Liebscher, Ines; Thor, Doreen; Zierau, Klaas; Römpler, Holger; Schulz, Angela

    2007-01-01

    Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members. PMID:18404440

  15. Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism

    SciTech Connect

    Shan, L.; Vincent, J.; Brunzelle, J.S.; Dussault, I.; Lin, M.; Ianculescu, I.; Sherman, M.A.; Forman, B.M.; Fernandez, E.

    2010-03-08

    The nuclear receptor CAR is a xenobiotic responsive transcription factor that plays a central role in the clearance of drugs and bilirubin while promoting cocaine and acetaminophen toxicity. In addition, CAR has established a 'reverse' paradigm of nuclear receptor action where the receptor is active in the absence of ligand and inactive when bound to inverse agonists. We now report the crystal structure of murine CAR bound to the inverse agonist androstenol. Androstenol binds within the ligand binding pocket, but unlike many nuclear receptor ligands, it makes no contacts with helix H12/AF2. The transition from constitutive to basal activity (androstenol bound) appears to be associated with a ligand-induced kink between helices H10 and H11. This disrupts the previously predicted salt bridge that locks H12 in the transcriptionally active conformation. This mechanism of inverse agonism is distinct from traditional nuclear receptor antagonists thereby offering a new approach to receptor modulation.

  16. Structural Overview of the Nuclear Receptor Superfamily: Insights into Physiology and Therapeutics

    PubMed Central

    Huang, Pengxiang; Chandra, Vikas; Rastinejad, Fraydoon

    2013-01-01

    As ligand-regulated transcription factors, the nuclear hormone receptors are nearly ideal drug targets, with internal pockets that bind to hydrophobic, drug-like molecules and well-characterized ligand-induced conformational changes that recruit transcriptional coregulators to promoter elements. Yet, due to the multitude of genes under the control of a single receptor, the major challenge has been the identification of ligands with gene-selective actions, impacting disease outcomes through a narrow subset of target genes and not across their entire gene-regulatory repertoire. Here, we summarize the concepts and work to date underlying the development of steroidal and nonsteroidal receptor ligands, including the use of crystal structures, high-throughput screens, and rational design approaches for finding useful therapeutic molecules. Difficulties in finding selective receptor modulators require a more complete understanding of receptor interdomain communications, posttranslational modifications, and receptor-protein interactions that could be exploited for target gene selectivity. PMID:20148675

  17. Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

    PubMed

    Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

    2013-01-01

    Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA. PMID:24020757

  18. Structure-Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics.

    PubMed

    Kaczor, Agnieszka A; Silva, Andrea G; Loza, María I; Kolb, Peter; Castro, Marián; Poso, Antti

    2016-04-01

    Structure-based virtual screening using a D2 receptor homology model was performed to identify dopamine D2 receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D2 ligands were identified (47.6 % success rate, among them D2 receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5-HT2A receptors. The affinity (Ki values) of the compounds ranged from 58 nm to about 24 μm. Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D2 receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D2 pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20-fold binding selectivity for the D2 receptor over the D3 receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction. PMID:26990027

  19. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

    PubMed

    Thal, David M; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C; Bures, Mark G; Evans, David A; Weis, William I; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M; Kobilka, Brian K; Christopoulos, Arthur

    2016-03-17

    Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  20. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

    PubMed Central

    Hopf, Thomas A.; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S.; Benton, Richard

    2015-01-01

    Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection. PMID:25584517

  1. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

    PubMed

    Kasuya, Go; Fujiwara, Yuichiro; Takemoto, Mizuki; Dohmae, Naoshi; Nakada-Nakura, Yoshiko; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-02-01

    P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors. PMID:26804916

  2. The avian beta-adrenergic receptor: primary structure and membrane topology.

    PubMed Central

    Yarden, Y; Rodriguez, H; Wong, S K; Brandt, D R; May, D C; Burnier, J; Harkins, R N; Chen, E Y; Ramachandran, J; Ullrich, A

    1986-01-01

    Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte beta-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membrane-spanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The beta-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins. Images PMID:3018746

  3. Structure and mechanism of activity-based inhibition of the EGF-Receptor by Mig6

    PubMed Central

    Ficarro, Scott B.; Zhang, Yi; Lee, Byung Il; Cho, Ahye; Kim, Kihong; Park, Angela K.J.; Park, Woong-Yang; Murray, Bradley; Meyerson, Matthew; Beroukhim, Rameen; Marto, Jarrod A.; Cho, Jeonghee; Eck, Michael J.

    2016-01-01

    Mig6 is a feedback inhibitor that directly binds, inhibits and drives internalization of ErbB-family receptors. Mig6 selectivity targets activated receptors. Here we find that the EGF receptor phosphorylates Mig6 on Tyr394, and that this phosphorylation is primed by prior phosphorylation of an adjacent residue, Tyr395, by Src. Crystal structures of human EGFR–Mig6 complexes reveal the structural basis for enhanced phosphorylation of primed Mig6 and show how Mig6 rearranges after phosphorylation by EGFR to effectively irreversibly inhibit the same receptor that catalyzed its phosphorylation. This dual phosphorylation site allows Mig6 to inactivate EGFR in a manner that requires activation of the target receptor and can be modulated by Src. Loss of Mig6 is a driving event in human cancer; analysis of 1057 gliomas reveals frequent focal deletions of ERRFI, the gene that encodes Mig6, in EGFR-amplified glioblastomas. PMID:26280531

  4. A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor

    PubMed Central

    VAN GALEN, PHILIP J. M.; VAN BERGEN, ANDREW H.; GALLO-RODRIGUEZ, CAROLA; MELMAN, NELI; OLAH, MARK E.; IJZERMAN, AD P.; STILES, GARY L.; JACOBSON, KENNETH A.

    2012-01-01

    SUMMARY A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at

  5. Structure-based drug design for G protein-coupled receptors.

    PubMed

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. PMID:24418607

  6. The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ.

    PubMed

    Smith, R A; Branca, A A; Reichert, L E

    1985-11-15

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate

  7. Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

    SciTech Connect

    Xu, Rui; McBride, Ryan; Nycholat, Corwin M.; Paulson, James C.; Wilson, Ian A.

    2012-02-13

    Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

  8. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

    PubMed

    Murthy, Shubha; Larson-Casey, Jennifer L; Ryan, Alan J; He, Chao; Kobzik, Lester; Carter, A Brent

    2015-08-01

    Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-β1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention. PMID:25953850

  9. Interferons and Their Receptors in Birds: A Comparison of Gene Structure, Phylogenetic Analysis, and Cross Modulation

    PubMed Central

    Zhou, Hao; Chen, Shun; Wang, Mingshu; Cheng, Anchun

    2014-01-01

    Interferon may be thought of as a key, with the interferon receptor as the signal lock: Crosstalk between them maintains their balance during viral infection. In this review, the protein structure of avian interferon and the interferon receptor are discussed, indicating remarkable similarity between different species. However, the structures of the interferon receptors are more sophisticated than those of the interferons, suggesting that the interferon receptor is a more complicated signal lock system and has considerable diversity in subtypes or structures. Preliminary evolutionary analysis showed that the subunits of the interferon receptor formed a distinct clade, and the orthologs may be derived from the same ancestor. Furthermore, the development of interferons and interferon receptors in birds may be related to an animal’s age and the maintenance of a balanced state. In addition, the equilibrium between interferon and its receptor during pathological and physiological states revealed that the virus and the host influence this equilibrium. Birds could represent an important model for studies on interferon’s antiviral activities and may provide the basis for new antiviral strategies. PMID:25405736

  10. Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis.

    PubMed Central

    Severne, Y; Wieland, S; Schaffner, W; Rusconi, S

    1988-01-01

    The glucocorticoid receptor and the other members of the steroid receptor super-family share a highly conserved, cysteine-rich region which coincides with the DNA binding/transactivating domain. It has been postulated that this region is folded into two 'zinc finger' structures, similar to those originally reported for the transcription factor TFIIIA. The first potential finger domain contains four conserved cysteines and one conserved histidine, while the second contains five conserved cysteines. Using site-directed mutagenesis, we have analysed the consequences of altering the proposed finger-like structures. Our results show that most of the mutations affecting the conserved cysteines result in a total loss of glucocorticoid receptor function. In one important exception, however, a conserved cysteine (Cys500) is dispensable for glucocorticoid receptor activity and therefore cannot be involved in complexing a metal ion to form a finger structure. Moreover, the replacement of either Cys476 or Cys482 by His residues maintains partial in vivo activity of the glucocorticoid receptor, while their exchange for an alanine or serine residue, respectively, eliminates receptor function. These results support, at a genetic level, the involvement of cysteines of the glucocorticoid receptor DNA binding domain in metal ion complexation and define the candidate residues involved in such coordination. Images PMID:3191912

  11. Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin

    SciTech Connect

    Chrencik, Jill E.; Orans, Jillian; Moore, Linda B.; Xue, Yu; Peng, Li; Collins, Jon L.; Wisely, G. Bruce; Lambert, Millard H.; Kliewer, Steven A.; Redinbo, Matthew R.

    2010-07-13

    The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 {angstrom} crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200-210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR's promiscuous response to xenobiotics.

  12. A Annealing Algorithm for Designing Ligands from Receptor Structures.

    NASA Astrophysics Data System (ADS)

    Zielinski, Peter J.

    DEenspace NOVO, a simulated annealing method for designing ligands is described. At a given temperature, ligand fragments are randomly selected and randomly placed within the given receptor cavity, often replacing existing ligand fragments. For each new ligand fragment combination, bonded, nonbonded, polarization and solvation energies of the new ligand-receptor system are compared to the previous system. Acceptance or rejection of the new system is decided using the Boltzmann distribution. Thus, energetically unfavorable fragment switches are sometimes accepted, sacrificing immediate energy gains in the interest of finding the system with the globally minimum energy. By lowering the temperature, the rate of unfavorable switches decreases and energetically favorable combinations become difficult to change. The process is halted when the frequency of switches becomes too small. As a test of the method, DEenspace NOVO predicted the positions of important ligand fragments for neuraminidase that are in accord with the natural ligand, sialic acid.

  13. Crystal structures of botulinum neurotoxin DC in complex with its protein receptors synaptotagmin I and II

    PubMed Central

    Berntsson, Ronnie Per-Arne; Peng, Lisheng; Svensson, Linda Marie; Dong, Min; Stenmark, Pål

    2013-01-01

    Summary Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a novel Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further show that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs. PMID:23932591

  14. Structural basis for receptor sharing and activation by interleukin-20 receptor-2 (IL-20R2) binding cytokines

    PubMed Central

    Logsdon, Naomi J.; Deshpande, Ashlesha; Harris, Bethany D.; Rajashankar, Kanagalaghatta R.; Walter, Mark R.

    2012-01-01

    Interleukin 20 (IL-20) is a pleotropic IL-10 family cytokine that protects epithelial surfaces from pathogens. However, dysregulated IL-20 signaling is implicated in several human pathologies including psoriasis, rheumatoid arthritis, atherosclerosis, and osteoporosis. IL-20, and related cytokines IL-19 and IL-24, designated IL-20 subfamily cytokines (IL-20SFCs), induce cellular responses through an IL-20R1/IL-20R2 (type I) receptor heterodimer, whereas IL-20 and IL-24 also signal through the IL-22R1/IL-20R2 (type II) receptor complex. The crystal structure of the IL-20/IL-20R1/IL-20R2 complex reveals how type I and II complexes discriminate cognate from noncognate ligands. The structure also defines how the receptor–cytokine interfaces are affinity tuned to allow distinct signaling through a receptor complex shared by three different ligands. Our results provide unique insights into the complexity of IL-20SFC signaling that may be critical in the design of mechanistic-based inhibitors of IL-20SFC–mediated inflammatory disease. PMID:22802649

  15. Muscarinic Receptors as Model Targets and Antitargets for Structure-Based Ligand Discovery

    PubMed Central

    Kruse, Andrew C.; Weiss, Dahlia R.; Rossi, Mario; Hu, Jianxin; Hu, Kelly; Eitel, Katrin; Gmeiner, Peter; Wess, Jürgen

    2013-01-01

    G protein–coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype–selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology. PMID:23887926

  16. Quantitative structure-activity relationship models with receptor-dependent descriptors for predicting peroxisome proliferator-activated receptor activities of thiazolidinedione and oxazolidinedione derivatives.

    PubMed

    Lather, Viney; Kairys, Visvaldas; Fernandes, Miguel X

    2009-04-01

    A quantitative structure-activity relationship study has been carried out, in which the relationship between the peroxisome proliferator-activated receptor alpha and the peroxisome proliferator-activated receptor gamma agonistic activities of thiazolidinedione and oxazolidinedione derivatives and quantitative descriptors, V(site) calculated in a receptor-dependent manner is modeled. These descriptors quantify the volume occupied by the optimized ligands in regions that are either common or specific to the superimposed binding sites of the targets under consideration. The quantitative structure-activity relationship models were built by forward stepwise linear regression modeling for a training set of 27 compounds and validated for a test set of seven compounds, resulting in a squared correlation coefficient value of 0.90 for peroxisome proliferator-activated receptor alpha and of 0.89 for peroxisome proliferator-activated receptor gamma. The leave-one-out cross-validation and test set predictability squared correlation coefficient values for these models were 0.85 and 0.62 for peroxisome proliferator-activated receptor alpha and 0.89 and 0.50 for peroxisome proliferator-activated receptor gamma respectively. A dual peroxisome proliferator-activated receptor model has also been developed, and it indicates the structural features required for the design of ligands with dual peroxisome proliferator-activated receptor activity. These quantitative structure-activity relationship models show the importance of the descriptors here introduced in the prediction and interpretation of the compounds affinity and selectivity. PMID:19243388

  17. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors.

    PubMed

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-09-15

    G protein-coupled receptors are divided into three classes (A, B and C) based on homology of their seven transmembrane domains. Class C is the smallest class with 22 human receptor subtypes including eight metabotropic glutamate (mGlu1-8) receptors, two GABAB receptors (GABAB1 and GABAB2), three taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we review the existence of post-translational modifications in class C G protein-coupled receptors and their regulatory roles, with particular focus on glycosylation, phosphorylation, ubiquitination, SUMOylation, disulphide bonding and lipidation. PMID:25981296

  18. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  19. Homology modeling and antagonist binding site study of the human histamine H2 receptor.

    PubMed

    Zhang, Jing; Qi, Tao; Wei, Jing

    2012-11-01

    On the basis of the high resolution crystal structures of bovine rhodopsin, human beta2-adrenergic receptor and human A(2a) adenosine receptor, three-dimensional structure of the human histamine H2 receptor (HHR2) was developed by homology modeling. Results of the evaluations suggest that a high quality homology model for HHR2 has been obtained. Three antagonists, cimetidine, ranitidine and nizatidine, were applied to binding site study with this model through molecular docking, molecular dynamics simulations and the molecular mechanics-Poisson-Boltzmann surface area calculations. One aspartic acid, Asp98 in transmembrane domain 7 (TM3), has been identified as major contributors to ligand binding by H-bond interactions. Asn159 in TM4 and Asp186 in TM5 are of great importance in stabilizing HHR2- antagonist complexes. Two hydrophobic sites especially two residues, Val99 in TM3 and Phe254 in TM6, were identified to be essential for their strong hydrophobic interactions with antagonists. The findings reported here are in agreement with available experimental mutagenesis data. This study should be very helpful for a better understanding of the action mode of the antagonist and for guiding further drug design and mutagenesis studies. PMID:22779803

  20. Structural basis for specificity of TGF[beta] family receptor small molecule inhibitors

    SciTech Connect

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2012-07-24

    Transforming growth factor-{beta} (TGF{beta}) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGF{beta}/Activin pathway receptors, but needs improvement of its EC{sub 50} (EC{sub 50} = 1 {mu}M) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGF{beta}/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGF{beta} type I receptor (T{beta}RI) kinase domain in complex with SB431542. We mutated T{beta}RI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated T{beta}RI. We discovered that a Ser280Thr mutation yielded a T{beta}RI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGF{beta}/Activin pathway.

  1. Structural Basis for Specificity of TGFβ Family Receptor Small Molecule Inhibitors

    PubMed Central

    Ogunjimi, Abiodun A.; Zeqiraj, Elton; Ceccarelli, Derek F.; Sicheri, Frank; Wrana, Jeffrey L.; David, Laurent

    2015-01-01

    Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC50 (EC50 = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimizing the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway. PMID:21983015

  2. Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists.

    PubMed

    Weichert, Dietmar; Stanek, Markus; Hübner, Harald; Gmeiner, Peter

    2016-06-15

    Aiming to discover dual-acting β2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β2 adrenergic antagonist/D2 receptor agonist with Ki values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders. PMID:27132867

  3. A conserved structural mechanism of NMDA receptor inhibition: A comparison of ifenprodil and zinc

    PubMed Central

    Sirrieh, Rita E.; MacLean, David M.

    2015-01-01

    N-methyl-d-aspartate (NMDA) receptors, one of the three main types of ionotropic glutamate receptors (iGluRs), are involved in excitatory synaptic transmission, and their dysfunction is implicated in various neurological disorders. NMDA receptors, heterotetramers typically composed of GluN1 and GluN2 subunits, are the only members of the iGluR family that bind allosteric modulators at their amino-terminal domains (ATDs). We used luminescence resonance energy transfer to characterize the conformational changes the receptor undergoes upon binding ifenprodil, a synthetic compound that specifically inhibits activation of NMDA receptors containing GluN2B. We found that ifenprodil induced an overall closure of the GluN2B ATD without affecting conformation of the GluN1 ATD or the upper lobes of the ATDs, the same mechanism whereby zinc inhibits GluN2A. These data demonstrate that the conformational changes induced by zinc and ifenprodil represent a conserved mechanism of NMDA receptor inhibition. Additionally, we compared the structural mechanism of zinc inhibition of GluN1–GluN2A receptors to that of ifenprodil inhibition of GluN1–GluN2B. The similarities in the conformational changes induced by inhibitor binding suggest a conserved structural mechanism of inhibition independent of the binding site of the modulator. PMID:26170175

  4. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  5. Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket.

    PubMed

    Mann, Rosalind J; Al-Sabah, Suleiman; de Maturana, Rakel López; Sinfield, John K; Donnelly, Dan

    2010-12-01

    G protein-coupled receptors (GPCRs) are seven transmembrane α-helical (7TM) integral membrane proteins that play a central role in both cell signaling and in the action of many pharmaceuticals. The crystal structures of several Family A GPCRs have shown the presence of a disulfide bond linking transmembrane helix 3 (TM3) to the second extracellular loop (ECL2), enabling ECL2 to stabilize and contribute to the ligand binding pocket. Family B GPCRs share no significant sequence identity with those in Family A but nevertheless share two conserved cysteines in topologically equivalent positions. Since there are no available crystal structures for the 7TM domain of any Family B GPCR, we used mutagenesis alongside pharmacological analysis to investigate the role of ECL2 and the conserved cysteine residues. We mutated Cys-226, at the extracellular end of TM3 of the glucagon-like peptide-1 (GLP-1) receptor, to alanine and observed a 38-fold reduction in GLP-1 potency. Interestingly, this potency loss was restored by the additional substitution of Cys-296 in ECL2 to alanine. Alongside the complete conservation of these cysteine residues in Family B GPCRs, this functional coupling suggested the presence of a disulfide bond. Further mutagenesis demonstrated that the low potency observed at the C226A mutant, compared with the C226A-C296A double mutant, was the result of the bulky nature of the released Cys-296 side chain. Since this suggested that ECL2 was in close proximity to the agonist activation pocket, an alanine scan of ECL2 was carried out which confirmed the important role of this loop in agonist-induced receptor activation. PMID:20869417

  6. Structural insights into ligand-induced activation of the insulin receptor

    SciTech Connect

    Ward, C.; Lawrence, M.; Streltsov, V.; Garrett, T.; McKern, N.; Lou, M.-Z.; Lovrecz, G.; Adams, T.

    2008-04-29

    The current model for insulin binding to the insulin receptor proposes that there are two binding sites, referred to as sites 1 and 2, on each monomer in the receptor homodimer and two binding surfaces on insulin, one involving residues predominantly from the dimerization face of insulin (the classical binding surface) and the other residues from the hexamerization face. High-affinity binding involves one insulin molecule using its two surfaces to make bridging contacts with site 1 from one receptor monomer and site 2 from the other. Whilst the receptor dimer has two identical site 1-site 2 pairs, insulin molecules cannot bridge both pairs simultaneously. Our structures of the insulin receptor (IR) ectodomain dimer and the L1-CR-L2 fragments of IR and insulin-like growth factor receptor (IGF-1R) explain many of the features of ligand-receptor binding and allow the two binding sites on the receptor to be described. The IR dimer has an unexpected folded-over conformation which places the C-terminal surface of the first fibronectin-III domain in close juxtaposition to the known L1 domain ligand-binding surface suggesting that the C-terminal surface of FnIII-1 is the second binding site involved in high-affinity binding. This is very different from previous models based on three-dimensional reconstruction from scanning transmission electron micrographs. Our single-molecule images indicate that IGF-1R has a morphology similar to that of IR. In addition, the structures of the first three domains (L1-CR-L2) of the IR and IGF-1R show that there are major differences in the two regions governing ligand specificity. The implications of these findings for ligand-induced receptor activation will be discussed. This review summarizes the key findings regarding the discovery and characterization of the insulin receptor, the identification and arrangement of its structural domains in the sequence and the key features associated with ligand binding. The remainder of the review

  7. Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

    SciTech Connect

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  8. Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin; Robinson, Howard; Varnum, Susan M.

    2010-10-28

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  9. Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex

    SciTech Connect

    Tan, Qiuxiang; Zhu, Ya; Li, Jian; Chen, Zhuxi; Han, Gye Won; Kufareva, Irina; Li, Tingting; Ma, Limin; Fenalti, Gustavo; Li, Jing; Zhang, Wenru; Xie, Xin; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Liu, Hong; Stevens, Raymond C.; Zhao, Qiang; Wu, Beili

    2013-10-21

    The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom–resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor–gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

  10. Synthesis and structure-activity relationships of novel indazolyl glucocorticoid receptor partial agonists.

    PubMed

    Gilmore, John L; Sheppeck, James E; Wang, Jim; Dhar, T G Murali; Cavallaro, Cullen; Doweyko, Arthur M; Mckay, Lorraine; Cunningham, Mark D; Habte, Sium F; Nadler, Steven G; Dodd, John H; Somerville, John E; Barrish, Joel C

    2013-10-01

    SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors. PMID:23916594

  11. Structural Determinants Underlying Constitutive Dimerization of Unoccupied Human Follitropin Receptors

    PubMed Central

    Guan, Rongbin; Wu, Xueqing; Feng, Xiuyan; Zhang, Meilin; Hébert, Terence E.; Segaloff, Deborah L.

    2009-01-01

    The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr110 in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr110 to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr110 of the ECD. PMID:19800402

  12. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2007-01-02

    Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

  13. VEGF receptor kinase inhibitors: phthalazines, anthranilamides and related structures.

    PubMed

    Dumas, Jacques; Dixon, Julie A

    2005-06-01

    Inhibition of vascular endothelial growth factor receptor (VEGFR) signalling, using either antibodies or small molecule inhibitors of the VEGFR kinase domain, has become a major area of research in oncology. The phthalazine PTK787/ZK222584, first published in the literature in 1998, is one of the most advanced VEGFR inhibitors in the clinic. This paper provides an update on the patenting activity related to the phthalazine class. In addition, newer kinase inhibitor pharmacophores derived from this class (e.g., anthranilamides) will be reviewed. PMID:20141503

  14. Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

    SciTech Connect

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Ho, Patricia W.M.; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, T. John; Parker, Michael W.

    2009-08-18

    Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1-108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an {alpha}-helical structure extending from residues 14-29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.

  15. Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

    PubMed Central

    Wang, Jin; Yu, Ye

    2016-01-01

    P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na+, K+ and Ca2+. Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors. PMID:26725734

  16. Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor*

    PubMed Central

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Ho, Patricia W. M.; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, T. John; Parker, Michael W.

    2009-01-01

    Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1–108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an α-helical structure extending from residues 14–29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor. PMID:19346515

  17. Secondary structure of corona proteins determines the cell surface receptors used by nanoparticles.

    PubMed

    Fleischer, Candace C; Payne, Christine K

    2014-12-11

    Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular receptors used by the protein-nanoparticle complexes. These results show that protein secondary structure is a key parameter in determining the cell surface receptor used by a protein-nanoparticle complex. We expect this link between protein structure and cellular outcomes will provide a molecular basis for the design of nanoparticles for use in biological and biomedical applications. PMID:24779411

  18. Structural activation pathways from dynamic olfactory receptor-odorant interactions.

    PubMed

    Lai, Peter C; Singer, Michael S; Crasto, Chiquito J

    2005-11-01

    We have simulated an odor ligand's dynamic behavior in the binding region of an olfactory receptor (OR). Our short timescale computational studies (up to 200 ps) have helped identify unprecedented postdocking ligand behavior of ligands. From in vacuo molecular dynamics simulations of interactions between models of rat OR I7 and 10 aldehyde ligands, we have identified a dissociative pathway along which the ligand exits and enters the OR-binding pocket--a transit event. The ligand's transit through the receptor's binding region may mark the beginning of a signal transduction cascade leading to odor recognition. We have graphically traced the rotameric changes in key OR amino acid side chains during the transit. Our results have helped substantiate or refute previously held notions of amino acid contribution to ligand stability in the binding pocket. Our observations of ligand activity when compared to those of experimental (electroolfactogram response) OR-activation studies provide a view to predicting the stability of ligands in the binding pocket as a precursor to OR activation by the ligand. PMID:16243965

  19. Neurosteroid Structure-Activity Relationships for Functional Activation of Extrasynaptic δGABA(A) Receptors.

    PubMed

    Carver, Chase Matthew; Reddy, Doodipala Samba

    2016-04-01

    Synaptic GABAA receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders. The δ-subunit GABAA receptors are expressed extrasynaptically in the dentate gyrus and contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. However, the neurosteroid structure-function relationship at δGABA(A) receptors within the native hippocampus neurons remains unclear. Here we report a structure-activity relationship for neurosteroid modulation of extrasynaptic GABAA receptor-mediated tonic inhibition in the murine dentate gyrus granule cells. We recorded neurosteroid allosteric potentiation of GABA as well as direct activation of tonic currents using a wide array of natural and synthetic neurosteroids. Our results shows that, for all neurosteroids, the C3α-OH group remains obligatory for extrasynaptic receptor functional activity, as C3β-OH epimers were inactive in activating tonic currents. Allopregnanolone and related pregnane analogs exhibited the highest potency and maximal efficacy in promoting tonic currents. Alterations at the C17 or C20 region of the neurosteroid molecule drastically altered the transduction kinetics of tonic current activation. The androstane analogs had the weakest modulatory response among the analogs tested. Neurosteroid potentiation of tonic currents was completely (approximately 95%) diminished in granule cells from δ-knockout mice, suggesting that δ-subunit receptors are essential for neurosteroid activity. The neurosteroid sensitivity of δGABA(A) receptors was confirmed at the systems level using a 6-Hz seizure test. A consensus neurosteroid pharmacophore model at extrasynaptic δGABA(A) receptors is proposed based on a structure-activity relationship for activation of tonic current and seizure protection. PMID:26857959

  20. NTB-A Receptor Crystal Structure: Insights into Homophilic Interactions in the Signaling Lymphocytic Activation Molecule Receptor Family

    SciTech Connect

    Cao,E.; Ramagopal, U.; Fedorov, A.; Fedorov, E.; Yan, Q.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2006-01-01

    The signaling lymphocytic activation molecule (SLAM) family includes homophilic and heterophilic receptors that regulate both innate and adaptive immunity. The ectodomains of most SLAM family members are composed of an N-terminal IgV domain and a C-terminal IgC2 domain. NK-T-B-antigen (NTB-A) is a homophilic receptor that stimulates cytotoxicity in natural killer (NK) cells, regulates bactericidal activities in neutrophils, and potentiates T helper 2 (Th2) responses. The 3.0 {angstrom} crystal structure of the complete NTB-A ectodomain revealed a rod-like monomer that self-associates to form a highly kinked dimer spanning an end-to-end distance of {approx}100 {angstrom}. The NTB-A homophilic and CD2-CD58 heterophilic dimers show overall structural similarities but differ in detailed organization and physicochemical properties of their respective interfaces. The NTB-A structure suggests a mechanism responsible for binding specificity within the SLAM family and imposes physical constraints relevant to the colocalization of SLAM-family proteins with other signaling molecules in the immunological synapse.

  1. Structural mechanism of ligand activation in human calcium-sensing receptor.

    PubMed

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P; Brennan, Sarah C; Mun, Hee-Chang; Bush, Martin; Chen, Yan; Nguyen, Trang X; Cao, Baohua; Chang, Donald D; Quick, Matthias; Conigrave, Arthur D; Colecraft, Henry M; McDonald, Patricia; Fan, Qing R

    2016-01-01

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. PMID:27434672

  2. Structural mechanism of ligand activation in human calcium-sensing receptor

    PubMed Central

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P; Brennan, Sarah C; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X; Cao, Baohua; Chang, Donald D; Quick, Matthias; Conigrave, Arthur D; Colecraft, Henry M; McDonald, Patricia; Fan, Qing R

    2016-01-01

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. DOI: http://dx.doi.org/10.7554/eLife.13662.001 PMID:27434672

  3. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor

    SciTech Connect

    Sobolevsky, Alexander I.; Rosconi, Michael P.; Gouaux, Eric

    2010-02-02

    Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact ionotropic glutamate receptor are unknown. Here we report the crystal structure of the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 {angstrom} resolution in complex with a competitive antagonist. The receptor harbours an overall axis of two-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting four-fold symmetry. A symmetry mismatch between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis, we suggest that GluN1 and GluN2A NMDA (N-methyl-D-aspartate) receptors have a similar architecture, with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers.

  4. Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus.

    PubMed

    Tzarum, Netanel; de Vries, Robert P; Zhu, Xueyong; Yu, Wenli; McBride, Ryan; Paulson, James C; Wilson, Ian A

    2015-03-11

    Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and human receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. This binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics. PMID:25766295

  5. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

    SciTech Connect

    Thompson, Aaron A.; Liu, Wei; Chun, Eugene; Katritch, Vsevolod; Wu, Huixian; Vardy, Eyal; Huang, Xi-Ping; Trapella, Claudio; Guerrini, Remo; Calo, Girolamo; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2012-07-11

    Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, {delta}, {kappa} and {mu} ({delta}-OR, {kappa}-OR and {mu}-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes ({approx}60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors {kappa} (ref. 5) and {mu} (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

  6. The role of antigenically different virus neuraminidases as structures implicated in receptor-binding processes.

    PubMed

    Coimbra, M V; Luiz, M O; Cabral, M C; Couceiro, J N

    1995-06-01

    Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits receptor-binding activity while neuraminidase develops sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neuraminidase (N1 to N9) structures on their surface. The objective of the present investigation was to study the role of N2, N8, and N9, antigenically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. Receptor-binding activity of N2 and N8 was analyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemagglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3N8,N8-H3N2). This previously demonstrated N9 hemagglutinating activity was analyzed for receptor-binding specificity using hemagglutination tests and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia). It exhibited receptor-binding specificity for NeuAc alpha2,3Gal sequences, a property similar to that observed in hemagglutinins from avian strains. These results indicate the importance of antigenically different neuraminidase structures as alternative agents for developing receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8547843

  7. Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

    PubMed Central

    Mayerhofer, Hubert; Panneerselvam, Saravanan; Kaljunen, Heidi; Tuukkanen, Anne; Mertens, Haydyn D. T.; Mueller-Dieckmann, Jochen

    2015-01-01

    Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants. PMID:25451923

  8. Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum

    SciTech Connect

    Hwang, J.; Menon, K.N.J.

    1986-05-29

    The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the /sup 125/I-hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum /sup 125/I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of /sup 125/I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14.

  9. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  10. New insights into the GABAA receptor structure and orthosteric ligand binding: Receptor modeling guided by experimental data

    PubMed Central

    Sander, Tommy; Frølund, Bente; Bruun, Anne Techau; Ivanov, Ivaylo; McCammon, J. Andrew; Balle, Thomas

    2011-01-01

    GABAA receptors (GABAARs) are ligand gated chloride ion channels that mediate overall inhibitory signaling in the CNS. A detailed understanding of their structure is important to gain insights in e.g. ligand binding and functional properties of this pharmaceutically important target. Homology modeling is a necessary tool in this regard because experimentally determined structures are lacking. Here we present an exhaustive approach for creating a high quality model of the α1β2γ2 subtype of the GABAAR ligand binding domain, and we demonstrate its usefulness in understanding details of orthosteric ligand binding. The model was constructed by using multiple templates and by incorporation of knowledge from biochemical/pharmacological experiments. It was validated on the basis of objective energy functions, its ability to account for available residue specific information, and its stability in molecular dynamics (MD) compared to that of two homologous crystal structures. We then combined the model with extensive structure-activity relationships available from two homologous series of orthosteric GABAAR antagonists to create a detailed hypothesis for their binding modes. Excellent agreement with key experimental data was found, including the ability of the model to accommodate and explain a previously developed pharmacophore model. A coupling to agonist binding was thereby established and discussed in relation to activation mechanisms. Our results highlight the importance of critical evaluation and optimization of each step in the homology modeling process. The approach taken here can greatly aid in increasing the understanding of GABAARs and related receptors where structural insight is limited and reliable models are difficult to obtain. PMID:21365676

  11. Structure and regulation of expression of the mouse GH receptor.

    PubMed

    Talamantes, F; Ortiz, R

    2002-10-01

    GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes

  12. Structure and functional interaction of the extracellular domain of human GABAB receptor GBR2

    PubMed Central

    Geng, Yong; Xiong, Dazhi; Mosyak, Lidia; Malito, David L.; Kniazeff, Julie; Chen, Yan; Burmakina, Svetlana; Quick, Matthias; Bush, Martin; Javitch, Jonathan A.; Pin, Jean-Philippe; Fan, Qing R.

    2012-01-01

    Inhibitory neurotransmission is mediated primarily by GABA. Metabotropic GABAB receptor is a G protein coupled receptor central to mammalian brain function. Malfunction of GABAB receptor has been implicated in a number of neurological disorders. GABAB receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABAB receptor that is unique to the GABAergic system. PMID:22660477

  13. Studies on the structure of the follicle-stimulating hormone receptor using photoaffinity labeling procedures

    SciTech Connect

    Smith, R.A.

    1985-01-01

    The general objective of this project was to study the structure of the follicle stimulating hormone (FSH) receptor using affinity labeling methods. A low density fraction derived from homogenates of bovine testis was found to contain high affinity and low capacity receptors specific for FSH. Electron microscopic examination of the fraction revealed structure resembling multilamellar membranous vesicles (MV). For photoaffinity labeling of the FSH receptors in MV, an azidobenzoyl-/sup 125/I-analog of human FSH was prepared (/sup 125/I-AB-hFSH) and binding of specific FSH receptors was studied. /sup 125/I-AB-hFSH binding of receptors was inhibited in a dose dependent manner by unlabeled hFSH, and binding was not prevented by structurally-related human chorionic gonadotropin (hCG). The formation of photocrosslinked protein of relative molecular mass (M/sub r/) 54,000, 64,000, 76,000, 84,000, 97,000 and 116,000 was found to be inhibited by unlabeled hFSH in a dose related manner, and to be dependent on photoactivation of the FSH derivative. The interpretation of the photoaffinity labeling experiments was that three proteins associated with the FSH receptor were photoaffinity labeled. Analysis by indirect means suggested that the three proteins were assembled to form oligomeric complexes, and based on the intensities and composition of the oligomeric species, spatial relationships of the polypeptides with respect to each other on the membrane surface were deduced. The results of photoaffinity labeling suggest the FSH receptor is composed of three subunits of M/sub r/ 38,000, 48,000, and 81,000 and exists in the membrane in part as a M/sub r/ 330,000 dimer.

  14. Crystal structure of the plant receptor-like kinase TDR in complex with the TDIF peptide

    PubMed Central

    Morita, Junko; Kato, Kazuki; Nakane, Takanori; Kondo, Yuki; Fukuda, Hiroo; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-01-01

    In plants, leucine-rich repeat receptor-like kinases (LRR-RKs) perceive ligands, including peptides and small molecules, to regulate various physiological processes. TDIF, a member of the CLE peptide family, specifically interacts with the LRR-RK TDR to inhibit meristem differentiation into tracheary elements, and promotes cell proliferation. Here we report the crystal structure of the extracellular domain of TDR in complex with the TDIF peptide. The extracellular domain of TDR adopts a superhelical structure comprising 22 LRRs, and specifically recognizes TDIF by its inner concave surface. Together with our biochemical and sequence analyses, our structure reveals a conserved TDIF-recognition mechanism of TDR among plant species. Furthermore, a structural comparison of TDR with other plant LRR-RKs suggested the activation mechanism of TDR by TDIF. The structure of this CLE peptide receptor provides insights into the recognition mechanism of the CLE family peptides. PMID:27498761

  15. From G Protein-coupled Receptor Structure Resolution to Rational Drug Design*

    PubMed Central

    Jazayeri, Ali; Dias, Joao M.; Marshall, Fiona H.

    2015-01-01

    A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design. PMID:26100628

  16. Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists.

    PubMed

    Caseley, Emily A; Muench, Stephen P; Fishwick, Colin W; Jiang, Lin-Hua

    2016-09-15

    The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3μM, 4.8±0.8μM and 3.2±0.2μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3μM and 1.2±0.1μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9μM, 1.0±0.1μM and 0.8±0.2μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation. PMID:27481062

  17. Discoidin domain receptor 1 (DDR1) kinase as target for structure-based drug discovery.

    PubMed

    Kothiwale, Sandeepkumar; Borza, Corina M; Lowe, Edward W; Pozzi, Ambra; Meiler, Jens

    2015-02-01

    Discoidin domain receptor (DDR) 1 and 2 are transmembrane receptors that belong to the family of receptor tyrosine kinases (RTK). Upon collagen binding, DDRs transduce cellular signaling involved in various cell functions, including cell adhesion, proliferation, differentiation, migration, and matrix homeostasis. Altered DDR function resulting from either mutations or overexpression has been implicated in several types of disease, including atherosclerosis, inflammation, cancer, and tissue fibrosis. Several established inhibitors, such as imatinib, dasatinib, and nilotinib, originally developed as Abelson murine leukemia (Abl) kinase inhibitors, have been found to inhibit DDR kinase activity. As we review here, recent discoveries of novel inhibitors and their co-crystal structure with the DDR1 kinase domain have made structure-based drug discovery for DDR1 amenable. PMID:25284748

  18. Discoidin domain receptor 1 (DDR1) kinase as target for structure-based drug discovery

    PubMed Central

    Kothiwale, Sandeepkumar; Borza, Corina M.; Lowe, Will; Pozzi, Ambra; Meiler, Jens

    2014-01-01

    Discoidin domain receptor (DDR) 1 and 2 are transmembrane receptors that belong to the family of receptor tyrosine kinases (RTK). Upon collagen binding, DDRs transduce cellular signaling involved in various cell functions, including cell adhesion, proliferation, differentiation, migration, and matrix homeostasis. Altered DDR function resulting from either mutations or overexpression has been implicated in several types of disease, including atherosclerosis, inflammation, cancer, and tissue fibrosis. Several established inhibitors, such as imatinib, dasatinib, and nilotinib, originally developed as Abelson murine leukemia (Abl) kinase inhibitors, have been found to inhibit DDR kinase activity. As we review here, recent discoveries of novel inhibitors and their co-crystal structure with the DDR1 kinase domain have made structure-based drug discovery for DDR1 amenable. PMID:25284748

  19. Structure-activity relationships of alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors.

    PubMed

    Millard, Emma L; Daly, Norelle L; Craik, David J

    2004-06-01

    alpha-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific alpha-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for alpha-conotoxins, CCX(m)CX(n)C, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity. PMID:15182347

  20. From Atomic Structures to Neuronal Functions of G Protein–Coupled Receptors

    PubMed Central

    Palczewski, Krzysztof; Orban, Tivadar

    2015-01-01

    G protein–coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, and binding of proteins, peptides, or small molecules such as neurotransmitters. GPCR dysfunction caused by receptor mutations and environmental challenges contributes to many neurological diseases. Moreover, modern genetic technology has helped identify a rich array of mono- and multigenic defects in humans and animal models that connect such receptor dysfunction with disease affecting neuronal function. The visual system is especially suited to investigate GPCR structure and function because advanced imaging techniques permit structural studies of photoreceptor neurons at both macro and molecular levels that, together with biochemical and physiological assessment in animal models, provide a more complete understanding of GPCR signaling. PMID:23682660

  1. Structure and chromosomal localization of the human antidiuretic hormone receptor gene

    SciTech Connect

    Seibold, A.; Brabet, P.; Rosenthal, W.; Birnbaumer, M. )

    1992-11-01

    Applying a genomic DNA-expression approach, the authors cloned the gene and cDNA coding for the human antidiuretic hormone receptor, also called vasopressin V2 receptor' (V2R). The nucleotide sequence of both cloned DNAs provided the information to elucidate the structure of the isolated transcriptional unit. The structure of this gene is unusual in that it is the first G protein-coupled receptor gene that contains two very small intervening sequences, the second of which separates the region encoding the seventh transmembrane region from the rest of the open reading frame. The sequence information was used to synthesize appropriate oligonucleotides to be used as primers in the PCR. The V2R gene was localized by PCR using DNA from hybrid cells as template. The gene was found to reside in the q28-qter portion of the human X chromosome, a region identified as the locus for congential nephrogenic diabetes insipidus. 27 refs., 4 figs.

  2. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors

    PubMed Central

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F.

    2016-01-01

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å2 of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition. PMID:26744311

  3. Primary structure of nicotinic acetylcholine receptor. Final report, 9 April 1989-6 April 1992

    SciTech Connect

    Patrick, J.W.

    1992-05-06

    Signals are transmitted between cells in the brain using neurotransmitters and neurotransmitter receptors. Poisons that interfere with this process stop normal brain function and often kill nerve cells. One of the neurotransmitters used in the mammalian brain is acetylcholine. We discovered that there is a large number of different nicotinic receptors for the neurotransmitter acetylcholine, each with its different properties. We used recombinant DNA technology to clone and sequence the gene transcripts that encode the subunits of these receptors. From these sequences we deduced the primary structures of the nicotinic receptor subunits. We also used the cDNA clones to determine which brain loci express the respective genes. We have expressed the clones in the Xenopus oocyte and have demonstrated that each functional combination of subunits has a unique pharmacology Unlike their homologs at the neuromuscular junction, the nicotinic acetylcholine receptors in the brain are exceptionally permeable to calcium. This property suggests that these receptors may play an important role in regulating calcium-dependent cytoplasmic processes and that they may be important contributors to use-dependent cell death.

  4. A Comparative Structural Bioinformatics Analysis of the Insulin Receptor Family Ectodomain Based on Phylogenetic Information

    PubMed Central

    Rentería, Miguel E.; Gandhi, Neha S.; Vinuesa, Pablo; Helmerhorst, Erik; Mancera, Ricardo L.

    2008-01-01

    The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals. PMID:18989367

  5. Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems.

    PubMed

    Muraki, Michiro

    2012-08-01

    The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals. PMID:22762186

  6. Human prostacyclin receptor structure and function from naturally-occurring and synthetic mutations.

    PubMed

    Stitham, Jeremiah; Arehart, Eric J; Gleim, Scott R; Douville, Karen L; Hwa, John

    2007-01-01

    Prostacyclin (PGI2) is released by vascular endothelial cells and serves as a potent vasodilator, inhibitor of platelet aggregation (anti-thrombotic), and moderator of vascular smooth muscle cell proliferation-migration-differentiation (anti-atherosclerotic). These actions are mediated via a seven transmembrane-spanning G-protein coupled receptor (GPCR), known as the human prostacyclin receptor or hIP. Animal studies using prostacyclin receptor knock-out (IP-/-) mice have revealed increased propensities towards thrombosis, intimal hyperplasia, atherosclerosis, restenosis, as well as reperfusion injury. Of further importance has been the world-wide withdrawal of selective COX-2 inhibitors, due to their discriminating suppression of COX-2-derived PGI2 and its cardioprotective effects, leading to increased cardiovascular events, including myocardial infarction and thrombotic stroke. Over the last decade, mutagenesis studies of the IP receptor, in conjunction with in vitro functional assays and molecular modeling, have provided critical insights into the molecular mechanisms of both agonist binding and receptor activation. Most recently, the discovery of naturally-occurring and dysfunctional mutations within the hIP has provided additional insights into the proposed cardioprotective role of prostacyclin. The aim of this review is to summarize the most recent findings regarding hIP receptor structure-function that have developed through the study of both synthetic and naturally-occurring mutations. PMID:17164137

  7. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    SciTech Connect

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James

    2012-02-21

    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  8. Structural Variation and Uniformity among Tetraloop-Receptor Interactions and Other Loop-Helix Interactions in RNA Crystal Structures

    PubMed Central

    Wu, Li; Chai, Dinggeng; Fraser, Marie E.; Zimmerly, Steven

    2012-01-01

    Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature. PMID:23152878

  9. Leptin receptor-positive and leptin receptor-negative proopiomelanocortin neurons innervate an identical set of brain structures.

    PubMed

    Lima, Leandro B; Metzger, Martin; Furigo, Isadora C; Donato, J

    2016-09-01

    Neurons that express the prohormone proopiomelanocortin (POMC) in the arcuate hypothalamic nucleus (Arc) are engaged in the regulation of energy balance and glucose homeostasis. Additionally, POMC neurons are considered key first-order cells regulated by leptin. Interestingly, in the Arc, POMC cells that express the leptin receptor (POMC/LepR+ cells) are found side by side with POMC cells not directly responsive to leptin (POMC/LepR- cells). However, it remains unknown whether these distinct populations innervate different target regions. Therefore, the objective of the present study was to compare the projections of POMC/LepR+ and POMC/LepR- neurons. Using genetically modified LepR-reporter mice to identify leptin receptor-expressing cells and immunohistochemistry to stain POMC-derived peptides (α-MSH or β-endorphin) we confirmed that approximately 80% of Arc β-endorphin-positive neurons co-expressed leptin receptors. POMC/LepR+ and POMC/LepR- axons were intermingled in all of their target regions. As revealed by confocal microscopy, we found an elevated degree of co-localization between α-MSH+ axons and the reporter protein (tdTomato) in all brain regions analyzed, with co-localization coefficients ranging from 0.889 to 0.701. Thus, these two populations of POMC neurons seem to project to the same set of brain structures, although one of the two subtypes of POMC axons was sometimes found to be more abundant than the other in distinct subregions of the same nucleus. Therefore, POMC/LepR+ and POMC/LepR- cells may target separate neuronal populations and consequently activate distinct neuronal circuits within some target nuclei. These findings contribute to unravel the neuronal circuits involved in the regulation of energy balance and glucose homeostasis. PMID:27321158

  10. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor

    PubMed Central

    Hennen, Stephanie; Kodra, János T.; Soroka, Vladyslav; Krogh, Berit O.; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G.; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S.; Reedtz-Runge, Steffen

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors. PMID:27196125

  11. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

    PubMed

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors. PMID:27196125

  12. The Structure of A Biologically Active Estrogen Receptor-Coactivator Complex on DNA

    PubMed Central

    Yi, Ping; Wang, Zhao; Feng, Qin; Pintilie, Grigore D.; Foulds, Charles E.; Lanz, Rainer B.; Ludtke, Steven J.; Schmid, Michael F.; Chiu, Wah; O’Malley, Bert W.

    2015-01-01

    SUMMARY Estrogen receptor (ER) is a transcription factor critical for development, reproduction, metabolism and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement pre-existing and sometimes controversial biochemical information is lacking. Here we use cryo-EM to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3) and a secondary coactivator (p300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment. PMID:25728767

  13. Structural Basis of M3 Muscarinic Receptor Dimer/Oligomer Formation*

    PubMed Central

    McMillin, Sara M.; Heusel, Moritz; Liu, Tong; Costanzi, Stefano; Wess, Jürgen

    2011-01-01

    Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the “outer” (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1–7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1–5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance. PMID:21685385

  14. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant

    NASA Astrophysics Data System (ADS)

    Yin, Jie; Mobarec, Juan Carlos; Kolb, Peter; Rosenbaum, Daniel M.

    2015-03-01

    The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

  15. Structural Basis for Platelet Collagen Responses by the Immune-type Receptor Glycoprotein VI

    SciTech Connect

    Horii,K.; Kahn, M.; Herr, A.

    2006-01-01

    Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of heart attack and stroke, and drugs to block platelet activation have successfully reduced cardiovascular morbidity and mortality. In humans and mice, collagen activation of platelets is mediated by glycoprotein VI (GPVI), a receptor that is homologous to immune receptors but bears little sequence similarity to known matrix protein adhesion receptors. Here we present the crystal structure of the collagen-binding domain of human GPVI and characterize its interaction with a collagen-related peptide. Like related immune receptors, GPVI contains 2 immunoglobulin-like domains arranged in a perpendicular orientation. Significantly, GPVI forms a back-to-back dimer in the crystal, an arrangement that could explain data previously obtained from cell-surface GPVI inhibition studies. Docking algorithms identify 2 parallel grooves on the GPVI dimer surface as collagen-binding sites, and the orientation and spacing of these grooves precisely match the dimensions of an intact collagen fiber. These findings provide a structural basis for the ability of an immunetype receptor to generate signaling responses to collagen and for the development of GPVI inhibitors as new therapies for human cardiovascular disease.

  16. Exploring the Role of Receptor Flexibility in Structure-Based Drug Discovery

    PubMed Central

    Feixas, Ferran; Lindert, Steffen; Sinko, William; McCammon, J. Andrew

    2015-01-01

    The proper understanding of biomolecular recognition mechanisms that take place in a drug target is of paramount importance to improve the efficiency of drug discovery and development. The intrinsic dynamic character of proteins has a strong influence on biomolecular recognition mechanisms and models such as conformational selection have been widely used to account for this dynamic association process. However, conformational changes occurring in the receptor prior and upon association with other molecules are diverse and not obvious to predict when only a few structures of the receptor are available. In view of the prominent role of protein flexibility in ligand binding and its implications for drug discovery, it is of great interest to identify receptor conformations that play a major role in biomolecular recognition before starting rational drug design efforts. In this review, we discuss a number of recent advances in computer-aided drug discovery techniques that have been proposed to incorporate receptor flexibility into structure-based drug design. The allowance for receptor flexibility provided by computational techniques such as molecular dynamics simulations or enhanced sampling techniques helps to improve the accuracy of methods used to estimate binding affinities and, thus, such methods can contribute to the discovery of novel drug leads. PMID:24332165

  17. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives

    PubMed Central

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn

    2013-01-01

    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r2 = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders. PMID:24250113

  18. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2008-01-01

    The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin–receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These “custom-designed” arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling. PMID:16460808

  19. Allosteric Modulation of Metabotropic Glutamate Receptors: Structural Insights and Therapeutic Potential

    PubMed Central

    Gregory, Karen J.; Dong, Elizabeth N.; Meiler, Jens; Conn, P. Jeffrey

    2010-01-01

    Allosteric modulation of G protein-coupled receptors (GPCRs) represents a novel approach to the development of probes and therapeutics that is expected to enable subtype-specific regulation of central nervous system target receptors. The metabotropic glutamate receptors (mGlus) are class C GPCRs that play important neuromodulatory roles throughout the brain, as such they are attractive targets for therapeutic intervention for a number of psychiatric and neurological disorders including anxiety, depression, Fragile X Syndrome, Parkinson’s disease and schizophrenia. Over the last fifteen years, selective allosteric modulators have been identified for many members of the mGlu family. The vast majority of these allosteric modulators are thought to bind within the transmembrane-spanning domains of the receptors to enhance or inhibit functional responses. A combination of mutagenesis-based studies and pharmacological approaches are beginning to provide a better understanding of mGlu allosteric sites. Collectively, when mapped onto a homology model of the different mGlu subtypes based on the β2-adrenergic receptor, the previous mutagenesis studies suggest commonalities in the location of allosteric sites across different members of the mGlu family. In addition, there is evidence for multiple allosteric binding pockets within the transmembrane region that can interact to modulate one another. In the absence of a class C GPCR crystal structure, this approach has shown promise with respect to the interpretation of mutagenesis data and understanding structure-activity relationships of allosteric modulator pharmacophores. PMID:20637216

  20. Structural differences between liver- and muscle-derived insulin receptors in rats

    SciTech Connect

    Burant, C.F.; Treutelaar, M.K.; Block, N.E.; Buse, M.G.

    1986-11-05

    The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The ..cap alpha.. subunit was identified by specific cross-linking to A14 /sup 125/I-insulin with disuccinimidyl suberate. Muscle- and liver-derived ..cap alpha.. subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a M/sub r/ of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the M/sub r/ of muscle- and liver-derived ..cap alpha.. subunits but did not affect the difference in M/sub r/. Autophosphorylated ..beta.. subunits migrated with a M/sub r/ of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at M/sub r/ 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that M/sub r/ heterogeneity was due in part to differences in the sialic acid content of ..beta.. subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action.

  1. Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

    SciTech Connect

    Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.; Miller, Laurence J.; Xu, H. Eric

    2010-06-25

    The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

  2. Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

    PubMed Central

    Liggett, S B; Freedman, N J; Schwinn, D A; Lefkowitz, R J

    1993-01-01

    The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In

  3. Structure-activity relationships of seco-prezizaane and picrotoxane/picrodendrane terpenoids by Quasar receptor-surface modeling.

    PubMed

    Schmidt, Thomas J; Gurrath, Marion; Ozoe, Yoshihisa

    2004-08-01

    The seco-prezizaane-type sesquiterpenes pseudoanisatin and parviflorolide from Illicium are noncompetitive antagonists at housefly (Musca domestica) gamma-aminobutyric acid (GABA) receptors. They show selectivity toward the insect receptor and thus represent new leads toward selective insecticides. Based on the binding data for 13 seco-prezizaane terpenoids and 17 picrotoxane and picrodendrane-type terpenoids to housefly and rat GABA receptors, a QSAR study was conducted by quasi-atomistic receptor-surface modeling (Quasar). The resulting models provide insight into the structural basis of selectivity and properties of the binding sites at GABA receptor-coupled chloride channels of insects and mammals. PMID:15246092

  4. Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

    SciTech Connect

    Karakas, E.; Simorowski, N; Furukawa, H

    2009-01-01

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

  5. Structural basis for bifunctional peptide recognition at human δ-opioid receptor.

    PubMed

    Fenalti, Gustavo; Zatsepin, Nadia A; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C H; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Gati, Cornelius; Yefanov, Oleksandr M; White, Thomas A; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W; Roth, Bryan L; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C; Cherezov, Vadim

    2015-03-01

    Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics. PMID:25686086

  6. Structural basis for bifunctional peptide recognition at human δ-opioid receptor

    DOE PAGESBeta

    Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; et al

    2015-02-16

    Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. In summary, the observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

  7. Structural basis for bifunctional peptide recognition at human δ-opioid receptor

    SciTech Connect

    Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; Giguere, Patrick; Han, Gye Won; Ishchenko, Andrii; Liu, Wei; Guillemyn, Karel; Zhang, Haitao; James, Daniel; Wang, Dingjie; Weierstall, Uwe; Spence, John C. H.; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J.; Gati, Cornelius; Yefanov, Oleksandr M.; White, Thomas A.; Oberthuer, Dominik; Metz, Markus; Yoon, Chun Hong; Barty, Anton; Chapman, Henry N.; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Fromme, Petra; Tourwé, Dirk; Schiller, Peter W.; Roth, Bryan L.; Ballet, Steven; Katritch, Vsevolod; Stevens, Raymond C.; Cherezov, Vadim

    2015-02-16

    Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. In summary, the observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

  8. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

    PubMed Central

    Wibowo, Ardian S.; Singh, Mirage; Reeder, Kristen M.; Carter, Joshua J.; Kovach, Alexander R.; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E.

    2013-01-01

    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases. PMID:23934049

  9. Structural Mechanisms of Voltage Sensing in G Protein-Coupled Receptors.

    PubMed

    Vickery, Owen N; Machtens, Jan-Philipp; Tamburrino, Giulia; Seeliger, Daniel; Zachariae, Ulrich

    2016-06-01

    G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins and one-third of all drug targets in humans. A number of recent studies have reported evidence for substantial voltage regulation of GPCRs. However, the structural basis of GPCR voltage sensing has remained enigmatic. Here, we present atomistic simulations on the δ-opioid and M2 muscarinic receptors, which suggest a structural and mechanistic explanation for the observed voltage-induced functional effects. The simulations reveal that the position of an internal Na(+) ion, recently detected to bind to a highly conserved aqueous pocket in receptor crystal structures, strongly responds to voltage changes. The movements give rise to gating charges in excellent agreement with previous experimental recordings. Furthermore, free energy calculations show that these rearrangements of Na(+) can be induced by physiological membrane voltages. Due to its role in receptor function and signal bias, the repositioning of Na(+) has important general implications for signal transduction in GPCRs. PMID:27210286

  10. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

    PubMed

    Busato, Mirko; Giorgetti, Alejandro

    2016-08-01

    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well. PMID:27102413

  11. A small difference in the molecular structure of angiotensin II receptor blockers induces AT1 receptor-dependent and -independent beneficial effects

    PubMed Central

    Fujino, Masahiro; Miura, Shin-ichiro; Kiya, Yoshihiro; Tominaga, Yukio; Matsuo, Yoshino; Karnik, Sadashiva S; Saku, Keijiro

    2013-01-01

    Angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) induce multiple pharmacological beneficial effects, but not all ARBs have the same effects and the molecular mechanisms underlying their actions are not certain. In this study, irbesartan and losartan were examined because of their different molecular structures (irbesartan has a cyclopentyl group whereas losartan has a chloride group). We analyzed the binding affinity and production of inositol phosphate (IP), monocyte chemoattractant protein-1 (MCP-1) and adiponectin. Compared with losartan, irbesartan showed a significantly higher binding affinity and slower dissociation rate from the AT1 receptor and a significantly higher degree of inverse agonism and insurmountability toward IP production. These effects of irbesartan were not seen with the AT1-Y113A mutant receptor. On the basis of the molecular modeling of the ARBs–AT1 receptor complex and a mutagenesis study, the phenyl group at Tyr113 in the AT1 receptor and the cyclopentyl group of irbesartan may form a hydrophobic interaction that is stronger than the losartan–AT1 receptor interaction. Interestingly, irbesartan inhibited MCP-1 production more strongly than losartan. This effect was mediated by the inhibition of nuclear factor-kappa B activation that was independent of the AT1 receptor in the human coronary endothelial cells. In addition, irbesartan, but not losartan, induced significant adiponectin production that was mediated by peroxisome proliferator-activated receptor-γ activation in 3T3-L1 adipocytes, and this effect was not mediated by the AT1 receptor. In conclusion, irbesartan induced greater beneficial effects than losartan due to small differences between their molecular structures, and these differential effects were both dependent on and independent of the AT1 receptor. PMID:20668453

  12. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    PubMed

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies. PMID:27335334

  13. Discovery of a Novel Accessory Structure of the Pitviper Infrared Receptor Organ (Serpentes: Viperidae)

    PubMed Central

    Bolívar-G, Wilmar; Antoniazzi, Marta M.; Grant, Taran; Jared, Carlos

    2014-01-01

    The facial pits of rattlesnakes, copperheads, lanceheads, bushmasters and other American and Asian pitvipers (Crotalinae) are highly innervated and densely vascularized infrared (IR) receptor organs. For over a century, studies have focused on a small sample of model species from North America and Asia. Based on an expanded survey of Central and South American crotalines, we report a conspicuous accessory structure composed of well-defined papillae that project from the anterior orbital adnexa. The papillae are continuous with the inner chamber of the IR receptor organ and our histological and ultrastructural data suggest that they possess a well-developed nervous network and extensive vascularization; however, they lack the characteristic IR-sensitive terminal nerve masses found in the IR-receptive pit membrane. The function of the IR receptor organ papillae is unknown. PMID:24595025

  14. Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket*

    PubMed Central

    Pedragosa-Badia, Xavier; Sliwoski, Gregory R.; Dong Nguyen, Elizabeth; Lindner, Diana; Stichel, Jan; Kaufmann, Kristian W.; Meiler, Jens; Beck-Sickinger, Annette G.

    2014-01-01

    Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr2.64 interacts with Tyr27 of hPP through hydrophobic contacts. Asn7.32 is affected by modifications on position Arg33 of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe7.35 is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6–TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect. PMID:24375409

  15. Recent advances in structure of progestins and their binding to progesterone receptors.

    PubMed

    Cabeza, Marisa; Heuze, Yvonne; Sánchez, Araceli; Garrido, Mariana; Bratoeff, Eugene

    2015-02-01

    The role of progesterone in women's cancers as well as the knowledge of the progesterone receptor (PR) structure has prompted the design of different therapies. The aim of this review is to describe the basic structure of PR agonists and antagonists as well as the recent treatments for illness associated with the progesterone receptor. The rational design for potent and effective drugs for the treatment of female cancer must consider the structural changes of the androgen and progestogen skeleton which are an indicator of their activity as progestins or antiprogestins. The presence of a hydroxyl group at C-17 in the progesterone skeleton brings about a loss of progestational activity whereas acetylation induces a progestational effect. The incorporation of an ethynyl functional group to the testosterone framework results in a loss of androgenic activity with a concomitant enhancement of the progestational effect. On the other hand, an ester function at C-3 of dehydroepiandrosterone skeleton induces partial antagonism to the PR. PMID:24666307

  16. Dynamics and structural determinants of ligand recognition of the 5-HT6 receptor.

    PubMed

    Vass, Márton; Jójárt, Balázs; Bogár, Ferenc; Paragi, Gábor; Keserű, György M; Tarcsay, Ákos

    2015-12-01

    In order to identify molecular models of the human 5-HT6 receptor suitable for virtual screening, homology modeling and membrane-embedded molecular dynamics simulations were performed. Structural requirements for robust enrichment were assessed by an unbiased chemometric analysis of enrichments from retrospective virtual screening studies. The two main structural features affecting enrichment are the outward movement of the second extracellular loop and the formation of a hydrophobic cavity deep in the binding site. These features appear transiently in the trajectories and furthermore the stretches of uniformly high enrichment may only last 4-10 ps. The formation of the inner hydrophobic cavity was also linked to the active-like to inactive-like transition of the receptor, especially the so-called connector region. The best structural models provided significant and robust enrichment over three independent ligand sets. PMID:26572911

  17. Crystal Structures of the Tie2 Receptor Ectodomain and the Angiopoietin-2-Tie2 Complex

    SciTech Connect

    Barton,W.; Tzvetkova-Robev, D.; Miranda, E.; Kolev, M.; Rajashankar, K.; Himanen, J.; Nikolov, D.

    2006-01-01

    The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition--unique for a receptor kinase--where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.

  18. Phytoestrogens and Mycoestrogens Induce Signature Structure Dynamics Changes on Estrogen Receptor α.

    PubMed

    Chen, Xueyan; Uzuner, Ugur; Li, Man; Shi, Weibing; Yuan, Joshua S; Dai, Susie Y

    2016-01-01

    Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER) and provided rich data in the literature. However, limited assays can offer structure information with regard to the ligand/ER complex. Our current study surveys the global structure dynamics changes for ERα ligand binding domain (LBD) when phytoestrogens and mycoestrogens bind. The assay is based on the structure dynamics information probed by hydrogen deuterium exchange mass spectrometry and offers a unique viewpoint to elucidate the mechanism how phytoestrogens and mycoestrogens interact with estrogen receptor. The cluster analysis based on the hydrogen deuterium exchange (HDX) assay data reveals a unique pattern when phytoestrogens and mycoestrogens bind with ERα LBD compared to that of estradiol and synthetic estrogen modulators. Our study highlights that structure dynamics could play an important role in the structure function relationship when endocrine disrupters interact with estrogen receptors. PMID:27589781

  19. Structural features of the C-terminus from the human neurokinin-1 receptor.

    PubMed

    Orel, Mikhail; Padrós, Esteve; Manyosa, Joan

    2012-07-01

    The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and β-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25% α-helix, 27% unordered structure and 48% β-sheets and β-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions. PMID:22530884

  20. Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity.

    PubMed

    Higgins, Matthew K; Tkachenko, Olga; Brown, Alan; Reed, Jenny; Raper, Jayne; Carrington, Mark

    2013-01-29

    African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake. PMID:23319650

  1. Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.

    PubMed

    Novikova, Irina V; Hennelly, Scott P; Sanbonmatsu, Karissa Y

    2012-06-01

    While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features. PMID:22362738

  2. Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

    PubMed Central

    Novikova, Irina V.; Hennelly, Scott P.; Sanbonmatsu, Karissa Y.

    2012-01-01

    While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features. PMID:22362738

  3. Artonin E and Structural Analogs from Artocarpus Species Abrogates Estrogen Receptor Signaling in Breast Cancer.

    PubMed

    Etti, Imaobong; Abdullah, Rasedee; Hashim, Najihah Mohd; Kadir, Arifah; Abdul, Ahmad Bustamam; Etti, Christopher; Malami, Ibrahim; Waziri, Peter; How, Chee Wun

    2016-01-01

    The increasing rate of mortality ensued from breast cancer has encouraged research into safer and efficient therapy. The human Estrogen receptor α has been implicated in the majority of reported breast cancer cases. Molecular docking employing Glide, Schrodinger suite 2015, was used to study the binding affinities of small molecules from the Artocarpus species after their drug-like properties were ascertained. The structure of the ligand-binding domain of human Estrogen receptor α was retrieved from Protein Data Bank while the structures of compounds were collected from PubChem database. The binding interactions of the studied compounds were reported as well as their glide scores. The best glide scored ligand, was Artonin E with a score of -12.72 Kcal when compared to other studied phytomolecules and it evoked growth inhibition of an estrogen receptor positive breast cancer cells in submicromolar concentration (3.8-6.9 µM) in comparison to a reference standard Tamoxifen (18.9-24.1 µM) within the tested time point (24-72 h). The studied ligands, which had good interactions with the target receptor, were also drug-like when compared with 95% of orally available drugs with the exception of Artoelastin, whose predicted physicochemical properties rendered it less drug-like. The in silico physicochemical properties, docking interactions and growth inhibition of the best glide scorer are indications of the anti-breast cancer relevance of the studied molecules. PMID:27367662

  4. Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

    NASA Astrophysics Data System (ADS)

    Ravetch, Jeffrey V.; Luster, Andrew D.; Weinshank, Richard; Kochan, Jarema; Pavlovec, Amalia; Portnoy, Daniel A.; Hulmes, Jeffrey; Pan, Yu-Ching E.; Unkeless, Jay C.

    1986-11-01

    Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule Eβ. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

  5. Structural and Biochemical Characterization of the KRLB Region in Insulin Receptor Substrate-2

    SciTech Connect

    Wu,J.; Tseng, Y.; Xu, C.; Neubert, T.; White, M.; Hubbard, S.

    2008-01-01

    Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2.

  6. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE PAGESBeta

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist withmore » residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.« less

  7. Structural prerequisites for G-protein activation by the neurotensin receptor

    SciTech Connect

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.

  8. Versatility and differential roles of cysteine residues in human prostacyclin receptor structure and function.

    PubMed

    Stitham, Jeremiah; Gleim, Scott R; Douville, Karen; Arehart, Eric; Hwa, John

    2006-12-01

    Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation). PMID:17015447

  9. Structure of the intact PPAR-Υ-RXR-α nuclear receptor complex on DNA

    SciTech Connect

    Chandra, Vikas; Huang, Pengxiang; Hamuro, Yoshitomo; Raghuram, Srilatha; Wang, Yongjun; Burris, Thomas P; Rastinejad, Fraydoon

    2009-01-09

    Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-{gamma} has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-{gamma} and RXR-{alpha} as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-{gamma} and RXR-{alpha} form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-{gamma} to contact multiple domains in both proteins. Three interfaces link PPAR-{gamma} and RXR-{alpha}, including some that are DNA dependent. The PPAR-{gamma} LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

  10. Structural basis of death domain signaling in the p75 neurotrophin receptor

    PubMed Central

    Lin, Zhi; Tann, Jason Y; Goh, Eddy TH; Kelly, Claire; Lim, Kim Buay; Gao, Jian Fang; Ibanez, Carlos F

    2015-01-01

    Death domains (DDs) mediate assembly of oligomeric complexes for activation of downstream signaling pathways through incompletely understood mechanisms. Here we report structures of complexes formed by the DD of p75 neurotrophin receptor (p75NTR) with RhoGDI, for activation of the RhoA pathway, with caspase recruitment domain (CARD) of RIP2 kinase, for activation of the NF-kB pathway, and with itself, revealing how DD dimerization controls access of intracellular effectors to the receptor. RIP2 CARD and RhoGDI bind to p75NTR DD at partially overlapping epitopes with over 100-fold difference in affinity, revealing the mechanism by which RIP2 recruitment displaces RhoGDI upon ligand binding. The p75NTR DD forms non-covalent, low-affinity symmetric dimers in solution. The dimer interface overlaps with RIP2 CARD but not RhoGDI binding sites, supporting a model of receptor activation triggered by separation of DDs. These structures reveal how competitive protein-protein interactions orchestrate the hierarchical activation of downstream pathways in non-catalytic receptors. DOI: http://dx.doi.org/10.7554/eLife.11692.001 PMID:26646181

  11. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

    PubMed

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. PMID:25497229

  12. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

    PubMed Central

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-01-01

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. DOI: http://dx.doi.org/10.7554/eLife.05553.001 PMID:25497229

  13. Structural prerequisites for G-protein activation by the neurotensin receptor

    PubMed Central

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-01-01

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

  14. Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity

    PubMed Central

    Lam, Jonathan; Nelson, Christopher A.; Ross, F. Patrick; Teitelbaum, Steven L.; Fremont, Daved H.

    2001-01-01

    RANK, the receptor activator of NF-κB, and its ligand RANKL (initially termed TRANCE, also termed ODF and OPGL), are a TNF superfamily receptor-ligand pair that govern the development and function of osteoclasts, lymphoid tissue, and mammary epithelium. While TNF family cytokines share a common structural scaffold, individual receptor-ligand pairs associate with high specificity. Given the low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To determine the elements of RANKL that mediate RANK activation, we have crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6 Å. RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family cytokines. Mutagenesis of selected residues in these loops significantly modulates RANK activation, as evidenced by in vitro osteoclastogenesis, thereby establishing their necessity in mediating the biological activities of RANKL. Such structural determinants of RANKL-RANK specificity may be of relevance in the pharmacologic design of compounds to ameliorate osteopenic disorders of bone. PMID:11581298

  15. Identification of Novel Liver X Receptor Activators by Structure-Based Modeling

    PubMed Central

    2012-01-01

    Liver X receptors (LXRs) are members of the nuclear receptor family. Activators of LXRs are of high pharmacological interest as LXRs regulate cholesterol, fatty acid, and carbohydrate metabolism as well as inflammatory processes. On the basis of different X-ray crystal structures, we established a virtual screening workflow for the identification of novel LXR modulators. A two-step screening concept to identify active compounds included 3D-pharmacophore filters and rescoring by shape alignment. Eighteen virtual hits were tested in vitro applying a reporter gene assay, where concentration-dependent activity was proven for four novel lead structures. The most active compound 10, a 1,4-naphthochinone, has an estimated EC50 of around 5 μM. PMID:22489742

  16. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  17. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon

    NASA Astrophysics Data System (ADS)

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome-nascent chain complex.

  18. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon

    PubMed Central

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome–nascent chain complex. PMID:26804923

  19. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon.

    PubMed

    Jomaa, Ahmad; Boehringer, Daniel; Leibundgut, Marc; Ban, Nenad

    2016-01-01

    Co-translational protein targeting to membranes is a universally conserved process. Central steps include cargo recognition by the signal recognition particle and handover to the Sec translocon. Here we present snapshots of key co-translational-targeting complexes solved by cryo-electron microscopy at near-atomic resolution, establishing the molecular contacts between the Escherichia coli translating ribosome, the signal recognition particle and the translocon. Our results reveal the conformational changes that regulate the latching of the signal sequence, the release of the heterodimeric domains of the signal recognition particle and its receptor, and the handover of the signal sequence to the translocon. We also observe that the signal recognition particle and the translocon insert-specific structural elements into the ribosomal tunnel to remodel it, possibly to sense nascent chains. Our work provides structural evidence for a conformational state of the signal recognition particle and its receptor primed for translocon binding to the ribosome-nascent chain complex. PMID:26804923

  20. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    SciTech Connect

    M Franklin; E Navarro; Y Wang; S Patel; P Singh; Y Zhang; K Persaud; A Bari; H Griffith; et al.

    2011-12-31

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  1. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    SciTech Connect

    Franklin, Matthew C.; Navarro, Elizabeth C.; Wang, Yujie; Patel, Sheetal; Singh, Pinki; Zhang, Yi; Persaud, Kris; Bari, Amtul; Griffith, Heather; Shen, Leyi; Balderes, Paul; Kussie, Paul

    2011-10-28

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  2. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    PubMed Central

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  3. Luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures.

    PubMed

    Venencie, P Y; Méduri, G; Pissard, S; Jolivet, A; Loosfelt, H; Milgrom, E; Misrahi, M

    1999-09-01

    Two different monoclonal antibodies recognizing different epitopes were used to study the localization of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in human skin. Immunolabelling was observed only in the epidermis and derived structures but not in the dermis. The basal, spinal and granular layers were stained, whereas no receptors were detected in the non-nucleated horny cells. In the growing (anagen) hair, immunostaining was found in the inner root sheath below the level of the sebaceous glands and in the outer root sheath above this level. In the resting (telogen) hair, only the latter staining was observed. In the sebaceous glands, only the thin cells close to the walls of the ducts were immunolabelled. In the eccrine sweat glands, the external clear cells were stained in the secretory portion of the gland, whereas only the cells close to the lumen were labelled in the ducts. The distribution of LH/hCG receptors was compared with that of steroidogenic enzymes (side chain cleavage cytochrome P450, adrenodoxin, 3-beta-hydroxy-5-ene steroid dehydrogenase Delta5-Delta4 isomerase, 17-hydroxylase cytochrome P450 and cytochrome P450 aromatase). Only partial overlaps were observed. The presence of LH receptor mRNA in the skin was confirmed by reverse transcription-polymerase chain reaction. Monoclonal antibodies raised against the human follicle-stimulating hormone receptor failed to detect the latter in the epidermal structures and in the dermis. The role of LH and hCG in skin modifications occurring during pregnancy and after the menopause is unknown. These hormones may possibly act by regulating steroidogenic enzymes or by modulating cell growth and differentiation. PMID:10583046

  4. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

    PubMed Central

    Fay, Jonathan F.; Farrens, David L.

    2015-01-01

    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling. PMID:26100912

  5. The dopamine D2 receptor dimer and its interaction with homobivalent antagonists: homology modeling, docking and molecular dynamics.

    PubMed

    Kaczor, Agnieszka A; Jörg, Manuela; Capuano, Ben

    2016-09-01

    In order to apply structure-based drug design techniques to G protein-coupled receptor complexes, it is essential to model their 3D structure and to identify regions that are suitable for selective drug binding. For this purpose, we have developed and tested a multi-component protocol to model the inactive conformation of the dopamine D2 receptor dimer, suitable for interaction with homobivalent antagonists. Our approach was based on protein-protein docking, applying the Rosetta software to obtain populations of dimers as present in membranes with all the main possible interfaces. Consensus scoring based on the values and frequencies of best interfaces regarding four scoring parameters, Rosetta interface score, interface area, free energy of binding and energy of hydrogen bond interactions indicated that the best scored dimer model possesses a TM4-TM5-TM7-TM1 interface, which is in agreement with experimental data. This model was used to study interactions of the previously published dopamine D2 receptor homobivalent antagonists based on clozapine,1,4-disubstituted aromatic piperidines/piperazines and arylamidoalkyl substituted phenylpiperazine pharmacophores. It was found that the homobivalent antagonists stabilize the receptor-inactive conformation by maintaining the ionic lock interaction, and change the dimer interface by disrupting a set of hydrogen bonds and maintaining water- and ligand-mediated hydrogen bonds in the extracellular and intracellular part of the interface. Graphical Abstract Structure of the final model of the dopamine D2 receptor homodimer, indicating the distancebetween Tyr37 and Tyr 5.42 in the apo form (left) and in the complex with the ligand (right). PMID:27491852

  6. Evaluation of Phytocannabinoids from High Potency Cannabis sativa using In Vitro Bioassays to Determine Structure-Activity Relationships for Cannabinoid Receptor 1 and Cannabinoid Receptor 2.

    PubMed

    Husni, Afeef S; McCurdy, Christopher R; Radwan, Mohamed M; Ahmed, Safwat A; Slade, Desmond; Ross, Samir A; ElSohly, Mahmoud A; Cutler, Stephen J

    2014-09-01

    Cannabis has been around for thousands of years and has been used recreationally, medicinally, and for fiber. Over 500 compounds have been isolated from Cannabis sativa with approximately 105 being cannabinoids. Of those 105 compounds, Δ(9)-tetrahydrocannabinol has been determined as the primary constituent, which is also responsible for the psychoactivity associated with Cannabis. Cannabinoid receptors belong to the large superfamily of G protein-coupled receptors. Targeting the cannabinoid receptors has the potential to treat a variety of conditions such as pain, neurodegeneration, appetite, immune function, anxiety, cancer, and others. Developing in vitro bioassays to determine binding and functional activity of compounds has the ability to lead researchers to develop a safe and effective drug that may target the cannabinoid receptors. Using radioligand binding and functional bioassays, a structure-activity relationship for major and minor cannabinoids was developed. PMID:25419092

  7. Evaluation of Phytocannabinoids from High Potency Cannabis sativa using In Vitro Bioassays to Determine Structure-Activity Relationships for Cannabinoid Receptor 1 and Cannabinoid Receptor 2

    PubMed Central

    Husni, Afeef S.; McCurdy, Christopher R.; Radwan, Mohamed M.; Ahmed, Safwat A.; Slade, Desmond; Ross, Samir A.; ElSohly, Mahmoud A.; Cutler, Stephen J.

    2014-01-01

    Cannabis has been around for thousands of years and has been used recreationally, medicinally, and for fiber. Over 500 compounds have been isolated from Cannabis sativa with approximately 105 being cannabinoids. Of those 105 compounds, Δ9-tetrahydrocannabinol has been determined as the primary constituent, which is also responsible for the psychoactivity associated with Cannabis. Cannabinoid receptors belong to the large superfamily of G protein-coupled receptors. Targeting the cannabinoid receptors has the potential to treat a variety of conditions such as pain, neurodegeneration, appetite, immune function, anxiety, cancer, and others. Developing in vitro bioassays to determine binding and functional activity of compounds has the ability to lead researchers to develop a safe and effective drug that may target the cannabinoid receptors. Using radioligand binding and functional bioassays, a structure-activity relationship for major and minor cannabinoids was developed. PMID:25419092

  8. The structural evolution of a P2Y-like G-protein-coupled receptor.

    PubMed

    Schulz, Angela; Schöneberg, Torsten

    2003-09-12

    Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution approximately 43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains. PMID:12835326

  9. Structural Basis of Interaction Between Urokinase-type Plasminogen Activator and its Receptor

    SciTech Connect

    Barinka,C.; Parry, G.; Callahan, J.; Shaw, D.; Kuo, A.; Cines, B.; Mazar, A.; Lubkowski, J.

    2006-01-01

    Recent studies indicate that binding of the urokinase-type plasminogen activator (uPA) to its high-affinity receptor (uPAR) orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes, including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known about the exact mode of uPAR/uPA interactions or the presumed conformational changes that accompany uPA/uPAR engagement. Here, we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell-surface anchoring sequence, in complex with the amino-terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 {angstrom}. We report the 1.9 {angstrom} crystal structure of free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR/uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding.

  10. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism.

    PubMed

    Chen, Lei; Dürr, Katharina L; Gouaux, Eric

    2014-08-29

    AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating. PMID:25103405

  11. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    SciTech Connect

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien

    2012-06-27

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  12. Understanding the structural specificity of Tn antigen for its receptor: an NMR solution study.

    PubMed

    D'Amelio, Nicola; Coslovi, Anna; Rossi, Marco; Uggeri, Fulvio; Paoletti, Sergio

    2012-04-01

    The present work aims at understanding the structural basis of the biological recognition of Tn antigen (GalNAc-α-O-L-Ser), a specific epitope expressed by tumor cells, and the role of its amino acidic moiety in the interaction with its receptor (the isolectin B4 extracted from Vicia villosa). An NMR structural characterization of the α and β anomers, based on J couplings and molecular modeling revealed a structure in very good agreement with data reported in literature for variants of the same molecules. In order to demonstrate the involvement of the amino acid in the ligand-receptor recognition, also GalNAc-α-O-D-Ser was studied; the change in the stereochemistry is in fact expected to impact on the interaction only in case the serine is part of the epitope. Relaxation properties in the presence of the receptor clearly indicated a selective recognition of the natural L form, probably due to the formation of a water-mediated hydrogen bond with Asn 129 of the protein. PMID:22341503

  13. The Polyomaviridae: Contributions of virus structure to our understanding of virus receptors and infectious entry

    SciTech Connect

    Neu, Ursula; Stehle, Thilo Atwood, Walter J.

    2009-02-20

    This review summarizes the field's major findings related to the characterization of polyomavirus structures and to the characterization of virus receptors and mechanisms of host cell invasion. The four members of the family that have received the most attention in this regard are the mouse polyomavirus (mPyV), the monkey polyomavirus SV40, and the two human polyomaviruses, JCV and BKV. The structures of both the mPyV and SV40 alone and in complex with receptor fragments have been solved to high resolution. The majority of polyomaviruses recognize terminal sialic acid in either an {alpha}2,3 linkage or an {alpha}2,6 linkage to the underlying galactose. Studies on virus structure, receptor utilization and mechanisms of entry have led to new insights into how these viruses interact in an active way with cells to ensure the nuclear delivery and expression of their genomes. Critical work on virus entry has led to the discovery of a pH neutral endocytic compartment that accepts cargo from caveolae and to novel roles for endoplasmic reticulum (ER) associated factors in virus uncoating and penetration of ER membranes. This review will summarize the major findings and compare and contrast the mechanisms used by these viruses to infect cells.

  14. Crystal Structure of the GerBC Component of a Bacillus Subtilis Spore Germinant Receptor

    SciTech Connect

    Li, Y.; Setlow, B; Setlow, P; Hao, B

    2010-01-01

    The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here, we report the crystal structure of the C protein of the GerB germinant receptor, so-called GerBC, of Bacillus subtilis spores at 2.3 {angstrom} resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a beta sheet surrounded by multiple alpha helices. Secondary-structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation, and function of nGRs.

  15. Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

    PubMed

    Hurt, Darrell E; Suzuki, Shigeru; Mayama, Takafumi; Charmandari, Evangelia; Kino, Tomoshige

    2016-02-01

    Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR. PMID:26745667

  16. Structures of CD200/CD200 Receptor Family and Implications for Topology, Regulation, and Evolution

    PubMed Central

    Hatherley, Deborah; Lea, Susan M.; Johnson, Steven; Barclay, A. Neil

    2013-01-01

    Summary CD200 is a widely distributed membrane glycoprotein that regulates myeloid cell activity through its interaction with an inhibitory receptor (CD200R). The interaction is of interest as a target for treating excessive inflammation and for treating leukemia. There are closely related proteins to CD200R that give activating signals making this a “paired receptor.” We report X-ray crystallography structures for the inhibitory CD200R, the activating receptor CD200RLa, and a complex between CD200R and CD200. Both CD200 and CD200R contain two Ig-like domains and interact through their NH2 terminal domains compatible with immunological synapse-like interactions occurring between myeloid cells and other CD200-expressing cells. The failure of the activating receptor to bind CD200 resides in subtle changes around the interface. CD200 has been acquired by herpes viruses to mimic the host interaction. CD200R has evolved rapidly presumably driven by pathogen pressure but it may also be important in homeostasis through interactions with commensal bacteria. PMID:23602662

  17. Development and Validation of Quantitative Structure-Activity Relationship Models for Compounds Acting on Serotoninergic Receptors

    PubMed Central

    Żydek, Grażyna; Brzezińska, Elżbieta

    2012-01-01

    A quantitative structure-activity relationship (QSAR) study has been made on 20 compounds with serotonin (5-HT) receptor affinity. Thin-layer chromatographic (TLC) data and physicochemical parameters were applied in this study. RP2 TLC 60F254 plates (silanized) impregnated with solutions of propionic acid, ethylbenzene, 4-ethylphenol, and propionamide (used as analogues of the key receptor amino acids) and their mixtures (denoted as S1–S7 biochromatographic models) were used in two developing phases as a model of drug-5-HT receptor interaction. The semiempirical method AM1 (HyperChem v. 7.0 program) and ACD/Labs v. 8.0 program were employed to calculate a set of physicochemical parameters for the investigated compounds. Correlation and multiple linear regression analysis were used to search for the best QSAR equations. The correlations obtained for the compounds studied represent their interactions with the proposed biochromatographic models. The good multivariate relationships (R2 = 0.78–0.84) obtained by means of regression analysis can be used for predicting the quantitative effect of biological activity of different compounds with 5-HT receptor affinity. “Leave-one-out” (LOO) and “leave-N-out” (LNO) cross-validation methods were used to judge the predictive power of final regression equations. PMID:22619602

  18. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

  19. Rat hepatic. beta. /sub 2/-adrenergic receptor: structural similarities to the rat fat cell. beta. /sub 1/-adrenergic receptor

    SciTech Connect

    Graziano, M.P.

    1984-01-01

    The mammalian ..beta../sub 2/-adrenergic receptor from rat liver has been purified by sequential cycles of affinity chromatography followed by steric-exclusion high performance liquid chromatography. Electrophoresis of highly purified receptor preparations on polyacrylamide gels in the presence of sodium dodecyl sulfate under reducing conditions reveals a single peptide M/sub r/ = 67,000, as judged by silver staining. Purified ..beta../sub 2/-adrenergic receptor migrates on steric-exclusion high performance liquid chromatography in two peaks, with M/sub r/ = 140,000 and 67,000. Specific binding of the high affinity, ..beta..-adrenergic receptor antagonists (-)(/sup 3/H)dihydroalprenolol and (-)(/sup 125/I)iodocyanopindolol to purified rat liver ..beta..-adrenergic receptor preparations displays stereoselectivity for (-)isomers of agonists and a rank order of potencies for agonists characteristics of a ..beta../sub 2/-adrenergic receptor. Radioiodinated, ..beta../sub 1/-adrenergic receptors from rat fat cells and ..beta../sub 2/-adrenergic receptors from rat liver purified in the presence of protease inhibitors comigrate in electrophoretic separations on polyacrylamide gels in the presence of sodium dodecyl sulfate as 67,000-M/sub r/ peptides. Autoradiograms of two dimensional partial proteolytic digests of the purified, radioiodinated rat liver ..beta../sub 2/-adrenergic receptor, generated with ..cap alpha..-chymotrypsin, S. aureus V8 protease and elastase reveal a pattern of peptide fragments essentially identical to those generated by partial proteolytic digests of the purified, radioiodinated ..beta../sub 1/-adrenergic receptor from rat fat cells, by these same proteases. These data indicate that a high degree of homology exists between these two pharmacologically distinct mammalian ..beta..-adrenergic receptor proteins.

  20. A Hybrid Approach to Structure and Function Modeling of G Protein-Coupled Receptors.

    PubMed

    Latek, Dorota; Bajda, Marek; Filipek, Sławomir

    2016-04-25

    The recent GPCR Dock 2013 assessment of serotonin receptor 5-HT1B and 5-HT2B, and smoothened receptor SMO targets, exposed the strengths and weaknesses of the currently used computational approaches. The test cases of 5-HT1B and 5-HT2B demonstrated that both the receptor structure and the ligand binding mode can be predicted with the atomic-detail accuracy, as long as the target-template sequence similarity is relatively high. On the other hand, the observation of a low target-template sequence similarity, e.g., between SMO from the frizzled GPCR family and members of the rhodopsin family, hampers the GPCR structure prediction and ligand docking. Indeed, in GPCR Dock 2013, accurate prediction of the SMO target was still beyond the capabilities of most research groups. Another bottleneck in the current GPCR research, as demonstrated by the 5-HT2B target, is the reliable prediction of global conformational changes induced by activation of GPCRs. In this work, we report details of our protocol used during GPCR Dock 2013. Our structure prediction and ligand docking protocol was especially successful in the case of 5-HT1B and 5-HT2B-ergotamine complexes for which we provide one of the most accurate predictions. In addition to a description of the GPCR Dock 2013 results, we propose a novel hybrid computational methodology to improve GPCR structure and function prediction. This computational methodology employs two separate rankings for filtering GPCR models. The first ranking is ligand-based while the second is based on the scoring scheme of the recently published BCL method. In this work, we prove that the use of knowledge-based potentials implemented in BCL is an efficient way to cope with major bottlenecks in the GPCR structure prediction. Thereby, we also demonstrate that the knowledge-based potentials for membrane proteins were significantly improved, because of the recent surge in available experimental structures. PMID:26978043

  1. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  2. Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein

    PubMed Central

    Moriel, Danilo G.; Paxman, Jason J.; Lo, Alvin W.; Tan, Lendl; Sullivan, Matthew J.; Dando, Samantha J.; Beatson, Scott A.

    2016-01-01

    ABSTRACT Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection. PMID:26980835

  3. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    PubMed Central

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners. PMID:22536173

  4. Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA.

    PubMed

    Muroi, Yukiko; Theusch, Cassandra M; Czajkowski, Cynthia; Jackson, Meyer B

    2009-01-01

    The barbiturate pentobarbital binds to gamma-aminobutyric acid type A (GABA(A)) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (approximately 10-100 microM), activate (approximately 100-800 microM), or block (approximately 1-10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABA(A) receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (F(TMRM)) of labels tethered to two residues in the extracellular domain (alpha(1)L127C and beta(2)L125C) that have been shown previously to produce an increase in F(TMRM) in response to GABA. Label at beta(2)K274C in the extracellular end of the M2 transmembrane helix reported a small but significant F(TMRM) increase during application of low modulating pentobarbital concentrations, and it showed a much greater F(TMRM) increase at higher concentrations. In contrast, GABA decreased F(TMRM) at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at alpha(1)L127C and beta(2)K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in F(TMRM) at these sites, and the pattern of F(TMRM) signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or

  5. Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation†

    PubMed Central

    Eilers, Markus; Hornak, Viktor; Smith, Steven O.; Konopka, James B.

    2005-01-01

    All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure–function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast α-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors. PMID:15966721

  6. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    SciTech Connect

    Nemčovičová, Ivana; Zajonc, Dirk M.

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor

  7. Structural Basis for Iloprost as a Dual Peroxisome Proliferator-activated Receptor [alpha/delta] Agonist

    SciTech Connect

    Jin, Lihua; Lin, Shengchen; Rong, Hui; Zheng, Songyang; Jin, Shikan; Wang, Rui; Li, Yong

    2012-03-15

    Iloprost is a prostacyclin analog that has been used to treat many vascular conditions. Peroxisome proliferator-activated receptors (PPARs) are ligand-regulated transcription factors with various important biological effects such as metabolic and cardiovascular physiology. Here, we report the crystal structures of the PPAR{alpha} ligand-binding domain and PPAR{delta} ligand-binding domain bound to iloprost, thus providing unambiguous evidence for the direct interaction between iloprost and PPARs and a structural basis for the recognition of PPAR{alpha}/{delta} by this prostacyclin analog. In addition to conserved contacts for all PPAR{alpha} ligands, iloprost also initiates several specific interactions with PPARs using its unique structural groups. Structural and functional studies of receptor-ligand interactions reveal strong functional correlations of the iloprost-PPAR{alpha}/{delta} interactions as well as the molecular basis of PPAR subtype selectivity toward iloprost ligand. As such, the structural mechanism may provide a more rational template for designing novel compounds targeting PPARs with more favorable pharmacologic impact based on existing iloprost drugs.

  8. Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models.

    PubMed

    Zhang, Xiaorui; Qi, Chunhui; Guo, Yan; Zhou, Wenxia; Zhang, Yongxiang

    2016-09-20

    Toll-like receptor (TLR) 4 is an important polysaccharide receptor; however, the relationships between the structures and biological activities of TLR4 and polysaccharides remain unknown. Many recent findings have revealed the primary structure of TLR4/MD-2-related polysaccharides, and several three-dimensional structure models of polysaccharide-binding proteins have been reported; and these models provide insights into the mechanisms through which polysaccharides interact with TLR4. In this review, we first discuss the origins of polysaccharides related to TLR4, including polysaccharides from higher plants, fungi, bacteria, algae, and animals. We then briefly describe the glucosidic bond types of TLR4-related heteroglycans and homoglycans and describe the typical molecular weights of TLR4-related polysaccharides. The primary structures and activity relationships of polysaccharides with TLR4/MD-2 are also discussed. Finally, based on the existing interaction models of LPS with TLR4/MD-2 and linear polysaccharides with proteins, we provide insights into the possible interaction models of polysaccharide ligands with TLR4/MD-2. To our knowledge, this review is the first to summarize the primary structures and activity relationships of TLR4-related polysaccharides and the possible mechanisms of interaction for TLR4 and TLR4-related polysaccharides. PMID:27261743

  9. Structure, signaling mechanism and regulation of natriuretic peptide receptor-guanylate cyclase

    PubMed Central

    Misono, Kunio S.; Philo, John S.; Arakawa, Tsutomu; Ogata, Craig M.; Qiu, Yue; Ogawa, Haruo; Young, Howard S.

    2011-01-01

    Summary Atrial natriuretic peptide (ANP) and homologous B-type natriuretic peptide (BNP) are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and BNP counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by the A-type natriuretic peptide receptor (NPRA), a single transmembrane segment, guanylate cyclase (GC) linked receptor that occurs as a homodimer. Here we present an overview of the structure, possible chloride-mediated regulation, and signaling mechanism of the NPRA and other receptor-GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacter GC Cya2 have been reported. These structures closely resemble that of the adenylate cyclase catalytic domain consisting of C1 and C2 subdomain heterodimer. AC is activated by binding of Gsα to C2 and ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer into a catalytically active conformation. We speculate that, in the NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity. PMID:21375693

  10. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S.

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  11. Structural insights into the dynamic process of β2-adrenergic receptor signaling

    PubMed Central

    Manglik, Aashish; Kim, Tae Hun; Masureel, Matthieu; Altenbach, Christian; Yang, Zhongyu; Hilger, Daniel; Lerch, Michael T.; Kobilka, Tong Sun; Thian, Foon Sun; Hubbell, Wayne L.; Prosser, R. Scott; Kobilka, Brian K.

    2015-01-01

    SUMMARY G protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium towards a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate and active states. Complete transition to the active conformation requires subsequent interaction with a G-protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs. PMID:25981665

  12. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    SciTech Connect

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  13. Molecular And Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System

    SciTech Connect

    LaPorte, S.L.; Juo, Z.S.; Vaclavikova, J.; Colf, L.A.; Qi, X.; Heller, N.M.; Keegan, A.D.; Garcia, K.C.

    2009-05-20

    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R{alpha}/{gamma}{sub c}/IL-4) and type II (IL-4R/IL-13R{alpha}1/IL-4, IL-4R{alpha}/IL-13R{alpha}1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for {gamma}{sub c}'s ability to recognize six different {gamma}{sub c}-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R{alpha}1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

  14. Structural Analysis of Sensor Domains from the TMAO-Responsive Histidine Kinase Receptor TorS.

    SciTech Connect

    Moore, J.; Hendrickson, W

    2009-01-01

    Histidine kinase receptors respond to diverse signals and mediate signal transduction across the plasma membrane in all prokaryotes and certain eukaryotes. Each receptor is part of a two-component system that regulates a particular cellular process. Organisms that use trimethylamine-N-oxide (TMAO) as a terminal electron acceptor typically control their anaerobic respiration through the TMAO reductase (Tor) pathway, which the TorS histidine kinase activates when sensing TMAO in the environment. We have determined crystal structures for the periplasmic sensor domains of TorS receptors from Escherichia coli and Vibrio parahaemolyticus. TorS sensor domains have a novel fold consisting of a membrane-proximal right-handed four-helical bundle and a membrane-distal left-handed four-helical bundle, but conformational dispositions differ significantly in the two structures. Isolated TorS sensor domains dimerize in solution; and from comparisons with dimeric NarX and Tar sensors, we postulate that signaling through TorS dimers involves a piston-type displacement between helices.

  15. Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies*

    PubMed Central

    Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

    2016-01-01

    Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229. PMID:27129274

  16. Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

    PubMed Central

    Maqbool, A; Saitoh, H; Franceschetti, M; Stevenson, CEM; Uemura, A; Kanzaki, H; Kamoun, S; Terauchi, R; Banfield, MJ

    2015-01-01

    Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001 PMID:26304198

  17. Crystal Structure of the β2Adrenergic Receptor-Gs protein complex

    PubMed Central

    Rasmussen, Søren G.F.; DeVree, Brian T.; Zou, Yaozhong; Kruse, Andrew C.; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M.; Shah, Syed T. A.; Lyons, Joseph A.; Caffrey, Martin; Gellman, Samuel H.; Steyaert, Jan; Skiniotis, Georgios; Weis, William I.; Sunahara, Roger K.; Kobilka, Brian K.

    2011-01-01

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signaling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signaling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino and carboxyl terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an alpha helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the alpha helical domain of Gαs relative to the ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signaling by a GPCR. PMID:21772288

  18. The Fas-FADD Death Domain Complex Structure Unravels Signalling by Receptor Clustering

    SciTech Connect

    Scott, F.; Stec, B; Pop, C; Dobaczewska, M; Lee, J; Monosov, E; Robinson, H; Salvesen, G; Schwarzenbacher, R; Riedl, S

    2009-01-01

    The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis1, 2, 3. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation4, 5. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

  19. Polymodal Transient Receptor Potential Vanilloid Type 1 Nocisensor: Structure, Modulators, and Therapeutic Applications.

    PubMed

    Cui, Minghua; Gosu, Vijayakumar; Basith, Shaherin; Hong, Sunhye; Choi, Sun

    2016-01-01

    Transient receptor potential (TRP) channels belong to a superfamily of sensory-related ion channels responding to a wide variety of thermal, mechanical, or chemical stimuli. In an attempt to comprehend the piquancy and pain mechanism of the archetypal vanilloids, transient receptor potential vanilloid (TRPV) 1 was discovered. TRPV1, a well-established member of the TRP family, is implicated in a range of functions including inflammation, painful stimuli sensation, and mechanotransduction. TRPV1 channels are nonselective cation receptors that are gated by a broad array of noxious ligands. Such polymodal-sensor aspect makes the TRPV1 channel extremely versatile and important for its role in sensing burning pain. Besides ligands, TRPV1 signaling can also be modulated by lipids, secondary messengers, protein kinases, cytoskeleton, and several other proteins. Due to its central role in hyperalgesia transduction and inflammatory processes, it is considered as the primary pharmacological pain target. Moreover, understanding the structural and functional intricacies of the channel is indispensable for the therapeutic intervention of TRPV1 in pain and other pathological disorders. In this chapter, we seek to give a mechanistic outlook on the TRPV1 channel. Specifically, we will explore the TRPV1 structure, activation, modulation, ligands, and its therapeutic targeting. However, the major objective of this review is to highlight the fact that TRPV1 channel can be treated as an effective therapeutic target for treating several pain- and nonpain-related physiological and pathological states. PMID:27038373

  20. Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies.

    PubMed

    Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

    2016-06-24

    Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229). PMID:27129274

  1. Amylin structure-function relationships and receptor pharmacology: implications for amylin mimetic drug development.

    PubMed

    Bower, Rebekah L; Hay, Debbie L

    2016-06-01

    Amylin is an important, but poorly understood, 37 amino acid glucoregulatory hormone with great potential to target metabolic diseases. A working example that the amylin system is one worth developing is the FDA-approved drug used in insulin-requiring diabetic patients, pramlintide. However, certain characteristics of pramlintide pharmacokinetics and formulation leave considerable room for further development of amylin-mimetic compounds. Given that amylin-mimetic drug design and development is an active area of research, surprisingly little is known about the structure/function relationships of amylin. This is largely due to the unfavourable aggregative and solubility properties of the native peptide sequence, which are further complicated by the composition of amylin receptors. These are complexes of the calcitonin receptor with receptor activity-modifying proteins. This review explores what is known of the structure-function relationships of amylin and provides insights that can be drawn from the closely related peptide, CGRP. We also describe how this information is aiding the development of more potent and stable amylin mimetics, including peptide hybrids. PMID:27061187

  2. Structure-based approach for the study of thyroid hormone receptor binding affinity and subtype selectivity.

    PubMed

    Wang, Fang-Fang; Yang, Wei; Shi, Yong-Hui; Cheng, Xiang-Rong; Le, Guo-Wei

    2016-10-01

    Thyroid hormone (TH) possesses the ability to lower cholesterol and improve cardiac performance, which have prompted the efforts to design analogs that can utilize the cholesterol-lowering property without adversely affecting heart function. In order to gain insights into the interaction mechanism for agonists at the active site of thyroid hormone receptor β (TRβ), quantitative structure-activity relationship (QSAR) models have been developed on TRβ agonists, significant statistical coefficients were obtained (CoMFA, R(2)cv, .732), (CoMSIA, R(2)cv, .853), indicating the internal consistency of the models, the obtained models were further validated using the test set, the acquired R(2)pred values .7054 and .7129 were in good agreement with the experimental results. The key amino acids affecting ligand binding were identified by molecular docking, and the detailed binding modes of the compounds with different activities were also determined. Furthermore, molecular dynamics (MD) simulations were conducted to assess the reliability of the derived models and the docking results. Moreover, TH exerts significant physiological effects through modulation of the two human thyroid hormone receptor subtypes. Because TRβ and TRα locate in different target cells, selective TR ligands would target specific tissues regulated by one receptor without affecting the other. Thus, the 3D information was analyzed to reveal the most relevant structural features involved in selectivity. The findings serve as the basis for further investigation into selective TRβ/TRα agonists. PMID:26510472

  3. Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

    SciTech Connect

    Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

    1986-05-01

    The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions and subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.

  4. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    PubMed Central

    Di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-01-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation. PMID:26567894

  5. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    NASA Astrophysics Data System (ADS)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-11-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  6. The crystal structure of a bimorphinan with highly selective kappa opioid receptor antagonist activity

    NASA Astrophysics Data System (ADS)

    Urbańczyk-Lipkowska, Zofia; Etter, Margaret C.; Lipkowski, Andrzej W.; Portoghese, Philip S.

    1987-07-01

    The crystal structure of the dihydrobromide heptahydrate of nor-binaltorphimine (17, 17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-4,5α: 4',5'α-diepoxy-6,6'-imino[7,7' bimorphinan]-3,3',14,14'-tetraol)is presented. This structure is the first reported structure of a rigid bivalent opioid ligand. Two morphinan pharmacophores are connected by a rigid spacer, the pyrrole ring. The nor-binaltorphimine structure itself shows unique, high selectivity as a kappa opioid receptor antagonist. Crystal data: P3 2, Z = 3, a = b = 20.223 (4), c = 9.541(7) Å, α = β = 90°, γ = 120°; R = 0.079 (1765 reflections, Fobs > 1σ( F)).

  7. Form follows function - the three-dimensional structure of antigen receptor gene loci.

    PubMed

    Fugmann, Sebastian D

    2014-04-01

    Antigen receptor genes are assembled during lymphocyte development from individual gene segments by a somatic gene rearrangement process named V(D)J recombination. This process is tightly regulated to ensure the generation of an unbiased broad primary repertoire of immunoglobulins and T cell receptors, and to prevent aberrant recombination products that could initiate lymphomagenesis. One important mode of regulation that has recently been discovered for the immunoglobulin heavy chain (IGH) gene locus is the adoption of distinct three-dimensional structures of the locus. Changes in the spatial conformation are thought to ensure the appropriate access of the V(D)J recombinase machinery at each developmental stage, and the formation of extensive chromosome loops has been implicated in allowing equal access to widely dispersed gene elements. PMID:24549092

  8. Structure-based design of eugenol analogs as potential estrogen receptor antagonists.

    PubMed

    Anita, Yulia; Radifar, Muhammad; Kardono, Leonardus Bs; Hanafi, Muhammad; Istyastono, Enade P

    2012-01-01

    Eugenol is an essential oil mainly found in the buds and leaves of clove (Syzygium aromaticum (L.) Merrill and Perry), which has been reported to have activity on inhibition of cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. This biological activity is correlated to its activity as an estrogen receptor antagonist. In this article, we present the construction and validation of structure-based virtual screening (SBVS) protocols to identify the potent estrogen receptor α (ER) antagonists. The selected protocol, which gave acceptable enrichment factors as a virtual screening protocol, subsequently used to virtually screen eugenol, its analogs and their dimers. Based on the virtual screening results, dimer eugenol of 4-[4-hydroxy-3-(prop-2-en-1- yl)phenyl]-2-(prop-2-en-1-yl)phenol is recommended to be developed further in order to discover novel and potent ER antagonists. PMID:23144548

  9. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  10. Glycine receptors: recent insights into their structural organization and functional diversity.

    PubMed

    Betz, Heinrich; Laube, Bodo

    2006-06-01

    Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds. PMID:16805771

  11. Structure of the measles virus hemagglutinin bound to the CD46 receptor.

    PubMed

    Santiago, César; Celma, María L; Stehle, Thilo; Casasnovas, José M

    2010-01-01

    The highly contagious measles virus infects millions of individuals worldwide, causing serious disease in children of developing countries. Infection is initiated by attachment of the measles virus hemagglutinin (MV-H), a glycoprotein anchored to the virus envelope, to the host cell receptors CD46 or signaling lymphocyte activation molecule (SLAM). Here we report the crystal structure of MV-H in complex with a CD46 protein spanning the two N-terminal domains. A unique groove at the side of the MV-H beta-propeller domain, which is absent in homologous paramyxovirus attachment proteins, engages residues in both CD46 domains. Key contacts involve a protruding loop in the N-terminal CD46 domain that carries two sequential proline residues (PP motif) and penetrates deeply into a hydrophobic socket in MV-H. We identify a similar PP motif in SLAM, defining a common measles virus recognition epitope in the CD46 and SLAM receptor proteins. PMID:20010840

  12. Structural model of ρ1 GABAC receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

    PubMed Central

    Adamian, Larisa; Gussin, Hélène A; Tseng, Yan Yuan; Muni, Niraj J; Feng, Feng; Qian, Haohua; Pepperberg, David R; Liang, Jie

    2009-01-01

    The homopentameric ρ1 GABAC receptor is a ligand-gated ion channel with a binding pocket for γ-aminobutyric acid (GABA) at the interfaces of N-terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABAC receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as ω-values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158–D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D–D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158–D204 salt bridge perhaps stabilizes the open state of the GABAC receptor. Our results emphasize the functional importance of highly conserved polar residues at the protein–protein interfaces in GABAC ρ1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions. PMID:19768800

  13. Structural insights into Resveratrol’s antagonist and partial agonist actions on estrogen receptor alpha

    PubMed Central

    2013-01-01

    Background Resveratrol, a naturally occurring stilbene, has been categorized as a phytoestrogen due to its ability to compete with natural estrogens for binding to estrogen receptor alpha (ERα) and modulate the biological responses exerted by the receptor. Biological effects of resveratrol (RES) on estrogen receptor alpha (ERα) remain highly controversial, since both estrogenic and anti-estrogenic properties were observed. Results Here, we provide insight into the structural basis of the agonist/antagonist effects of RES on ERα ligand binding domain (LBD). Using atomistic simulation, we found that RES bound ERα monomer in antagonist conformation, where Helix 12 moves away from the ligand pocket and orients into the co-activator binding groove of LBD, is more stable than RES bound ERα in agonist conformation, where Helix 12 lays over the ligand binding pocket. Upon dimerization, the agonistic conformation of RES-ERα dimer becomes more stable compared to the corresponding monomer but still remains less stable compared to the corresponding dimer in antagonist conformation. Interestingly, while the binding pocket and the binding contacts of RES to ERα are similar to those of pure agonist diethylstilbestrol (DES), the binding energy is much less and the hydrogen bonding contacts also differ providing clues for the partial agonistic character of RES on ERα. Conclusions Our Molecular Dynamics simulation of RES-ERα structures with agonist and antagonist orientations of Helix 12 suggests RES action is more similar to Selective Estrogen Receptor Modulator (SERM) opening up the importance of cellular environment and active roles of co-regulator proteins in a given system. Our study reveals that potential co-activators must compete with the Helix 12 and displace it away from the activator binding groove to enhance the agonistic activity. PMID:24160181

  14. Structure of the human [kappa]-opioid receptor in complex with JDTic

    SciTech Connect

    Wu, Huixian; Wacker, Daniel; Mileni, Mauro; Katritch, Vsevolod; Han, Gye Won; Vardy, Eyal; Liu, Wei; Thompson, Aaron A.; Huang, Xi-Ping; Carroll, F. Ivy; Mascarella, S. Wayne; Westkaemper, Richard B.; Mosier, Philip D.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2013-04-25

    Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.

  15. Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment*

    PubMed Central

    Kofoed, Eric M.; Guerbadot, Martin; Schaufele, Fred

    2010-01-01

    An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor α-isoform (ERα) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ERα with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ERαs at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ERα monomer at higher SRC-RID concentrations (∼10 nm). The methods also showed that only a subpopulation of ERα was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment. PMID:19926790

  16. Probing Insect Odorant Receptors with their Cognate Ligands: Insights into Structural Features

    PubMed Central

    Xu, Pingxi; Leal, Walter S.

    2013-01-01

    Oodorant receptors (ORs) are essential for insect survival in the environment and thus are ideal molecular targets for the design of insect-inspired modern green chemicals to control populations of agricultural pests and insects of medical importance. Although insect ORs are known for more than a decade, their structural biology is still in its infancy. Here, we unravel the first structural features of ORs from the malaria mosquito, the Southern house mosquito and the silkworm moth. The second extracellular loops (ECL-2s) of their predicted structures are much longer than ECL-1s and ECL-3s. The 27 amino-acid-residue-long of the ECL-2s in mosquito and the 43 amino-acid-residue-long ECL2s in moth ORs are well-conserved. About one-third of the residues are identical, including 3-4 Pro residues. Thorough examination of well-conserved residues in these structures, by point mutation and functional assay with the Xenopus oocyte recording system, strongly suggest that these “loops” include three β-turns and some degree of folding. In the Southern house mosquito three Pro residues in ECL-2 are essential for full activation of the receptor, which is finely tuned to the oviposition attractant 3-methylindole. Additionally, the “corner residues” of prolines, including Gly, Tyr, and Leu are functionally important thus suggesting that turns are stabilized not only by backbone hydrogen bonds, but also by side-chain interactions. Examination of ECL-2s from a distant taxonomical group suggests these ECL-2 loops might be functionally important in all insect ORs. Two of the four Pro residues in the predicted ECL-2 of the bombykol receptor in the silkworm moth, BmorOR1, are essential for function. Experimental evidence indicates that these loops may not be specificity determinants, but they may form a cover to the yet-to-be-identified membrane embedded binding cavities of insect ORs. PMID:23673297

  17. Structural Basis of Activation of Bitter Taste Receptor T2R1 and Comparison with Class A G-protein-coupled Receptors (GPCRs)*

    PubMed Central

    Singh, Nisha; Pydi, Sai Prasad; Upadhyaya, Jasbir; Chelikani, Prashen

    2011-01-01

    The human bitter taste receptors (T2Rs) are non-Class A members of the G-protein-coupled receptor (GPCR) superfamily, with very limited structural information. Amino acid sequence analysis reveals that most of the important motifs present in the transmembrane helices (TM1–TM7) of the well studied Class A GPCRs are absent in T2Rs, raising fundamental questions regarding the mechanisms of activation and how T2Rs recognize bitter ligands with diverse chemical structures. In this study, the bitter receptor T2R1 was used to systematically investigate the role of 15 transmembrane amino acids in T2Rs, including 13 highly conserved residues, by amino acid replacements guided by molecular modeling. Functional analysis of the mutants by calcium imaging analysis revealed that replacement of Asn-662.65 and the highly conserved Asn-241.50 resulted in greater than 90% loss of agonist-induced signaling. Our results show that Asn-241.50 plays a crucial role in receptor activation by mediating an hydrogen bond network connecting TM1-TM2-TM7, whereas Asn-662.65 is essential for binding to the agonist dextromethorphan. The interhelical hydrogen bond between Asn-241.50 and Arg-552.54 restrains T2R receptor activity because loss of this bond in I27A and R55A mutants results in hyperactive receptor. The conserved amino acids Leu-1975.50, Ser-2005.53, and Leu-2015.54 form a putative LXXSL motif which performs predominantly a structural role by stabilizing the helical conformation of TM5 at the cytoplasmic end. This study provides for the first time mechanistic insights into the roles of the conserved transmembrane residues in T2Rs and allows comparison of the activation mechanisms of T2Rs with the Class A GPCRs. PMID:21852241

  18. A Hybrid Structural Approach to Analyze Ligand Binding by the Serotonin Type 4 Receptor (5-HT4)*

    PubMed Central

    Padayatti, Pius S.; Wang, Liwen; Gupta, Sayan; Orban, Tivadar; Sun, Wenyu; Salom, David; Jordan, Steven R.; Palczewski, Krzysztof; Chance, Mark R.

    2013-01-01

    Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography or NMR data on the protein of interest has been limited. For G protein-coupled receptors (GPCRs), high resolution structures of native structural forms other than rhodopsin have not yet been achieved; gaps in our knowledge have been filled by creative crystallography studies that have developed stable forms of receptors by multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting many physiological manifestations in humans ranging from mood and anxiety to bowel function. However, a high resolution structure of any of the serotonin receptors has not yet been solved. Here, we used structural mass spectrometry along with theoretical computations, modeling, and other biochemical methods to develop a structured model for human serotonin receptor subtype 4(b) in the presence and absence of its antagonist GR125487. Our data confirmed the overall structure predicted by the model and revealed a highly conserved motif in the ligand-binding pocket of serotonin receptors as an important participant in ligand binding. In addition, identification of waters in the transmembrane region provided clues as to likely paths mediating intramolecular signaling. Overall, this study reveals the potential of hybrid structural methods, including mass spectrometry, to probe physiological and functional GPCR-ligand interactions with purified native protein. PMID:23378516

  19. Modeling, Molecular Dynamics Simulation, and Mutation Validation for Structure of Cannabinoid Receptor 2 Based on Known Crystal Structures of GPCRs

    PubMed Central

    2015-01-01

    The cannabinoid receptor 2 (CB2) plays an important role in the immune system. Although a few of GPCRs crystallographic structures have been reported, it is still challenging to obtain functional transmembrane proteins and high resolution X-ray crystal structures, such as for the CB2 receptor. In the present work, we used 10 reported crystal structures of GPCRs which had high sequence identities with CB2 to construct homology-based comparative CB2 models. We applied these 10 models to perform a prescreen by using a training set consisting of 20 CB2 active compounds and 980 compounds randomly selected from the National Cancer Institute (NCI) database. We then utilized the known 170 cannabinoid receptor 1 (CB1) or CB2 selective compounds for further validation. Based on the docking results, we selected one CB2 model (constructed by β1AR) that was most consistent with the known experimental data, revealing that the defined binding pocket in our CB2 model was well-correlated with the training and testing data studies. Importantly, we identified a potential allosteric binding pocket adjacent to the orthosteric ligand-binding site, which is similar to the reported allosteric pocket for sodium ion Na+ in the A2AAR and the δ-opioid receptor. Our studies in correlation of our data with others suggested that sodium may reduce the binding affinities of endogenous agonists or its analogs to CB2. We performed a series of docking studies to compare the important residues in the binding pockets of CB2 with CB1, including antagonist, agonist, and our CB2 neutral compound (neutral antagonist) XIE35-1001. Then, we carried out 50 ns molecular dynamics (MD) simulations for the CB2 docked with SR144528 and CP55940, respectively. We found that the conformational changes of CB2 upon antagonist/agonist binding were congruent with recent reports of those for other GPCRs. Based on these results, we further examined one known residue, Val1133.32, and predicted two new residues, Phe183 in

  20. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

    SciTech Connect

    Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles; Lazear, Eric; Connolly, Sarah A.; Eisenberg, Roselyn J.; Cohen, Gary H.; Wiley, Don C.; Carfi, Andrea

    2010-07-19

    Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

  1. Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

    PubMed Central

    Krieger, James; Bahar, Ivet; Greger, Ingo H.

    2015-01-01

    Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment. PMID:26255587

  2. Structural insights and functional implications of inter-individual variability in β2-adrenergic receptor

    PubMed Central

    Tandale, Aditi; Joshi, Manali; Sengupta, Durba

    2016-01-01

    The human β2-adrenergic receptor (β2AR) belongs to the G protein-coupled receptor (GPCR) family and due to its central role in bronchodilation, is an important drug target. The inter-individual variability in β2AR has been implicated in disease susceptibility and differential drug response. In this work, we identified nine potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) using a consensus approach. The deleterious nsSNPs were found to cluster near the ligand binding site and towards the G-protein binding site. To assess their molecular level effects, we built structural models of these receptors and performed atomistic molecular dynamics simulations. Most notably, in the Phe290Ser variant we observed the rotameric flip of Trp2866.48, a putative activation switch that has not been reported in β2AR thus far. In contrast, the variant Met82Lys was found to be the most detrimental to epinephrine binding. Additionally, a few of the nsSNPs were seen to cause perturbations to the lipid bilayer, while a few lead to differences at the G-protein coupling site. We are thus able to classify the variants as ranging from activating to damaging, prioritising them for experimental studies. PMID:27075228

  3. Steroid hormone receptors and prostate cancer: role of structural dynamics in therapeutic targeting.

    PubMed

    Kumar, Raj

    2016-01-01

    Steroid hormone receptors (SHRs) act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator (SRM) ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain (LBD)/AF2 and neglect intrinsically disordered (ID) N-terminal domain (NTD)/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor's (AR's) ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR's structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer. PMID:27364545

  4. Steroid hormone receptors and prostate cancer: role of structural dynamics in therapeutic targeting

    PubMed Central

    Kumar, Raj

    2016-01-01

    Steroid hormone receptors (SHRs) act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator (SRM) ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain (LBD)/AF2 and neglect intrinsically disordered (ID) N-terminal domain (NTD)/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor's (AR's) ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR's structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer. PMID:27364545

  5. Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

    PubMed Central

    Hirano, Seishiro; Kanno, Sanae

    2015-01-01

    The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways. PMID:26545255

  6. Structural Recognition of an Optimized Substrate for the Ephrin family of Receptor Tyrosine Kinases

    PubMed Central

    Davis, Tara L.; Walker, John R.; Allali-Hassani, Abdellah; Parker, Sirlester A.; Turk, Benjamin E.; Dhe-Paganon, Sirano

    2010-01-01

    Summary Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor- expressing cells. Consequently the ephrins and ephrin RTKs often mediate processes involving cell:cell contact, including cellular adhesion or repulsion, developmental remodeling, and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and that is suitable for cocrystallization studies. Complex structures show multiple contacts between kinase and substrates, and in particular two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes. PMID:19678838

  7. Structure and receptor binding preferences of recombinant human A(H3N2) virus hemagglutinins.

    PubMed

    Yang, Hua; Carney, Paul J; Chang, Jessie C; Guo, Zhu; Villanueva, Julie M; Stevens, James

    2015-03-01

    A(H3N2) influenza viruses have circulated in humans since 1968, and antigenic drift of the hemagglutinin (HA) protein continues to be a driving force that allows the virus to escape the human immune response. Since the major antigenic sites of the HA overlap into the receptor binding site (RBS) of the molecule, the virus constantly struggles to effectively adapt to host immune responses, without compromising its functionality. Here, we have structurally assessed the evolution of the A(H3N2) virus HA RBS, using an established recombinant expression system. Glycan binding specificities of nineteen A(H3N2) influenza virus HAs, each a component of the seasonal influenza vaccine between 1968 and 2012, were analyzed. Results suggest that while its receptor-binding site has evolved from one that can bind a broad range of human receptor analogs to one with a more restricted binding profile for longer glycans, the virus continues to circulate and transmit efficiently among humans. PMID:25617824

  8. The structure of the atypical killer cell immunoglobulin-like receptor, KIR2DL4.

    PubMed

    Moradi, Shoeib; Berry, Richard; Pymm, Phillip; Hitchen, Corinne; Beckham, Simone A; Wilce, Matthew C J; Walpole, Nicholas G; Clements, Craig S; Reid, Hugh H; Perugini, Matthew A; Brooks, Andrew G; Rossjohn, Jamie; Vivian, Julian P

    2015-04-17

    The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor. PMID:25759384

  9. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.

    PubMed

    Anderson, K; Lai, F A; Liu, Q Y; Rousseau, E; Erickson, H P; Meissner, G

    1989-01-15

    Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle. PMID:2463249

  10. Kinetic and Structural Analysis of Coxsackievirus B3 Receptor Interactions and Formation of the A-Particle

    PubMed Central

    Organtini, Lindsey J.; Makhov, Alexander M.; Conway, James F.

    2014-01-01

    ABSTRACT The coxsackievirus and adenovirus receptor (CAR) has been identified as the cellular receptor for group B coxsackieviruses, including serotype 3 (CVB3). CAR mediates infection by binding to CVB3 and catalyzing conformational changes in the virus that result in formation of the altered, noninfectious A-particle. Kinetic analyses show that the apparent first-order rate constant for the inactivation of CVB3 by soluble CAR (sCAR) at physiological temperatures varies nonlinearly with sCAR concentration. Cryo-electron microscopy (cryo-EM) reconstruction of the CVB3-CAR complex resulted in a 9.0-Å resolution map that was interpreted with the four available crystal structures of CAR, providing a consensus footprint for the receptor binding site. The analysis of the cryo-EM structure identifies important virus-receptor interactions that are conserved across picornavirus species. These conserved interactions map to variable antigenic sites or structurally conserved regions, suggesting a combination of evolutionary mechanisms for receptor site preservation. The CAR-catalyzed A-particle structure was solved to a 6.6-Å resolution and shows significant rearrangement of internal features and symmetric interactions with the RNA genome. IMPORTANCE This report presents new information about receptor use by picornaviruses and highlights the importance of attaining at least an ∼9-Å resolution for the interpretation of cryo-EM complex maps. The analysis of receptor binding elucidates two complementary mechanisms for preservation of the low-affinity (initial) interaction of the receptor and defines the kinetics of receptor-catalyzed conformational change to the A-particle. PMID:24623425

  11. Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies.

    PubMed Central

    Careaga, C L; Falke, J J

    1992-01-01

    Cysteine residues introduced by site-directed mutagenesis have been used to probe the conformation and dynamics of two receptors in the E. coli chemotaxis pathway. (a) Thermal motions of the polypeptide backbone were investigated in the periplasmic D-galactose and D-glucose receptor, a globular protein of known structure. Disulfide bond formation between pairs of engineered sulfhydryls were used to trap collisions during the relative motions of surface alpha-helices I and X. Motions with amplitudes ranging from 4.5 to 15.2 A were detected on timescales ranging from 10(-4) to 10(-1) s, respectively. These results suggest that thermal backbone motions may have larger amplitudes than previously thought. (b) Conformational features of the transmembrane aspartate transducer have been investigated. Engineered sulfhydryls were used to ascertain the location and orientations of two putative transmembrane alpha-helices in the primary structure, to investigate the packing of these helices, to determine the oligomer and surface structures, and to detect thermal and ligand-induced dynamics of the polypeptide backbone. A model for the folded conformation of the transducer oligomer is reviewed. Images FIGURE 4 PMID:1318100

  12. Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

    PubMed Central

    Rahmeh, Rita; Damian, Marjorie; Cottet, Martin; Orcel, Hélène; Mendre, Christiane; Durroux, Thierry; Sharma, K. Shivaji; Durand, Grégory; Pucci, Bernard; Trinquet, Eric; Zwier, Jurriaan M.; Deupi, Xavier; Bron, Patrick; Banères, Jean-Louis; Mouillac, Bernard; Granier, Sébastien

    2012-01-01

    G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs. PMID:22493271

  13. In silico modeling techniques for predicting the tertiary structure of human H4 receptor.

    PubMed

    Zaid, Hilal; Raiyn, Jamal; Osman, Midhat; Falah, Mizied; Srouji, Samer; Rayan, Anwar

    2016-01-01

    First cloned in 2000, the human Histamine H4 Receptor (hH4R) is the last member of the histamine receptors family discovered so far, it belongs to the GPCR super-family and is involved in a wide variety of immunological and inflammatory responses. Potential hH4R antagonists are proposed to have therapeutic potential for the treatment of allergies, inflammation, asthma and colitis. So far, no hH4R ligands have been successfully introduced to the pharmaceutical market, which creates a strong demand for new selective ligands to be developed. in silico techniques and structural based modeling are likely to facilitate the achievement of this goal. In this review paper we attempt to cover the fundamental concepts of hH4R structure modeling and its implementations in drug discovery and development, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of hH4R and GPCRs, in regards to computerized techniques for 3-D structure modeling. PMID:26709794

  14. Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

    PubMed Central

    Roudnitzky, Natacha; Behrens, Maik; Engel, Anika; Kohl, Susann; Thalmann, Sophie; Hübner, Sandra; Lossow, Kristina; Wooding, Stephen P.; Meyerhof, Wolfgang

    2015-01-01

    The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were

  15. Structure-Based Ligand Discovery Targeting Orthosteric and Allosteric Pockets of Dopamine Receptors

    PubMed Central

    Lane, J. Robert; Chubukov, Pavel; Liu, Wei; Canals, Meritxell; Cherezov, Vadim; Abagyan, Ruben; Stevens, Raymond C.

    2013-01-01

    Small molecules targeting allosteric pockets of G protein–coupled receptors (GPCRs) have a great therapeutic potential for the treatment of neurologic and other chronic disorders. Here we performed virtual screening for orthosteric and putative allosteric ligands of the human dopamine D3 receptor (D3R) using two optimized crystal-structure–based models: the receptor with an empty binding pocket (D3RAPO), and the receptor complex with dopamine (D3RDopa). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 μM in the D3RAPO candidate list (56% hit rate), and 8 novel ligands in the D3RDopa list (32% hit rate). Most ligands in the D3RAPO model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC50 = 7 nM). In contrast, compounds identified by the D3RDopa model are predicted to occupy an allosteric site at the extracellular extension of the pocket, and they all lack the anchoring amino group. Compounds targeting the allosteric site display a variety of functional activity profiles, where behavior of at least two compounds (23 and 26) is consistent with noncompetitive allosteric modulation of dopamine signaling in the extracellular signal-regulated kinase 1 and 2 phosphorylation and β-arrestin recruitment assays. The high affinity and ligand efficiency of the chemically diverse hits identified in this study suggest utility of structure-based screening targeting allosteric sites of GPCRs. PMID:24021214

  16. A structural perspective on nuclear receptors as targets of environmental compounds

    PubMed Central

    Delfosse, Vanessa; Maire, Albane le; Balaguer, Patrick; Bourguet, William

    2015-01-01

    Nuclear receptors (NRs) are members of a large superfamily of evolutionarily related transcription factors that control a plethora of biological processes. NRs orchestrate complex events such as development, organ homeostasis, metabolism, immune function, and reproduction. Approximately one-half of the 48 human NRs have been shown to act as ligand-regulated transcription factors and respond directly to a large variety of endogenous hormones and metabolites that are generally hydrophobic and small in size (eg, retinoic acid or estradiol). The second half of the NR family comprises the so-called orphan receptors, for which regulatory ligands are still unknown or may not exist despite the presence of a C-terminal ligand-binding domain, which is the hallmark of all NRs. Several chemicals released into the environment (eg, bisphenols, phthalates, parabens, etc) share some physicochemical properties with natural ligands, allowing them to bind to NRs and activate or inhibit their action. Collectively referred to as endocrine disruptors or endocrine-disrupting chemicals (EDCs), these environmental pollutants are highly suspected to cause a wide range of developmental, reproductive, neurological, or metabolic defects in humans and wildlife. Crystallographic studies are revealing unanticipated mechanisms by which chemically diverse EDCs interact with the ligand-binding domain of NRs. These studies thereby provide a rational basis for designing novel chemicals with lower impacts on human and animal health. In this review, we provide a structural and mechanistic view of endocrine disrupting action using estrogen receptors α and β, (ERα/β), peroxisome proliferator activated receptor γ (PPARγ), and their respective environmental ligands as representative examples. PMID:25500867

  17. FUNCTIONAL ANALYSIS OF A NOVEL POSITIVE ALLOSTERIC MODULATOR OF AMPA RECEPTORS DERIVED FROM A STRUCTURE-BASED DRUG DESIGN STRATEGY

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Maclean, John K. F.; Partin, Kathryn M.; Jamieson, Craig

    2012-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors facilitate synaptic plasticity and can improve various forms of learning and memory. These modulators show promise as therapeutic agents for the treatment of neurological disorders such as schizophrenia, ADHD, and mental depression. Three classes of positive modulator, the benzamides, the thiadiazides, and the biarylsulfonamides differentially occupy a solvent accessible binding pocket at the interface between the two subunits that form the AMPA receptor ligand-binding pocket. Here, we describe the electrophysiological properties of a new chemotype derived from a structure-based drug design strategy (SBDD), which makes similar receptor interactions compared to previously reported classes of modulator. This pyrazole amide derivative, JAMI1001A, with a promising developability profile, efficaciously modulates AMPA receptor deactivation and desensitization of both flip and flop receptor isoforms. PMID:22735771

  18. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

    SciTech Connect

    Lentz, T.L. )

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similar segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.

  19. Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2.

    PubMed

    Johnston, Christopher A; Kimple, Adam J; Giguère, Patrick M; Siderovski, David P

    2008-07-01

    A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta. PMID:18611381

  20. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs

    PubMed Central

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-01-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an “Ω”-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

  1. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs.

    PubMed

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-05-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an "Ω"-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

  2. Functional Importance of a Structurally Distinct Homodimeric Complex of the Family B G Protein-Coupled Secretin Receptor

    PubMed Central

    Gao, Fan; Harikumar, Kaleeckal G.; Dong, Maoqing; Lam, Polo C.-H.; Sexton, Patrick M.; Christopoulos, Arthur; Bordner, Andrew; Abagyan, Ruben; Miller, Laurence J.

    2009-01-01

    Oligomerization of G protein-coupled receptors has been described, but its structural basis and functional importance have been inconsistent. Here, we demonstrate that the agonist occupied wild-type secretin receptor is predominantly in a guanine nucleotide-sensitive high-affinity state and exhibits negative cooperativity, whereas the monomeric receptor is primarily in a guanine nucleotide-insensitive lower affinity state. We previously demonstrated constitutive homodimerization of this receptor through the lipid-exposed face of transmembrane (TM) IV. We now use cysteine-scanning mutagenesis of 14 TM IV residues, bioluminescence resonance energy transfer (BRET), and functional analysis to map spatial approximations and functional importance of specific residues in this complex. All, except for three helix-facing mutants, trafficked to the cell surface, where secretin was shown to bind and elicit cAMP production. Cells expressing complementary-tagged receptors were treated with cuprous phenanthroline to establish disulfide bonds between spatially approximated cysteines. BRET was measured as an indication of receptor oligomerization and was repeated after competitive disruption of oligomers with TM IV peptide to distinguish covalent from noncovalent associations. Although all constructs generated a significant BRET signal, this was disrupted by peptide in all except for single-site mutants replacing five residues with cysteine. Of these, covalent stabilization of receptor homodimers through positions of Gly243, Ile247, and Ala250 resulted in a GTP-sensitive high-affinity state of the receptor, whereas the same procedure with Ala246 and Phe240 mutants resulted in a GTP-insensitive lower affinity state. We propose the existence of a functionally important, structurally specific high-affinity dimeric state of the secretin receptor, which may be typical of family B G protein-coupled receptors. PMID:19429716

  3. sup 19 F NMR studies of the D-galactose chemosensory receptor. (1) Sugar binding yields a global structural change

    SciTech Connect

    Luck, L.A.; Falke, J.J. )

    1991-04-30

    The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enable docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, the authors have used {sup 19}F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting {sup 19}F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylaline. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via {sup 19}F NMR frequency shifts at 10 of the 12 labeled sites. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.

  4. Serotonin 5-HT7 receptor agents: structure-activity relationships and potential therapeutic applications in central nervous system disorders

    PubMed Central

    Leopoldo, Marcello; Lacivita, Enza; Berardi, Francesco; Perrone, Roberto; Hedlund, Peter B.

    2010-01-01

    Since its discovery in the 1940s in serum, the mammalian intestinal mucosa, and in the central nervous system, serotonin (5-HT) has been shown to be involved in virtually all cognitive and behavioral human functions, and alterations in its neurochemistry have been implicated in the etiology of a plethora of neuropsychiatric disorders. The cloning of 5-HT receptor subtypes has been of importance in enabling them to be classified as specific protein molecules encoded by specific genes. The 5-HT7 receptor is the most recently classified member of the serotonin receptor family. Since its identification, it has been the subject of intense research efforts driven by its presence in functionally relevant regions of the brain. The availability of some selective antagonists and agonists, in combination with genetically modified mice lacking the 5-HT7 receptor, has allowed for a better understanding of the pathophysiological role of this receptor. This paper reviews data on localization and pharmacological properties of the 5-HT7 receptor, and summarizes the results of structure-activity relationship studies aimed at the discovery of selective 5-HT7 receptor ligands. Additionally, an overview of the potential therapeutic applications of 5-HT7 receptor agonists and antagonists in central nervous system disorders is presented. PMID:20923682

  5. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding.

    PubMed

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  6. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding

    PubMed Central

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H.; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  7. Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

    SciTech Connect

    Li,H.; van Vranken, S.; Zhao, Y.; Li, Z.; Guo, Y.; Eisele, L.; Li, Y.

    2005-01-01

    The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.

  8. DOR – a Database of Olfactory Receptors – Integrated Repository for Sequence and Secondary Structural Information of Olfactory Receptors in Selected Eukaryotic Genomes

    PubMed Central

    Nagarathnam, Balasubramanian; Karpe, Snehal D; Harini, Krishnan; Sankar, Kannan; Iftekhar, Mohammed; Rajesh, Durairaj; Giji, Sadasivam; Archunan, Govidaraju; Balakrishnan, Veluchamy; Gromiha, M Michael; Nemoto, Wataru; Fukui, Kazhuhiko; Sowdhamini, Ramanathan

    2014-01-01

    Olfaction is the response to odors and is mediated by a class of membrane-bound proteins called olfactory receptors (ORs). An understanding of these receptors serves as a good model for basic signal transduction mechanisms and also provides important clues for the strategies adopted by organisms for their ultimate survival using chemosensory perception in search of food or defense against predators. Prior research on cross-genome phylogenetic analyses from our group motivated the addressal of conserved evolutionary trends, clustering, and ortholog prediction of ORs. The database of olfactory receptors (DOR) is a repository that provides sequence and structural information on ORs of selected organisms (such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, and Homo sapiens). Users can download OR sequences, study predicted membrane topology, and obtain cross-genome sequence alignments and phylogeny, including three-dimensional (3D) structural models of 100 selected ORs and their predicted dimer interfaces. The database can be accessed from http://caps.ncbs.res.in/DOR. Such a database should be helpful in designing experiments on point mutations to probe into the possible dimerization modes of ORs and to even understand the evolutionary changes between different receptors. PMID:25002814

  9. No evidence of association between structural polymorphism at the dopamine D3 receptor locus and alcoholism in the Japanese

    SciTech Connect

    Higuchi, Susumu; Muramatsu, Taro; Matsushita, Sachio; Murayama, Masanobu

    1996-07-26

    Dopaminergic systems mediate reward mechanisms and are involved in reinforcing self-administration of dependence-forming substances, including alcohol. Studies have reported that polymorphisms of the dopamine D2 receptor, whose structure and function are similar to those of the dopamine D3 receptor, increase the susceptibility to alcoholism. The observations led to the examination of the possible association between a structural polymorphism of the D3 receptor gene and alcoholism. Genotyping results, employing a PCR-RFLP method, showed no difference in allele and genotype frequencies of the D3 BalI polymorphism (Ser{sup 9}/Gly{sup 9}) between Japanese alcoholics and controls. Moreover, these frequencies were not altered in alcoholics with inactive aldehyde dehydrogenase-2 (ALDH2), a well-defined negative risk factor for alcoholism. These results strongly suggest that the dopamine D3 receptor is not associated with alcoholism. 19 refs., 1 fig., 1 tab.

  10. Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

    PubMed Central

    Kmiecik, Sebastian; Jamroz, Michal; Kolinski, Michal

    2014-01-01

    G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs. PMID:24896119

  11. Disulfide Trapping for Modeling and Structure Determination of Receptor:Chemokine Complexes

    PubMed Central

    Kufareva, Irina; Gustavsson, Martin; Holden, Lauren G.; Qin, Ling; Zheng, Yi; Handel, Tracy M.

    2016-01-01

    Despite the recent breakthrough advances in GPCR crystallography, structure determination of protein-protein complexes involving chemokine receptors and their endogenous chemokine ligands remains challenging. Here we describe disulfide trapping, a methodology for generating irreversible covalent binary protein complexes from unbound protein partners by introducing two cysteine residues, one per interaction partner, at selected positions within their interaction interface. Disulfide trapping can serve at least two distinct purposes: (i) stabilization of the complex to assist structural studies, and/or (ii) determination of pairwise residue proximities to guide molecular modeling. Methods for characterization of disulfide-trapped complexes are described and evaluated in terms of throughput, sensitivity, and specificity towards the most energetically favorable cross-links. Due to abundance of native disulfide bonds at receptor:chemokine interfaces, disulfide trapping of their complexes can be associated with intramolecular disulfide shuffling and result in misfolding of the component proteins; because of this, evidence from several experiments is typically needed to firmly establish a positive disulfide crosslink. An optimal pipeline that maximizes throughput and minimizes time and costs by early triage of unsuccessful candidate constructs is proposed. PMID:26921956

  12. Genomic and supragenomic structure of the nucleotide-like G-protein-coupled receptor GPR34.

    PubMed

    Engemaier, Eva; Römpler, Holger; Schöneberg, Torsten; Schulz, Angela

    2006-02-01

    Directed cloning approaches and large-scale sequencing of several vertebrate genomes unveiled many new members of the G-protein-coupled receptor (GPCR) superfamily, among them GPR34. Initial studies showed that GPR34 is an evolutionarily old GPCR structurally related to a group of ADP-like receptors. To gain insight into the genomic organization, regulation of expression, and supragenomic diversification of GPR34 several vertebrate species were analyzed. In contrast to the obviously intronless coding region GPR34 displays an evolutionary preserved 5' noncoding intron-exon structure. Further, an alternatively used cryptic intron was identified within the coding region, which shortens the N terminus by 47 amino acids. Ubiquitous expression of GPR34 is driven by genomic sequences upstream of at least two transcriptional start regions in mouse and rat but only one region in human. In rodents, both promoters are active in all tissues investigated, but the level of activity is tissue-specific. At the translational level, several conserved in-frame AUGs within the first 150 bp of the coding region may serve as start points for translation in human and other mammals. Combinatory mutagenesis and expression of reporter constructs confirmed these multiple translational start points and revealed a preference for the second in-frame AUG in human GPR34. Our data show that multiple translation initiation starts and alternative splicing contribute to the supragenomic diversification of GPR34. PMID:16338117

  13. Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

    SciTech Connect

    Abraham, Jonathan; Corbett, Kevin D.; Farzan, Michael; Choe, Hyeryun; Harrison, Stephen C.

    2010-08-18

    New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

  14. Loss of Insulin Receptor in Osteoprogenitor Cells Impairs Structural Strength of Bone

    PubMed Central

    Thrailkill, Kathryn; Bunn, R. Clay; Lumpkin, Charles; Cockrell, Gael; Kahn, C. Ronald; Fowlkes, John; Nyman, Jeffry S.

    2014-01-01

    Type 1 diabetes mellitus (T1D) is associated with decreased bone mineral density, a deficit in bone structure, and subsequently an increased risk of fragility fracture. These clinical observations, paralleled by animal models of T1D, suggest that the insulinopenia of T1D has a deleterious effect on bone. To further examine the action of insulin signaling on bone development, we generated mice with an osteoprogenitor-selective (osterix-Cre) ablation of the insulin receptor (IR), designated OIRKO. OIRKO mice exhibited an 80% decrease in IR in osteoblasts. Prenatal elimination of IR did not affect fetal survival or gross morphology. However, loss of IR in mouse osteoblasts resulted in a postnatal growth-constricted phenotype. By 10–12 weeks of age, femurs of OIRKO mice were more slender, with a thinner diaphyseal cortex and, consequently, a decrease in whole bone strength when subjected to bending. In male mice alone, decreased metaphyseal trabecular bone, with thinner and more rodlike trabeculae, was also observed. OIRKO mice did not, however, exhibit abnormal glucose tolerance. The skeletal phenotype of the OIRKO mouse appeared more severe than that of previously reported bone-specific IR knockdown models, and confirms that insulin receptor expression in osteoblasts is critically important for proper bone development and maintenance of structural integrity. PMID:24963495

  15. NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor

    PubMed Central

    O’Connor, Casey; White, Kate L.; Doncescu, Nathalie; Didenko, Tatiana; Roth, Bryan L.; Czaplicki, Georges; Stevens, Raymond C.; Wüthrich, Kurt; Milon, Alain

    2015-01-01

    The structure of the dynorphin (1–13) peptide (dynorphin) bound to the human kappa opioid receptor (KOR) has been determined by liquid-state NMR spectroscopy. 1H and 15N chemical shift variations indicated that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM KOR. Radioligand binding indicated an intermediate-affinity interaction, with a Kd of ∼200 nM. Transferred nuclear Overhauser enhancement spectroscopy was used to determine the structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly disordered, the central part of the peptide from L5 to R9 to form a helical turn, and the C-terminal segment from P10 to K13 to be flexibly disordered in this intermediate-affinity bound state. Combining molecular modeling with NMR provided an initial framework for understanding multistep activation of a G protein-coupled receptor by its cognate peptide ligand. PMID:26372966

  16. Structural basis for integration of GluD receptors within synaptic organizer complexes.

    PubMed

    Elegheert, Jonathan; Kakegawa, Wataru; Clay, Jordan E; Shanks, Natalie F; Behiels, Ester; Matsuda, Keiko; Kohda, Kazuhisa; Miura, Eriko; Rossmann, Maxim; Mitakidis, Nikolaos; Motohashi, Junko; Chang, Veronica T; Siebold, Christian; Greger, Ingo H; Nakagawa, Terunaga; Yuzaki, Michisuke; Aricescu, A Radu

    2016-07-15

    Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function. PMID:27418511

  17. Structure and Dimerization Properties of the Aryl Hydrocarbon Receptor PAS-A Domain

    PubMed Central

    Wu, Dalei; Potluri, Nalini; Kim, Youngchang

    2013-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds to xenobiotics and responds by regulating the expression of gene programs required for detoxification and metabolism. AHR and its heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the basic helix-loop-helix (bHLH)–PER-ARNT-SIM (PAS) family of transcription factors. Here we report the 2.55-Å-resolution crystal structure of the mouse AHR PAS-A domain, which represents the first AHR-derived protein structure. The AHR PAS-A domain forms a helix-swapped homodimer in the crystal and also in solution. Through a detailed mutational analysis of all interface residues, we identified several hydrophobic residues that are important for AHR dimerization and function. Our crystallographic visualization of AHR PAS-A dimerization leads us to propose a mode of heterodimerization with ARNT that is supported by both biochemical and cell-based data. Our studies also highlight the residues of other mammalian bHLH-PAS proteins that are likely involved in their homo- or heterodimerization. PMID:24001774

  18. Improvement in Aqueous Solubility of Retinoic Acid Receptor (RAR) Agonists by Bending the Molecular Structure.

    PubMed

    Hiramatsu, Michiaki; Ichikawa, Yuki; Tomoshige, Shusuke; Makishima, Makoto; Muranaka, Atsuya; Uchiyama, Masanobu; Yamaguchi, Takao; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-08-01

    Aqueous solubility is a key requirement for many functional molecules, e. g., drug candidates. Decrease of the partition coefficient (log P) by chemical modification, i.e., introduction of hydrophilic group(s) into molecules, is a classical strategy for improving aqueous solubility. We have been investigating alternative strategies for improving the aqueous solubility of pharmaceutical compounds by disrupting intermolecular interactions. Here, we show that introducing a bend into the molecular structure of retinoic acid receptor (RAR) agonists by changing the substitution pattern from para to meta or ortho dramatically enhances aqueous solubility by up to 890-fold. We found that meta analogs exhibit similar hydrophobicity to the parent para compound, and have lower melting points, supporting the idea that the increase of aqueous solubility was due to decreased intermolecular interactions in the solid state as a result of the structural changes. PMID:27378357

  19. Structure of the extracellular domains of the human interleukin-6 receptor alpha -chain.

    PubMed

    Varghese, J N; Moritz, R L; Lou, M-Z; Van Donkelaar, A; Ji, H; Ivancic, N; Branson, K M; Hall, N E; Simpson, R J

    2002-12-10

    Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex. PMID:12461182

  20. Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen

    PubMed Central

    Velikovsky, C Alejandro; Deng, Lu; Tasumi, Satoshi; Iyer, Lakshminarayan M; Kerzic, Melissa C; Aravind, L; Pancer, Zeev; Mariuzza, Roy A

    2009-01-01

    Variable lymphocyte receptors (VLRs) are leucine-rich repeat (LRR) proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLRB, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure, combined with sequence analysis, revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications. PMID:19543291

  1. Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

    PubMed

    Grinnell, Steven G; Ansonoff, Michael; Marrone, Gina F; Lu, Zhigang; Narayan, Ankita; Xu, Jin; Rossi, Grace; Majumdar, Susruta; Pan, Ying-Xian; Bassoni, Daniel L; Pintar, John; Pasternak, Gavril W

    2016-10-01

    Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors. PMID:27223691

  2. Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement

    PubMed Central

    Gruszczyk, Jakub; Lim, Nicholas T. Y.; Arnott, Alicia; He, Wen-Qiang; Nguitragool, Wang; Roobsoong, Wanlapa; Mok, Yee-Foong; Murphy, James M.; Smith, Katherine R.; Lee, Stuart; Bahlo, Melanie; Mueller, Ivo; Barry, Alyssa E.

    2016-01-01

    Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5—the essential invasion ligand in Plasmodium falciparum—its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection. PMID:26715754

  3. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  4. Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction

    PubMed Central

    Rose, Alexander S.; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W.

    2015-01-01

    GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen–deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket. PMID:26606751

  5. Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction.

    PubMed

    Rose, Alexander S; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W

    2015-01-01

    GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket. PMID:26606751

  6. Enhanced liquid-liquid anion exchange using macrocyclic anion receptors: effect of receptor structure on sulphate-nitrate exchange selectivity

    SciTech Connect

    Moyer, Bruce A; Sloop Jr, Frederick {Fred} V; Fowler, Christopher J; Haverlock, Tamara; Kang, Hyun Ah; Delmau, Laetitia Helene; Bau, Diadra; Hossain, Alamgir; Bowman-James, Kristin; Shriver, James A.; Gross, Mr. Dustin E.; Bill, Nathan; Marquez, Manuel; Lynch, Vincent M.; Sessler, Jonathan L.

    2010-01-01

    When certain macrocyclic anion receptors are added to a chloroform solution of the nitrate form of a lipophilic quaternary ammonium salt (methyltri-C8,10-ammonium nitrate, Aliquat 336N), the extraction of sulphate from an aqueous sodium nitrate solution via exchange with the organic-phase nitrate is significantly enhanced. Eight macrocycles were surveyed, including two derivatives of a tetraamide macrocycle, five derivatives of calix[4]pyrrole and -decafluorocalix[5]pyrrole. Under the hypothesis that the enhancement originates from sulphate binding by the anion receptors in the chloroform phase, it was possible to obtain reasonable fits to the sulphate distribution survey data based on the formation of 1:1 and 2:1 receptor:sulphate complexes in the chloroform phase. Apparent 1:1 sulphate-binding constants obtained from the model in this system fell in the range . Comparison of the results for the various anion receptors included in this study reveals that sulphate binding is sensitive to the nature of the substituents on the parent macrocycle scaffolds in a way that does not follow straightforwardly from simple chemical expectations, such as electron-withdrawing effects on hydrogen-bond donor strength.

  7. Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

    PubMed

    Sablin, Elena P; Blind, Raymond D; Uthayaruban, Rubatharshini; Chiu, Hsiu-Ju; Deacon, Ashley M; Das, Debanu; Ingraham, Holly A; Fletterick, Robert J

    2015-12-01

    The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1. PMID:26416531

  8. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    SciTech Connect

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  9. Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK.

    PubMed

    Stiegler, Amy L; Burden, Steven J; Hubbard, Stevan R

    2009-10-16

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 A resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ. PMID:19664639

  10. Structure of a variable lymphocyte receptor-like protein from the amphioxus Branchiostoma floridae.

    PubMed

    Cao, Dong-Dong; Liao, Xin; Cheng, Wang; Jiang, Yong-Liang; Wang, Wen-Jie; Li, Qiong; Chen, Jun-Yuan; Chen, Yuxing; Zhou, Cong-Zhao

    2016-01-01

    Discovery of variable lymphocyte receptors (VLRs) in agnathans (jawless fish) has brought the origin of adaptive immunity system (AIS) forward to 500 million years ago accompanying with the emergence of vertebrates. Previous findings indicated that amphioxus, a representative model organism of chordate, also possesses some homologs of the basic components of TCR/BCR-based AIS, but it remains unknown if there exist any components of VLR-based AIS in amphioxus. Bioinformatics analyses revealed the amphioxus Branchiostoma floridae encodes a group of putative VLR-like proteins. Here we reported the 1.79 Å crystal structure of Bf66946, which forms a crescent-shaped structure of five leucine-rich repeats (LRRs). Structural comparisons indicated that Bf66946 resembles the lamprey VLRC. Further electrostatic potential analyses showed a negatively-charged patch at the concave of LRR solenoid structure that might be responsible for antigen recognition. Site-directed mutagenesis combined with bacterial binding assays revealed that Bf66946 binds to the surface of Gram-positive bacteria Staphylococcus aureus and Streptococcus pneumonia via a couple of acidic residues at the concave. In addition, the closest homolog of Bf66946 is highly expressed in the potential immune organ gill of Branchiostoma belcheri. Altogether, our findings provide the first structural evidence for the emergence of VLR-like molecules in the basal chordates. PMID:26821753

  11. Structure of a variable lymphocyte receptor-like protein from the amphioxus Branchiostoma floridae

    PubMed Central

    Cao, Dong-Dong; Liao, Xin; Cheng, Wang; Jiang, Yong-Liang; Wang, Wen-Jie; Li, Qiong; Chen, Jun-Yuan; Chen, Yuxing; Zhou, Cong-Zhao

    2016-01-01

    Discovery of variable lymphocyte receptors (VLRs) in agnathans (jawless fish) has brought the origin of adaptive immunity system (AIS) forward to 500 million years ago accompanying with the emergence of vertebrates. Previous findings indicated that amphioxus, a representative model organism of chordate, also possesses some homologs of the basic components of TCR/BCR-based AIS, but it remains unknown if there exist any components of VLR-based AIS in amphioxus. Bioinformatics analyses revealed the amphioxus Branchiostoma floridae encodes a group of putative VLR-like proteins. Here we reported the 1.79 Å crystal structure of Bf66946, which forms a crescent-shaped structure of five leucine-rich repeats (LRRs). Structural comparisons indicated that Bf66946 resembles the lamprey VLRC. Further electrostatic potential analyses showed a negatively-charged patch at the concave of LRR solenoid structure that might be responsible for antigen recognition. Site-directed mutagenesis combined with bacterial binding assays revealed that Bf66946 binds to the surface of Gram-positive bacteria Staphylococcus aureus and Streptococcus pneumonia via a couple of acidic residues at the concave. In addition, the closest homolog of Bf66946 is highly expressed in the potential immune organ gill of Branchiostoma belcheri. Altogether, our findings provide the first structural evidence for the emergence of VLR-like molecules in the basal chordates. PMID:26821753

  12. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells★

    PubMed Central

    Engelstoft, Maja S.; Park, Won-mee; Sakata, Ichiro; Kristensen, Line V.; Husted, Anna Sofie; Osborne-Lawrence, Sherri; Piper, Paul K.; Walker, Angela K.; Pedersen, Maria H.; Nøhr, Mark K.; Pan, Jie; Sinz, Christopher J.; Carrington, Paul E.; Akiyama, Taro E.; Jones, Robert M.; Tang, Cong; Ahmed, Kashan; Offermanns, Stefan; Egerod, Kristoffer L.; Zigman, Jeffrey M.; Schwartz, Thue W.

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell. PMID:24327954

  13. Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors.

    PubMed

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2014-05-01

    Current methods of G protein coupled receptors (GPCRs) phylogenetic classification are sequence based and therefore inappropriate for highly divergent sequences, sharing low sequence identity. In this study, sequence structure profile based alignment generated by PROMALS3D was used to understand the GPCR Class A Rhodopsin superfamily evolution using the MEGA 5 software. Phylogenetic analysis included a combination of Neighbor-Joining method and Maximum Likelihood method, with 1000 bootstrap replicates. Our study was able to identify potential ligand association for Class A Orphans and putative/unclassified Class A receptors with no cognate ligand information: GPR21 and GPR52 with fatty acids; GPR75 with Neuropeptide Y; GPR82, GPR18, GPR141 with N-arachidonylglycine; GPR176 with Free fatty acids, GPR10 with Tachykinin & Neuropeptide Y; GPR85 with ATP, ADP & UDP glucose; GPR151 with Galanin; GPR153 and GPR162 with Adrenalin, Noradrenalin; GPR146, GPR139, GPR142 with Neuromedin, Ghrelin, Neuromedin U-25 & Thyrotropin-releasing hormone; GPR171 with ATP, ADP & UDP Glucose; GPR88, GPR135, GPR161, GPR101with 11-cis-retinal; GPR83 with Tackykinin; GPR148 with Prostanoids, GPR109b, GPR81, GPR31with ATP & UTP and GPR150 with GnRH I & GnRHII. Furthermore, we suggest that this study would prove useful in re-classification of receptors, selecting templates for homology modeling and identifying ligands which may show cross reactivity with other GPCRs as signaling via multiple ligands play a significant role in disease modulation. PMID:24503482

  14. Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Bound β1-Adrenergic Receptor

    PubMed Central

    Sato, Tomomi; Baker, Jillian; Warne, Tony; Brown, Giles A.; Leslie, Andrew G.W.; Congreve, Miles

    2015-01-01

    Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser5.46, coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol–bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol–bound β1AR and the hydroxyl group of Ser5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser5.46 side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists. PMID:26385885

  15. Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound β1-Adrenergic Receptor.

    PubMed

    Sato, Tomomi; Baker, Jillian; Warne, Tony; Brown, Giles A; Leslie, Andrew G W; Congreve, Miles; Tate, Christopher G

    2015-12-01

    Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol-bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound β1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists. PMID:26385885

  16. Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.

    PubMed

    Shibata, Yoko; Gvozdenovic-Jeremic, Jelena; Love, James; Kloss, Brian; White, Jim F; Grisshammer, Reinhard; Tate, Christopher G

    2013-04-01

    Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination. PMID:23337476

  17. N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor.

    PubMed

    Bouley, R; Pérodin, J; Plante, H; Rihakova, L; Bernier, S G; Maletínská, L; Guillemette, G; Escher, E

    1998-02-19

    The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV > Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1

  18. Structural snapshots of full-length Jak1, a transmembrane gp130/IL-6/IL-6Rα cytokine receptor complex, and the receptor-Jak1 holocomplex

    PubMed Central

    Lupardus, Patrick J.; Skiniotis, Georgios; Rice, Amanda J.; Thomas, Christoph; Fischer, Suzanne; Walz, Thomas; Garcia, K. Christopher

    2011-01-01

    Summary The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to rigidify the connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three-lobed structure comprised of FERM-SH2, pseudokinase and kinase modules possessing extensive inter-segmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment. PMID:21220115

  19. Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer▿

    PubMed Central

    Watts, Kylie J.; Johnson, Mark S.; Taylor, Barry L.

    2008-01-01

    Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed. PMID:18203838

  20. Crystal structure of a TSH receptor monoclonal antibody: insight into Graves' disease pathogenesis.

    PubMed

    Chen, Chun-Rong; Hubbard, Paul A; Salazar, Larry M; McLachlan, Sandra M; Murali, Ramachandran; Rapoport, Basil

    2015-01-01

    The TSH receptor (TSHR) A-subunit is more effective than the holoreceptor in inducing thyroid-stimulating antibodies (TSAb) that cause Graves' disease. A puzzling phenomenon is that 2 recombinant, eukaryotic forms of A-subunits (residues 22-289), termed active and inactive, are recognized mutually exclusively by pathogenic TSAb and mouse monoclonal antibody 3BD10, respectively. Understanding the structural difference between these TSHR A-subunit forms could provide insight into Graves' disease pathogenesis. The 3-dimensional structure of the active A-subunit (in complex with a human TSAb Fab, M22) is known, but the structural difference with inactive A-subunits is unknown. We solved the 3BD10 Fab 3-dimensional crystal structure. Guided by prior knowledge of a portion of its epitope, 3BD10 docked in silico with the known active TSHR-289 monomeric structure. Because both TSAb and 3BD10 recognize the active TSHR A-subunit monomer, this form of the molecule can be excluded as the basis for the active-inactive dichotomy, suggesting, instead a role for A-subunit quaternary structure. Indeed, in silico analysis revealed that M22, but not 3BD10, bound to a TSHR-289 trimer. In contrast, 3BD10, but not M22, bound to a TSHR-289 dimer. The validity of these models is supported experimentally by the temperature-dependent balance between active and inactive TSHR-289. In summary, we provide evidence for a structural basis to explain the conformational heterogeneity of TSHR A-subunits (TSHR-289). The pathophysiologic importance of these findings is that affinity maturation of pathogenic TSAb in Graves' disease is likely to involve a trimer of the shed TSHR A-subunit. PMID:25419797

  1. Structure-Activity Relationships for the Antifungal Activity of Selective Estrogen Receptor Antagonists Related to Tamoxifen

    PubMed Central

    Butts, Arielle; Martin, Jennifer A.; DiDone, Louis; Bradley, Erin K.; Mutz, Mitchell; Krysan, Damian J.

    2015-01-01

    Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold. PMID:26016941

  2. Uncoupling the Structure-Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding.

    PubMed

    Dickson, Callum J; Hornak, Viktor; Velez-Vega, Camilo; McKay, Daniel J J; Reilly, John; Sandham, David A; Shaw, Duncan; Fairhurst, Robin A; Charlton, Steven J; Sykes, David A; Pearlstein, Robert A; Duca, Jose S

    2016-06-23

    Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions. PMID:27239696

  3. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  4. Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

    PubMed Central

    Pandhare, Akash; Grozdanov, Petar N.; Jansen, Michaela

    2016-01-01

    In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap. PMID:27045630

  5. Computation identifies structural features that govern neuronal firing properties in slowly adapting touch receptors

    PubMed Central

    Lesniak, Daine R; Marshall, Kara L; Wellnitz, Scott A; Jenkins, Blair A; Baba, Yoshichika; Rasband, Matthew N; Gerling, Gregory J; Lumpkin, Ellen A

    2014-01-01

    Touch is encoded by cutaneous sensory neurons with diverse morphologies and physiological outputs. How neuronal architecture influences response properties is unknown. To elucidate the origin of firing patterns in branched mechanoreceptors, we combined neuroanatomy, electrophysiology and computation to analyze mouse slowly adapting type I (SAI) afferents. These vertebrate touch receptors, which innervate Merkel cells, encode shape and texture. SAI afferents displayed a high degree of variability in touch-evoked firing and peripheral anatomy. The functional consequence of differences in anatomical architecture was tested by constructing network models representing sequential steps of mechanosensory encoding: skin displacement at touch receptors, mechanotransduction and action-potential initiation. A systematic survey of arbor configurations predicted that the arrangement of mechanotransduction sites at heminodes is a key structural feature that accounts in part for an afferent’s firing properties. These findings identify an anatomical correlate and plausible mechanism to explain the driver effect first described by Adrian and Zotterman. DOI: http://dx.doi.org/10.7554/eLife.01488.001 PMID:24448409

  6. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  7. Evaluation of N-substituent structural variations in opioid receptor profile of LP1.

    PubMed

    Pasquinucci, Lorella; Turnaturi, Rita; Aricò, Giuseppina; Parenti, Carmela; Pallaki, Paschalina; Georgoussi, Zafiroula; Ronsisvalle, Simone

    2016-06-15

    The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9-15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (Ki(MOR)=38±4nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA2) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD50=2.0mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function. PMID:27234885

  8. Computation identifies structural features that govern neuronal firing properties in slowly adapting touch receptors.

    PubMed

    Lesniak, Daine R; Marshall, Kara L; Wellnitz, Scott A; Jenkins, Blair A; Baba, Yoshichika; Rasband, Matthew N; Gerling, Gregory J; Lumpkin, Ellen A

    2014-01-01

    Touch is encoded by cutaneous sensory neurons with diverse morphologies and physiological outputs. How neuronal architecture influences response properties is unknown. To elucidate the origin of firing patterns in branched mechanoreceptors, we combined neuroanatomy, electrophysiology and computation to analyze mouse slowly adapting type I (SAI) afferents. These vertebrate touch receptors, which innervate Merkel cells, encode shape and texture. SAI afferents displayed a high degree of variability in touch-evoked firing and peripheral anatomy. The functional consequence of differences in anatomical architecture was tested by constructing network models representing sequential steps of mechanosensory encoding: skin displacement at touch receptors, mechanotransduction and action-potential initiation. A systematic survey of arbor configurations predicted that the arrangement of mechanotransduction sites at heminodes is a key structural feature that accounts in part for an afferent's firing properties. These findings identify an anatomical correlate and plausible mechanism to explain the driver effect first described by Adrian and Zotterman. DOI: http://dx.doi.org/10.7554/eLife.01488.001. PMID:24448409

  9. Synthesis and Structure-Activity Relationships of Substituted Urea Derivatives on Mouse Melanocortin Receptors.

    PubMed

    Singh, Anamika; Kast, Johannes; Dirain, Marvin L S; Huang, Huisuo; Haskell-Luevano, Carrie

    2016-02-17

    The melanocortin system is involved in the regulation of several complex physiological functions. In particular, the melanocortin-3 and -4 receptors (MC3R/MC4R) have been demonstrated to regulate body weight, energy homeostasis, and feeding behavior. Synthetic and endogenous melanocortin agonists have been shown to be anorexigenic in rodent models. Herein, we report synthesis and structure-activity relationship (SAR) studies of 27 nonpeptide small molecule ligands based on an unsymmetrical substituted urea core. Three templates containing key residues from the lead compounds, showing diversity at three positions (R(1), R(2), R(3)), were designed and synthesized. The syntheses were optimized for efficient microwave-assisted chemistry that significantly reduced total syntheses time compared to a previously reported room temperature method. The pharmacological characterization of the compounds on the mouse melanocortin receptors identified compounds 1 and 12 with full agonist activity at the mMC4R, but no activity was observed at the mMC3R when tested up to 100 μM concentrations. The SAR identified compounds possessing aliphatic or saturated cyclic amines at the R(1) position, bulky aromatic groups at the R(2) position, and benzyl group at the R(3) position resulted in mMC4R selectivity over the mMC3R. The small molecule template and SAR knowledge from this series may be helpful in further design of MC3R/MC4R selective small molecule ligands. PMID:26645732

  10. Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions

    PubMed Central

    Chavent, Matthieu; Seiradake, Elena; Jones, E. Yvonne; Sansom, Mark S.P.

    2016-01-01

    Summary Ephs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459–461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations. PMID:26724997

  11. Structurally Flexible C₃-Symmetric Receptors for Molecular Recognition and Their Self-Assembly Properties.

    PubMed

    Singh, Ashutosh S; Sun, Shih-Sheng

    2015-12-01

    The bioinspired design and synthesis of building blocks and their assemblies by the supramolecular approach has ever fascinated scientists to utilize such artificial systems for numerous purposes. Flexibility is a basic feature of natural systems. However, in artificial systems this is difficult to control, especially if there is no preorganization of the component(s) of a system. We have designed and synthesized a series of C3 -symmetric N-bridged flexible receptors and successfully utilized them to selectively entrap the notorious and toxic nitrate anion in aqueous medium. This was the first report of highest binding affinity for the nitrate anion in aqueous medium. An impressive self-sorting phenomenon of reversibly formed hydrogen-bonded capsules, which self-assembled from flexible tripodal receptors having branches of similar size and bearing the same amide functionality, has been disclosed. Encapsulated nitrate anion has been further utilized for the photochemical [2+2] cycloaddition reaction for the synthesis of strained four-membered ring structures through dynamic self-assembly. In this Personal Account, we summarize these results showing the utility of naturally inspired flexibility in artificial systems. PMID:26202256

  12. Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors

    SciTech Connect

    Chang, C.; Kokontis, J.; Liao, S. )

    1988-10-01

    Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural Ars. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

  13. Mouse model recapitulating human Fcγ receptor structural and functional diversity

    PubMed Central

    Smith, Patrick; DiLillo, David J.; Bournazos, Stylianos; Li, Fubin; Ravetch, Jeffrey V.

    2012-01-01

    The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies. PMID:22474370

  14. Neural-Network Scoring Functions Identify Structurally Novel Estrogen-Receptor Ligands.

    PubMed

    Durrant, Jacob D; Carlson, Kathryn E; Martin, Teresa A; Offutt, Tavina L; Mayne, Christopher G; Katzenellenbogen, John A; Amaro, Rommie E

    2015-09-28

    The magnitude of the investment required to bring a drug to the market hinders medical progress, requiring hundreds of millions of dollars and years of research and development. Any innovation that improves the efficiency of the drug-discovery process has the potential to accelerate the delivery of new treatments to countless patients in need. "Virtual screening," wherein molecules are first tested in silico in order to prioritize compounds for subsequent experimental testing, is one such innovation. Although the traditional scoring functions used in virtual screens have proven useful, improved accuracy requires novel approaches. In the current work, we use the estrogen receptor to demonstrate that neural networks are adept at identifying structurally novel small molecules that bind to a selected drug target, ultimately allowing experimentalists to test fewer compounds in the earliest stages of lead identification while obtaining higher hit rates. We describe 39 novel estrogen-receptor ligands identified in silico with experimentally determined Ki values ranging from 460 nM to 20 μM, presented here for the first time. PMID:26286148

  15. Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

    PubMed Central

    Snyder, Greg A.; Brooks, Andrew G.; Sun, Peter D.

    1999-01-01

    Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface. PMID:10097129

  16. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding

    SciTech Connect

    Thompson, T.B.; Lerch, T.F.; Cook, R.W.; Woodruff, T.K.; Jardetzky, T.S.

    2010-03-08

    TGF-{beta} ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-{beta} ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

  17. Grid inhomogeneous solvation theory: hydration structure and thermodynamics of the miniature receptor cucurbit[7]uril.

    PubMed

    Nguyen, Crystal N; Young, Tom Kurtzman; Gilson, Michael K

    2012-07-28

    The displacement of perturbed water upon binding is believed to play a critical role in the thermodynamics of biomolecular recognition, but it is nontrivial to unambiguously define and answer questions about this process. We address this issue by introducing grid inhomogeneous solvation theory (GIST), which discretizes the equations of inhomogeneous solvation theory (IST) onto a three-dimensional grid situated in the region of interest around a solute molecule or complex. Snapshots from explicit solvent simulations are used to estimate localized solvation entropies, energies, and free energies associated with the grid boxes, or voxels, and properly summing these thermodynamic quantities over voxels yields information about hydration thermodynamics. GIST thus provides a smoothly varying representation of water properties as a function of position, rather than focusing on hydration sites where solvent is present at high density. It therefore accounts for full or partial displacement of water from sites that are highly occupied by water, as well as for partly occupied and water-depleted regions around the solute. GIST can also provide a well-defined estimate of the solvation free energy and therefore enables a rigorous end-states analysis of binding. For example, one may not only use a first GIST calculation to project the thermodynamic consequences of displacing water from the surface of a receptor by a ligand, but also account, in a second GIST calculation, for the thermodynamics of subsequent solvent reorganization around the bound complex. In the present study, a first GIST analysis of the molecular host cucurbit[7]uril is found to yield a rich picture of hydration structure and thermodynamics in and around this miniature receptor. One of the most striking results is the observation of a toroidal region of high water density at the center of the host's nonpolar cavity. Despite its high density, the water in this toroidal region is disfavored energetically and

  18. Discovery of Novel Human Epidermal Growth Factor Receptor-2 Inhibitors by Structure-based Virtual Screening

    PubMed Central

    Shi, Zheng; Yu, Tian; Sun, Rong; Wang, Shan; Chen, Xiao-Qian; Cheng, Li-Jia; Liu, Rong

    2016-01-01

    Background: Human epidermal growth factor receptor-2 (HER2) is a trans-membrane receptor like protein, and aberrant signaling of HER2 is implicated in many human cancers, such as ovarian cancer, gastric cancer, and prostate cancer, most notably breast cancer. Moreover, it has been in the spotlight in the recent years as a promising new target for therapy of breast cancer. Objective: Since virtual screening has become an integral part of the drug discovery process, it is of great significant to identify novel HER2 inhibitors by structure-based virtual screening. Materials and Methods: In this study, we carried out a series of elegant bioinformatics approaches, such as virtual screening and molecular dynamics (MD) simulations to identify HER2 inhibitors from Food and Drug Administration-approved small molecule drug as potential “new use” drugs. Results: Molecular docking identified top 10 potential drugs which showed spectrum affinity to HER2. Moreover, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) might exert potential inhibitory effects against HER2-targeted anti-breast cancer therapeutics. Conclusion: Together, our findings may provide successful application of virtual screening studies in the lead discovery process, and suggest that our discovered small molecules could be effective HER2 inhibitor candidates for further study. SUMMARY A series of elegant bioinformatics approaches, including virtual screening and molecular dynamics (MD) simulations were took advantage to identify human epidermal growth factor receptor-2 (HER2) inhibitors. Molecular docking recognized top 10 candidate compounds, which showed spectrum affinity to HER2. Further, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) in candidate compounds were identified as potential “new use” drugs against HER2-targeted anti-breast cancer therapeutics. Abbreviations used: HER2: Human epidermal growth factor receptor-2

  19. Grid inhomogeneous solvation theory: Hydration structure and thermodynamics of the miniature receptor cucurbit[7]uril

    NASA Astrophysics Data System (ADS)

    Nguyen, Crystal N.; Kurtzman Young, Tom; Gilson, Michael K.

    2012-07-01

    The displacement of perturbed water upon binding is believed to play a critical role in the thermodynamics of biomolecular recognition, but it is nontrivial to unambiguously define and answer questions about this process. We address this issue by introducing grid inhomogeneous solvation theory (GIST), which discretizes the equations of inhomogeneous solvation theory (IST) onto a three-dimensional grid situated in the region of interest around a solute molecule or complex. Snapshots from explicit solvent simulations are used to estimate localized solvation entropies, energies, and free energies associated with the grid boxes, or voxels, and properly summing these thermodynamic quantities over voxels yields information about hydration thermodynamics. GIST thus provides a smoothly varying representation of water properties as a function of position, rather than focusing on hydration sites where solvent is present at high density. It therefore accounts for full or partial displacement of water from sites that are highly occupied by water, as well as for partly occupied and water-depleted regions around the solute. GIST can also provide a well-defined estimate of the solvation free energy and therefore enables a rigorous end-states analysis of binding. For example, one may not only use a first GIST calculation to project the thermodynamic consequences of displacing water from the surface of a receptor by a ligand, but also account, in a second GIST calculation, for the thermodynamics of subsequent solvent reorganization around the bound complex. In the present study, a first GIST analysis of the molecular host cucurbit[7]uril is found to yield a rich picture of hydration structure and thermodynamics in and around this miniature receptor. One of the most striking results is the observation of a toroidal region of high water density at the center of the host's nonpolar cavity. Despite its high density, the water in this toroidal region is disfavored energetically and

  20. Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino terminal domains

    PubMed Central

    Kumar, Janesh; Mayer, Mark L.

    2010-01-01

    Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into the AMPA, kainate and NMDA receptor subtypes is regulated by their extracellular amino terminal domains (ATD). Kainate receptors are further classified into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptor families based on their affinity for the neurotoxin kainic acid. These two families share 42% sequence identity for the intact receptor but only 28% sequence identity at the level of ATD. We have determined for the first time high-resolution crystal structures for the GluK3 and GluK5 ATDs, both of which crystallize as dimers, but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5 the R2 domain dimer assembly is similar to that reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result from both extensive intramolecular contacts between domains R1 and R2, and from their assembly as dimers which interact at both the R1 and R2 domains. Our results provide the first insights into the structure and function for GluK4-5, the least understood family of iGluRs. PMID:20951142

  1. Functional organization and conformational dynamics of the nicotinic receptor: a plausible structural interpretation of myasthenic mutations.

    PubMed

    Taly, Antoine; Changeux, Jean-Pierre

    2008-01-01

    To understand the structural causes of myasthenic mutations, molecular models of the nicotinic acetycholine receptor were first constructed. Then, the gating transition between resting and open conformation was investigated in silico by normal mode analysis using a physically meaningful description of protein flexibility. This analysis revealed a global quaternary twist motion that opens the ion pore. Second, it was found that most (24/27) of the spontaneous mutations known to cause congenital myasthenia (and autosomal dominant nocturnal frontal lobe epilepsy) are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode together with significant changes of the tertiary organization of the subunits. These data provide a qualitative interpretation of the molecular phenotype of pathological mutations responsible for congenital myasthenia. PMID:18567852

  2. Structural modifications of salicylates: inhibitors of human CD81-receptor HCV-E2 interaction.

    PubMed

    Holzer, Marcel; Ziegler, Sigrid; Neugebauer, Alexander; Kronenberger, Bernd; Klein, Christian D; Hartmann, Rolf W

    2008-08-01

    Starting point of the present paper was the result of a virtual screening using the open conformation of the large extracellular loop (LEL) of the CD81-receptor (crystal structure: PDB-ID: 1G8Q). After benzyl salicylate had been experimentally validated to be a moderate inhibitor of the CD81-LEL-HCV-E2 interaction, further optimization was performed and heterocyclic-substituted benzyl salicylate derivatives were synthesized. The compounds were tested for their ability to inhibit the interaction of a fluorescence-labeled antibody to CD81-LEL using HUH7.5 cells. No compound showed an increase concerning the inhibition of the protein-protein interaction compared to benzyl salicylate. PMID:18618487

  3. [Structure and function of the T-lymphocyte antigen receptor and its role in infectious diseases].

    PubMed

    Peralta Zaragoza, O; Sánchez Magaña, T; Barrera Rodriguez, R; Madrid Marina, V

    1996-01-01

    The T lymphocytes recognize antigens through antigen receptors (TcRs) and the major histocompatibility complex (MHC) molecules: they lysate the cells that bear the antigen, or release cytokines that are mediators of the immune response. The TcRs recognize antigens in the form of short peptides bound to MHC molecules. So far, there are two isotypes of TcR: gamma/delta and alpha/beta, which appear in the sequence during T-cell ontogeny. The process of selection of TcRs during thymic ontogeny obeys to molecular mechanisms which generate intracellular events that will participate in the gene expression of the TcR. The aim of the present paper is to review the molecular, structural, and functional aspects of the TcRs, and their role in human autoimmune infectious disease. PMID:8815490

  4. Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors

    PubMed Central

    Verdiyan, Ekaterina E.; Allakhverdiev, Elvin S.; Maksimov, Georgy V.

    2016-01-01

    In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the “axon-SC” interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+—influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization. PMID:27455410

  5. COUP-TF gene: a structure unique for the steroid/thyroid receptor superfamily.

    PubMed Central

    Ritchie, H H; Wang, L H; Tsai, S; O'Malley, B W; Tsai, M J

    1990-01-01

    Two different genomic genes for the COUP-transcription factor, COUP-TF I and COUP-TF II, have been isolated from a human cosmid genomic library using a [32P]-labeled cDNA probe. Data obtained from Southern blot analysis of these cosmid clones indicated that two closely related genes exist in the human genome and have a similar genomic organization. The genes are similar in the hormone and DNA binding domains but diverge from one another in the N-terminal region. Using DNA sequencing and polymerase chain reaction (PCR) techniques we have determined that the structure of COUP-TF I consists only of three exons and two introns. Surprisingly, both zinc fingers (i.e., F1 and F2) are located in the first exon. Therefore, COUP-TF I is unique among the members of the steroid/thyroid hormone receptor superfamily which have been described to date. Images PMID:2263450

  6. Human population genetic structure detected by pain-related mu opioid receptor gene polymorphisms

    PubMed Central

    López Soto, Eduardo Javier; Catanesi, Cecilia Inés

    2015-01-01

    Several single nucleotide polymorphisms (SNPs) in the Mu Opioid Receptor gene (OPRM1) have been identified and associated with a wide variety of clinical phenotypes related both to pain sensitivity and analgesic requirements. The A118G and other potentially functional OPRM1 SNPs show significant differences in their allele distributions among populations. However, they have not been properly addressed in a population genetic analysis. Population stratification could lead to erroneous conclusions when they are not taken into account in association studies. The aim of our study was to analyze OPRM1 SNP variability by comparing population samples of the International Hap Map database and to analyze a new population sample from the city of Corrientes, Argentina. The results confirm that OPRM1 SNP variability differs among human populations and displays a clear ancestry genetic structure, with three population clusters: Africa, Asia, and Europe-America. PMID:26273217

  7. In vivo determination of the structure of oligomers of a G protein-coupled receptor

    NASA Astrophysics Data System (ADS)

    Rath, Sasmita; Raicu, Valerica

    2009-03-01

    Resonance Energy Transfer (RET) is a process of nonradiative energy transfer between a donor and an acceptor molecule, which is widely used for studies of protein-protein interactions in living cells. Here we report on the results of a spectrally-resolved two-photon microscopy study of image pixel-level RET in yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor called Sterile 2 α factor protein (Ste2P). The number of pixels showing RET were plotted against the RET efficiency to obtain distributions of RET efficiency in the cells. These distributions were simulated with models for plausible geometries and sizes of protein complexes (V. Raicu, 2007, J. Biol. Phys. 33:109--127). From all the models tested we found that a parallelogram-shaped tetramer is the most likely structure for the Ste2p oligomers.

  8. Agonist-bound structure of the human P2Y12 receptor

    PubMed Central

    Zhang, Jin; Zhang, Kaihua; Gao, Zhan-Guo; Paoletta, Silvia; Zhang, Dandan; Han, Gye Won; Li, Tingting; Ma, Limin; Zhang, Wenru; Müller, Christa E.; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Katritch, Vsevolod; Jacobson, Kenneth A.; Stevens, Raymond C.; Wu, Beili; Zhao, Qiang

    2014-01-01

    The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs. PMID:24784220

  9. Structure and diversity of the T-cell receptor alpha chain in the Mexican axolotl.

    PubMed

    Fellah, J S; Kerfourn, F; Dumay, A M; Aubet, G; Charlemagne, J

    1997-01-01

    Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) alpha chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRalpha-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRalpha-V segments were all provided by sequences belonging to the human TCRalpha-V1 family and the mouse TCRalpha-V3 and TCRalpha-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 alpha chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 alpha chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Calpha domain had the typical structure of mammalian and avian Calpha domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. PMID:9002443

  10. The Natural Product Magnolol as a Lead Structure for the Development of Potent Cannabinoid Receptor Agonists

    PubMed Central

    Müller, Christa E.

    2013-01-01

    Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenyl)phenol), the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB) receptors. We now investigated the structure-activity relationships of (tetrahydro)magnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB1/CB2 full agonist 2-(2-methoxy-5-propyl-phenyl)-4-hexylphenol (61a, Ki CB1∶0.00957 µM; Ki CB2∶0.0238 µM), and the CB2-selective partial agonist 2-(2-hydroxy-5-propylphenyl)-4-pentylphenol (60, Ki CB1∶0.362 µM; Ki CB2∶0.0371 µM), which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC50 value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies. PMID:24204944

  11. Structure-Activity Relationships of Constrained Phenylethylamine Ligands for the Serotonin 5-HT2 Receptors

    PubMed Central

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L.; Gloriam, David E.

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9–11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9–11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9–11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands. PMID:24244317

  12. Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene

    SciTech Connect

    Kandil, Eman; Ishibashi, Teruo; Kasahara, Masanori

    1995-06-01

    The intestinal epithelium of neonatal mice and rats expresses an Fc receptor that mediates selective uptake of IgG in mothers`milk. This receptor (FcRn), which helps newborn animals to acquire passive immunity, is an MHC class I-like heterodimer made up of a heavy chain and {beta}{sub 2}-microglobulin. In the present study, we determined the genomic structure of a mouse gene (FcRn) encoding the heavy of FcRn. The overall exon-intron organization of the Fcrn gene was similar to that of the Fcrn gene, thus providing structural evidence that Fcrn os a bona fide class I gene. The 5{prime}-flanking region of the Fcrn gene contained the binding motifs for two cytokine-inducible transcription factors, NF-IL6 and NF1. However, regulatory elements found in MHC class I genes (enhancer A, enhancer B, and the IFN response element) were absent. Phylogenetic tree analysis suggested that, like the MICA, AZGP1, and CD1 genes, the Fcrn gene diverged form MHC class I genes after the emergence of amphibians but before the split of placental and marsupial mammals. Consistent with this result, Southern blot analysis with a mouse Fcrn cDNA probe detected cross-hybridizing bands in various mammalian species and chickens. Sequence analysis of the Fcrn gene isolated from eight mouse strains showed that the membrane-distal domain of FcRn has at least three amino acid variants. The fact that Fcrn is a single copy gene indicates that it is expressed in both the neonatal intestine and the fetal yolk sac. 74 refs., 7 figs., 2 tabs.

  13. Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

    PubMed

    Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-08-29

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

  14. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis

    PubMed Central

    Hausmann, Ralf; Kless, Achim; Schmalzing, Günther

    2015-01-01

    P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-Å crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures. PMID:25439586

  15. Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

    SciTech Connect

    Geng, Yong; Xiong, Dazhi; Mosyak, Lidia; Malito, David L.; Kniazeff, Julie; Chen, Yan; Burmakina, Svetlana; Quick, Matthias; Bush, Martin; Javitch, Jonathan A.; Pin, Jean-Philippe; Fan, Qing R.

    2012-10-24

    Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.

  16. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes

    PubMed Central

    Dawe, G. Brent; Musgaard, Maria; Aurousseau, Mark R.P.; Nayeem, Naushaba; Green, Tim; Biggin, Philip C.; Bowie, Derek

    2016-01-01

    Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  17. Specific increase in potency via structure-based design of a T cell receptor

    PubMed Central

    Malecek, Karolina; Grigoryan, Arsen; Zhong, Shi; Gu, Wei Jun; Johnson, Laura A.; Rosenberg, Steven A.; Cardozo, Timothy; Krogsgaard, Michelle

    2014-01-01

    Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are non-reactive “self” proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable anti-tumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related antigens. Our results showed that the mutated TCRs had improved T cell activation potency, while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. And even though of limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects. PMID:25070852

  18. Structure-Activity Relationships in Toll-like Receptor 2-Agonists Leading to Simplified Monoacyl Lipopeptides

    PubMed Central

    Agnihotri, Geetanjali; Crall, Breanna M.; Lewis, Tyler C.; Day, Timothy P.; Balakrishna, Rajalakshmi; Warshakoon, Hemamali J.; Malladi, Subbalakshmi S.; David, Sunil A.

    2011-01-01

    Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM2CS) compounds are potential vaccine adjuvants. In continuation of previously reported structure-activity relationships on this chemotype, we have determined that at least one acyl group of optimal length (C16) and an appropriately orientated ester carbonyl group is essential for TLR2-agonistic activity. The spacing between one of the palmitoyl ester carbonyl and the thioether is crucial to allow for an important H-bond, which observed in the crystal structure of the lipopeptide:TLR2 complex; consequently, activity is lost in homologated compounds. Penicillamine-derived analogues are also inactive, likely due to unfavorable steric interactions with the carbonyl of Ser 12 in TLR2. The thioether in this chemotype can be replaced with a selenoether. Importantly, the thioglycerol motif can be dispensed with altogether, and can be replaced with a thioethanol bridge. These results have led to a structurally simpler, synthetically more accessible, and water-soluble analogue possessing strong TLR2-agonistic activities in human blood. PMID:22007676

  19. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes.

    PubMed

    Dawe, G Brent; Musgaard, Maria; Aurousseau, Mark R P; Nayeem, Naushaba; Green, Tim; Biggin, Philip C; Bowie, Derek

    2016-03-16

    Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  20. Structural Studies of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Tetramer in Complex with Its Receptor, Sialyllactose

    SciTech Connect

    Yuan, Ping; Thompson, Thomas B.; Wurzburg, Beth A.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2010-03-08

    The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to be the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.

  1. Phocid seal leptin: tertiary structure and hydrophobic receptor binding site preservation during distinct leptin gene evolution.

    PubMed

    Hammond, John A; Hauton, Chris; Bennett, Kimberley A; Hall, Ailsa J

    2012-01-01

    The cytokine hormone leptin is a key signalling molecule in many pathways that control physiological functions. Although leptin demonstrates structural conservation in mammals, there is evidence of positive selection in primates, lagomorphs and chiropterans. We previously reported that the leptin genes of the grey and harbour seals (phocids) have significantly diverged from other mammals. Therefore we further investigated the diversification of leptin in phocids, other marine mammals and terrestrial taxa by sequencing the leptin genes of representative species. Phylogenetic reconstruction revealed that leptin diversification was pronounced within the phocid seals with a high dN/dS ratio of 2.8, indicating positive selection. We found significant evidence of positive selection along the branch leading to the phocids, within the phocid clade, but not over the dataset as a whole. Structural predictions indicate that the individual residues under selection are away from the leptin receptor (LEPR) binding site. Predictions of the surface electrostatic potential indicate that phocid seal leptin is notably different to other mammalian leptins, including the otariids. Cloning the grey seal leptin binding domain of LEPR confirmed that this was structurally conserved. These data, viewed in toto, support a hypothesis that phocid leptin divergence is unlikely to have arisen by random mutation. Based upon these phylogenetic and structural assessments, and considering the comparative physiology and varying life histories among species, we postulate that the unique phocid diving behaviour has produced this selection pressure. The Phocidae includes some of the deepest diving species, yet have the least modified lung structure to cope with pressure and volume changes experienced at depth. Therefore, greater surfactant production is required to facilitate rapid lung re-inflation upon surfacing, while maintaining patent airways. We suggest that this additional surfactant requirement

  2. Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

    PubMed

    Balsera, Beatriz; Mulet, José; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Ferrer-Montiel, Antonio; Hernández-Jiménez, José G; Estévez-Herrera, Judith; Borges, Ricardo; Freitas, Andiara E; López, Manuela G; García-López, M Teresa; González-Muñiz, Rosario; Pérez de Vega, María Jesús; Valor, Luis M; Svobodová, Lucie; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2014-10-30

    The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure. PMID:25232969

  3. Ancient Evolutionary Origin of Diversified Variable Regions Demonstrated by Crystal Structures of an Immune-Type Receptor in Amphioxus

    SciTech Connect

    Prada,J.; Haire, R.; Allaire, M.; Jakoncic, J.; Stojanoff, V.; Cannon, J.; Litman, G.; Ostrov, D.

    2006-01-01

    Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region-containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 Angstroms) and a pair of V-type domains (to 1.85 Angstroms) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.

  4. Analysis of the murine Dtk gene identifies conservation of genomic structure within a new receptor tyrosine kinase subfamily

    SciTech Connect

    Lewis, P.M.; Crosier, K.E.; Crosier, P.S.

    1996-01-01

    The receptor tyrosine kinase Dtk/Tyro 3/Sky/rse/brt/tif is a member of a new subfamily of receptors that also includes Axl/Ufo/Ark and Eyk/Mer. These receptors are characterized by the presence of two immunoglobulin-like loops and two fibronectin type III repeats in their extracellular domains. The structure of the murine Dtk gene has been determined. The gene consists of 21 exons that are distributed over 21 kb of genomic DNA. An isoform of Dtk is generated by differential splicing of exons from the 5{prime} region of the gene. The overall genomic structure of Dtk is virtually identical to that determined for the human UFO gene. This particular genomic organization is likely to have been duplicated and closely maintained throughout evolution. 38 refs., 3 figs., 1 tab.

  5. Brainstem metabotropic glutamate receptors reduce food intake and activate dorsal pontine and medullar structures after peripheral bacterial lipopolysaccharide administration.

    PubMed

    Chaskiel, Léa; Paul, Flora; Gerstberger, Rüdiger; Hübschle, Thomas; Konsman, Jan Pieter

    2016-08-01

    During infection-induced inflammation food intake is reduced. Vagal and brainstem pathways are important both in feeding regulation and immune-to-brain communication. Glutamate is released by vagal afferent terminals in the nucleus of the solitary tract and by its neurons projecting to the parabrachial nuclei. We therefore studied the role of brainstem glutamate receptors in spontaneous food intake of healthy animals and during sickness-associated hypophagia after peripheral administration of bacterial lipopolysaccharides or interleukin-1beta. Brainstem group I and II metabotropic, but not ionotropic, glutamate receptor antagonism increased food intake both in saline- and lipopolysaccharide-treated rats. In these animals, expression of the cellular activation marker c-Fos in the lateral parabrachial nuclei and lipopolysaccharide-induced activation of the nucleus of the solitary tract rostral to the area postrema were suppressed. Group I metabotropic glutamate receptors did not colocalize with c-Fos or neurons regulating gastric function in these structures. Group I metabotropic glutamate receptors were, however, found on raphé magnus neurons that were part of the brainstem circuit innervating the stomach and on trigeminal and hypoglossal motor neurons. In conclusion, our findings show that brainstem metabotropic glutamate receptors reduce food intake and activate the lateral parabrachial nuclei as well as the rostral nucleus of the solitary tract after peripheral bacterial lipopolysaccharide administration. They also provide insight into potential group I metabotropic glutamate receptor-dependent brainstem circuits mediating these effects. PMID:27016016

  6. Structural/functional relationships between internal and external MSH receptors: modulation of expression in Cloudman melanoma cells by UVB radiation

    SciTech Connect

    Chakraborty, A.K.; Orlow, S.J.; Bolognia, J.L.; Pawelek, J.M. )

    1991-04-01

    Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells. Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs. Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.

  7. Molecular Evolution of the Transmembrane Domains of G Protein-Coupled Receptors

    PubMed Central

    Fatakia, Sarosh N.; Costanzi, Stefano; Chow, Carson C.

    2011-01-01

    G protein-coupled receptors (GPCRs) are a superfamily of integral membrane proteins vital for signaling and are important targets for pharmaceutical intervention in humans. Previously, we identified a group of ten amino acid positions (called key positions), within the seven transmembrane domain (7TM) interhelical region, which had high mutual information with each other and many other positions in the 7TM. Here, we estimated the evolutionary selection pressure at those key positions. We found that the key positions of receptors for small molecule natural ligands were under strong negative selection. Receptors naturally activated by lipids had weaker negative selection in general when compared to small molecule-activated receptors. Selection pressure varied widely in peptide-activated receptors. We used this observation to predict that a subgroup of orphan GPCRs not under strong selection may not possess a natural small-molecule ligand. In the subgroup of MRGX1-type GPCRs, we identified a key position, along with two non-key positions, under statistically significant positive selection. PMID:22132149

  8. Crystal structure of a complex between β-glucopyranose and a macrocyclic receptor with dendritic multicharged water solubilizing chains.

    PubMed

    Mandal, Pradeep K; Kauffmann, Brice; Destecroix, Harry; Ferrand, Yann; Davis, Anthony P; Huc, Ivan

    2016-08-01

    Using commercial screens for crystallization of biomolecules and taking advantage of the use of racemic crystallography allowed the production of X-ray quality single crystals and the elucidation at 1.08 Å resolution of the solid state structure of a difficult target: the complex between glucopyranose and a water soluble macrocyclic receptor equipped with dendritic multianionic solubilizing chains. PMID:27373805

  9. SPECIES DIFFERENCES IN ANDROGEN AND ESTROGEN RECEPTOR STRUCTURE AND FUNCTION AMONG VERTEBRATES AND INVERTEBRATES: INTERSPECIES EXTRAPOLATIONS REGARDING ENDOCRINE DISRUPTING CHEMICALS

    EPA Science Inventory

    Species Differences in Androgen and Estrogen Receptor Structure and Function Among Vertebrates and Invertebrates: Interspecies Extrapolations regarding Endocrine Disrupting Chemicals
    VS Wilson1, GT Ankley2, M Gooding 1,3, PD Reynolds 1,4, NC Noriega 1, M Cardon 1, P Hartig1,...

  10. Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations

    SciTech Connect

    Singla, Nikhil; Goldgur, Yehuda; Xu, Kai; Paavilainen, Sari; Nikolov, Dimitar B.; Himanen, Juha P.

    2010-09-08

    Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3 {angstrom} resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.

  11. Interaction between glycated serum albumin and AGE-receptors depends on structural changes and the glycation reagent.

    PubMed

    Indurthi, Venkata S K; Leclerc, Estelle; Vetter, Stefan W

    2012-12-15

    Physiologically relevant reactive carbonyl compounds vary greatly in their glycation reactivity and the resulting advanced glycation endproducts (AGE) are likely to have distinct structural and biological properties. We characterized a panel of twenty AGE-BSA preparations in terms of (i) their biophysical properties, (ii) their binding to the receptor for advanced glycation endproducts (RAGE) and galectin-3, and (iii) their effects on cellular proliferation. We could establish correlations between lysine glycation and changes in secondary structure. Circular dichroism and differential scanning calorimetry experiments showed that glycation causes albumin to adopt folding properties of a molten globule. Binding studies between AGE-albumin and RAGE or galectin-3 indicate that binding to the isolated receptor domains was weak. Only AGE compounds derived by glycation with ribose were able to bind tightly (K(d) < 10 μM) to both AGE receptors. Cell based assays using an engineered melanoma cell line demonstrated correlations between the extent of (i) lysine side chain modification, (ii) β-sheet content and (iii) albumin multimerization with stimulation of cell proliferation. However, in addition to structural properties of the protein, the chemical structures of the AGE-modifications were important for receptor binding and biological activity as well. PMID:23079546

  12. Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia.

    PubMed

    Shpilman, Michal; Hollander-Cohen, Lian; Ventura, Tomer; Gertler, Arieh; Levavi-Sivan, Berta

    2014-10-01

    Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The full-length cDNA of tLepR was 3423bp, encoding a protein of 1140 amino acid (aa) which contained all functionally important domains conserved among vertebrate leptin receptors. The cDNAs of tLepA and tLepB were 486bp and 459bp in length, encoding proteins of 161 aa and 152 aa, respectively. Modeling the three-dimensional structures of tLepA and tLepB predicted strong conservation of tertiary structure with that of human leptin, comprised of four helixes. Using synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression in Escherichia coli was prepared according to the cloned sequence. The tLepA- and tLepB-expressing plasmids were transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography. In the case of tLepA, the fraction eluted contained a mixture of monomers and dimers. The purified tLepA and tLepB monomers and tLepA dimer showed a single band of ∼15kDa on an SDS-polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer showed one band of ∼30kDa in the absence of reducing agent, indicating its formation by S-S bonds. The three tLeps were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the three tLeps were biologically active in promoting STAT-LUC activation in COS7 cells transfected with the identified tLepR but not in cells transfected with hLepR. tLepA was more active than tLepB. Low or no activity

  13. Rational Quantitative Structure-Activity Relationship (RQSAR) Screen for PXR and CAR Isoform-Specific Nuclear Receptor Ligands

    PubMed Central

    Dring, Ann M.; Anderson, Linnea E.; Qamar, Saima; Stoner, Matthew A.

    2010-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets. PMID:20869355

  14. Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice

    PubMed Central

    van Os, Ronald P.; Dethmers-Ausema, Albertina; Bos, I. Sophie T.; Hylkema, Machteld N.; van den Berge, Maarten; Hiemstra, Pieter S.; Wess, Jürgen; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    2014-01-01

    Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M3 receptor-deficient mice (M3R−/−) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M3 receptors are present on almost all cell types, and in this study we investigated the relative contribution of M3 receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M3R−/− bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M3R−/− animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-α and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M3R−/− animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6–2.5 fold). These findings indicate that the M3 receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M3 receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-α and CD177. PMID:25381025

  15. Structure of the active core of human stem cell factor and analysis of binding to its receptor Kit

    PubMed Central

    Jiang, Xuliang; Gurel, Ogan; Mendiaz, Elizabeth A.; Stearns, George W.; Clogston, Christi L.; Lu, Hsieng S.; Osslund, Timothy D.; Syed, Rashid S.; Langley, Keith E.; Hendrickson, Wayne A.

    2000-01-01

    Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 Å resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric ‘head-to-head’ association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor. PMID:10880433

  16. Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants.

    PubMed

    Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul; Yu, Wenli; Sasisekharan, Ram; Wilson, Ian A

    2015-11-24

    Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses. PMID:26586437

  17. Structure of macrophage colony stimulating factor bound to FMS: Diverse signaling assemblies of class III receptor tyrosine kinases

    SciTech Connect

    Chen, Xiaoyan; Liu, Heli; Focia, Pamela J.; Shim, Ann Hye-Ryong; He, Xiaolin

    2009-06-12

    Macrophage colony stimulating factor (M-CSF), through binding to its receptor FMS, a class III receptor tyrosine kinase (RTK), regulates the development and function of mononuclear phagocytes, and plays important roles in innate immunity, cancer and inflammation. We report a 2.4 {angstrom} crystal structure of M-CSF bound to the first 3 domains (D1-D3) of FMS. The ligand binding mode of FMS is surprisingly different from KIT, another class III RTK, in which the major ligand-binding domain of FMS, D2, uses the CD and EF loops, but not the {beta}-sheet on the opposite side of the Ig domain as in KIT, to bind ligand. Calorimetric data indicate that M-CSF cannot dimerize FMS without receptor-receptor interactions mediated by FMS domains D4 and D5. Consistently, the structure contains only 1 FMS-D1-D3 molecule bound to a M-CSF dimer, due to a weak, hydrophilic M-CSF:FMS interface, and probably a conformational change of the M-CSF dimer in which binding to the second site is rendered unfavorable by FMS binding at the first site. The partial, intermediate complex suggests that FMS may be activated in two steps, with the initial engagement step distinct from the subsequent dimerization/activation step. Hence, the formation of signaling class III RTK complexes can be diverse, engaging various modes of ligand recognition and various mechanistic steps for dimerizing and activating receptors.

  18. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  19. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors.

    PubMed

    Homan, Kristoff T; Larimore, Kelly M; Elkins, Jonathan M; Szklarz, Marta; Knapp, Stefan; Tesmer, John J G

    2015-01-16

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors. PMID:25238254

  20. Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A

    PubMed Central

    2016-01-01

    Background Botulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A–H). Currently, botulinum neurotoxin serotype A (BoNT⁄A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT⁄A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT⁄A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT⁄A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures. Results 1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT⁄A. 2) TRPV1 colocalized with both BoNT⁄A and cleaved SNAP-25 when BoNT⁄A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT⁄A treatment (1 nmol⁄l), both TRPV1 and BoNT⁄A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT⁄A. Conclusion BoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT⁄A to mediate pain relief. PMID:26745805

  1. Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli

    PubMed Central

    Lonardi, Emanuela; Moonens, Kristof; Buts, Lieven; de Boer, Arjen R.; Olsson, Johan D. M.; Weiss, Manfred S.; Fabre, Emeline; Guérardel, Yann; Deelder, André M.; Oscarson, Stefan; Wuhrer, Manfred; Bouckaert, Julie

    2013-01-01

    Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcβ1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract. PMID:24833052

  2. The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21).

    PubMed

    Hannan, Jonathan Paul

    2016-01-01

    Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity. PMID:26916158

  3. Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli.

    PubMed

    Lonardi, Emanuela; Moonens, Kristof; Buts, Lieven; de Boer, Arjen R; Olsson, Johan D M; Weiss, Manfred S; Fabre, Emeline; Guérardel, Yann; Deelder, André M; Oscarson, Stefan; Wuhrer, Manfred; Bouckaert, Julie

    2013-01-01

    Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract. PMID:24833052

  4. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

    PubMed Central

    Wu, Jianping; Li, Zhangqiang; Xie, Tian; Peng, Wei; Yin, Changcheng; Li, Xueming; Scheres, Sjors H.W.; Shi, Yigong; Yan, Nieng

    2014-01-01

    The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The fou