In silico analysis of Human and Zebrafish ?-2 Adrenergic Receptors (original) (raw)
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In silico analysis of Human and Zebrafish α-2 Adrenergic Receptors
2012
α-2 adrenoceptors, belong to class of Rhodopsin-like G-protein coupled receptors. Proteins of the G-protein coupled receptor (GPCR) family are involved in many pathophysiological conditions and hence are targets for various drug discovery methods. The current information on the structure of GPCRs is limited to few structures like Rhodopsin, β adrenergic receptors, adenosine A2A receptors, Human Dopamine D3 and Chemokine receptor. In our study α-2 adrenergic receptors of Human and Zebrafish were modeled using MODELLER with Human Dopamine D3 receptor (PDB ID: 3PBL) as template. Through our modeling studies we have identified the critical role played by Proline residues (2.38, 2.59, 4.39, 4.59, 4.60, 7.50) of transmembrane helices and extracellular loop in stabilizing structural deviations in the transmembrane. Novel ligand binding residues S/T (6.56) and F (7.35) along with the positional significance of Y (3.28), Y (6.55) in regulating function were identified. Our models have shown that the Phenylalanine at 7.39 in TM7 can favourably interact with positively charged N-methyl group of the catecholamine ligands via hydrophobic contacts rather than 7.38 as reported previously. Furthermore, we are able to correctly show the orientation of Serine at 5.42 and 5.46 and discuss the relevance of residues at position 3.37 and 5.43 in the receptor regulation. We also demonstrate and propose that the orientation of V (2.61)/S should be taken into account in drug/ pharmacophore design specific for α-2 adrenergic receptors. We believe that these findings will open new lead for ligand/ pharmacophore design, in silico leading further to experimental validation using Zebrafish as experimental model.
In silico analysis of the binding of agonists and blockers to the β2-adrenergic receptor
Journal of Molecular Graphics and Modelling, 2011
Activation of G protein-coupled receptors (GPCRs) is a complex phenomenon. Here, we applied Induced Fit Docking (IFD) in tandem with linear discriminant analysis (LDA) to generate hypotheses on the conformational changes induced to the  2 -adrenergic receptor by agonist binding, preliminary to the sequence of events that characterize activation of the receptor. This analysis, corroborated by a follow-up molecular dynamics study, suggested that agonists induce subtle movements to the fifth transmembrane domain (TM5) of the receptor. Furthermore, molecular dynamics also highlighted a correlation between movements of TM5 and the second extracellular loop (EL2), suggesting that freedom of motion of EL2 is required for the agonist-induced TM5 displacement. Importantly, we also showed that the IFD/LDA procedure can be used as a computational means to distinguish agonists from blockers on the basis of the differential conformational changes induced to the receptor. In particular, the two most predictive models obtained are based on the RMSD induced to Ser207 and on the counterclockwise rotation induced to TM5.
FEBS Letters, 1996
Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the ale-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the ale-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the CQ-AR thus providing new insights into the structure/ function relationship of G protein-coupled receptors.
British Journal of Pharmacology, 2009
Zebrafish has five distinct a 2-adrenoceptors. Two of these, a 2Da and a 2Db , represent a duplicated, fourth a 2-adrenoceptor subtype, while the others are orthologue of the human a 2A-, a 2B-and a 2Cadrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish a 2-adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [ 35 S]GTPgS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3 The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human a 2-adrenoceptors, separated by B350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish a 2-adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4 The a 2A orthologues and the zebrafish a 2D duplicates clustered as close pairs, but the relationships between the orthologues of a 2B and a 2C were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the a 2-adrenoceptors, we suggest that the second extracellular loop-where significant sequence variability is located-might contribute significantly to the observed affinity differences.
Journal of Biological Chemistry, 2001
To investigate their role in receptor coupling to G q , we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣ 1badrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the ␣ 1b -AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg 254 and Lys 258 in the third intracellular loop and Lys 291 at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val 147 and Leu 151 in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg 254 , Lys 258 , as well as Leu 151 are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the ␣ 1b -AR. These findings contribute to the delineation of the molecular determinants of the ␣ 1b -AR/G q interface.
Biochemical and Biophysical Research Communications, 2004
The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length a 1a adrenergic receptor (a 1a-AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of a 1a-AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands.
Structure of a β1-adrenergic G-protein-coupled receptor
Nature, 2008
G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a b 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane a-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the b 1 -adrenergic receptor and binding of carazolol to the b 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the b 2 -adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
Journal of Structural Biology, 2005
Antagonist binding to-2 adrenoceptors (2-ARs) is not well understood. Structural models were constructed for the three human 2-AR subtypes based on the bovine rhodopsin X-ray structure. Twelve antagonist ligands (including covalently binding phenoxybenzamine) were automatically docked to the models. A hallmark of agonist binding is the electrostatic interaction between a positive charge on the agonist and the negatively charged side chain of D3.32. For antagonist binding, ion-pair formation would require deviations of the models from the rhodopsin structural template, e.g., a rotation of TM3 to relocate D3.32 more centrally within the binding cavity, and/or creation of new space near TM2/TM7 such that antagonists would be shifted away from TM5. Thus, except for the quinazolines, antagonist ligands automatically docked to the model structures did not form ion-pairs with D3.32. This binding mode represents a valid alternative, whereby the positive charge on the antagonists is stabilized by cation-interactions with aromatic residues (e.g., F6.51) and antagonists interact with D3.32 via carboxylate-aromatic interactions. This binding mode is in good agreement with maps derived from a molecular interaction library that predicts favorable atomic contacts; similar interaction environments are seen for unrelated proteins in complex with ligands sharing similarities with the 2-AR antagonists.
Structure of a beta1-adrenergic G-protein-coupled receptor
Nature, 2008
G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a b 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane a-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the b 1 -adrenergic receptor and binding of carazolol to the b 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the b 2 -adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
Biochemical and biophysical research communications, 2004
The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length alpha(1a) adrenergic receptor (alpha(1a)-AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of alpha(1a)-AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands.