Modelling and mutation studies on the histamine H1-receptor agonist binding site reveal different binding modes for H1-agonists: Asp116 (TM3) has a constitutive role in receptor stimulation

Summary A modelling study has been carried out, investigating the binding of histamine (Hist), 2-methylhistamine (2-MeHist) and 2-phenylhistamine (2-PhHist) at two postulated agonistic binding sites on transmembrane domain 5 (TM5) of the histamine H₁-receptor. For this purpose a conformational analysis study was performed on three particular residues of TM5, i.e., Lys²⁰⁰, Thr²⁰³ and Asn²⁰⁷, for which a functional role in binding has been proposed. The most favourable results were obtained for the interaction between Hist and the Lys²⁰⁰/Asn²⁰⁷ pair. Therefore, Lys²⁰⁰ was subsequently mutated and converted to an alanine, resulting in a 50-fold decrease of H₁-receptor stimulation by histamine. Altogether, the data suggest that the Lys²⁰⁰/Asn²⁰⁷ pair is important for activation of the H₁-receptor by histamine. In contrast, analogues of 2-PhHist seem to belong to a distinct subclass of histamine agonists and an alternative mode of binding is proposed in which the 2-phenyl ring binds to the same receptor location as one of the aromatic rings of classical histamine H₁-antagonists. Subsequently, the binding modes of the agonists Hist, 2-MeHist and 2-PhHist and the H₁-antagonist cyproheptadine were evaluated in three different seven--helical models of the H₁-receptor built in homology with bacteriorhodopsin, but using three different alignments. Our findings suggest that the position of the carboxylate group of Asp¹¹⁶ (TM3) within the receptor pocket depends on whether an agonist or an antagonist binds to the protein; a conformational change of this aspartate residue upon agonist binding is expected to play an essential role in receptor stimulation.Abbreviations 2-MeHist 2-methylhistamine - 2-PEA 2-pyridyl-ethylamine - 2-PhHist 2-phenylhistamine - CHO Chinese hamster ovary - E_int interaction energy - E_str strain energy - GES global energy structure - gpH₁R guinea pig H₁-receptor - GPCR G-protein coupled receptor - Hist histamine - N proximal nitrogen - N tele nitrogen - TM transmembrane domain - WT wild type     

Phosphomimetic sulfonamide and sulfonamidoxy analogues of (Lyso)phosphatidic acid

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are potent bioactive lipid mediators of signal transduction and are inactivated by phosphatases. To obtain receptor-isoform selective ligands with neutral phosphomimetic head groups, we performed the stereoselective synthesis of LPA and PA analogues with trifluoromethanesulfonamide and trifluoromethanesulfonamidoxy moieties replacing the phosphomonoester.     

Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors

Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintain cellular homeostasis, and transduce signals. G protein-coupled receptors (GPCRs) constitute a family of receptors with seven transmembrane alpha-helical domains (7TM receptors) expressed at the cell surface, where they regulate physiological and pathological cellular processes. Several herpesviruses encode receptors (vGPCRs) which benefits the virus by avoiding host immune surveillance, supporting viral dissemination, and thereby establishing widespread and lifelong infection, processes where receptor signaling and/or endocytosis seem central. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been exploited for selective drug delivery in virus infected cells. Therefore, studying GPCR trafficking is of great importance. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using such novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis.     

Classifying G-protein coupled receptors with bagging classification tree

G-protein coupled receptors (GPCRs) play a key role in different biological processes, such as regulation of growth, death and metabolism of cells. They are major therapeutic targets of numerous prescribed drugs. However, the ligand specificity of many receptors is unknown and there is little structural information available. Bioinformatics may offer one approach to bridge the gap between sequence data and functional knowledge of a receptor. In this paper, we use a bagging classification tree algorithm to predict the type of the receptor based on its amino acid composition. The prediction is performed for GPCR at the sub-family and sub-sub-family level. In a cross-validation test, we achieved an overall predictive accuracy of 91.1% for GPCR sub-family classification, and 82.4% for sub-sub-family classification. These results demonstrate the applicability of this relative simple method and its potential for improving prediction accuracy.     

Modeling Yeast Cell Polarization Induced by Pheromone Gradients

Yeast cells respond to spatial gradients of mating pheromones by polarizing and projecting up the gradient toward the source. It is thought that they employ a spatial sensing mechanism in which the cell compares the concentration of pheromone at different points on the cell surface and determines the maximum point, where the projection forms. Here we constructed the first spatial mathematical model of the yeast pheromone response that describes the dynamics of the heterotrimeric and Cdc42p G-protein cycles, which are linked in a cascade. Two key performance objectives of this system are (1) amplification—converting a shallow external gradient of ligand to a steep internal gradient of protein components and (2) tracking—following changes in gradient direction. We used simulations to investigate amplification mechanisms that allow tracking. We identified specific strategies for regulating the spatial dynamics of the protein components (i.e. their changing location in the cell) that would enable the cell to achieve both objectives.     

Pharmacophore model for bile acids recognition by the FPR receptor

Formyl-peptide receptors (FPRs) belong to the family A of the G-protein coupled receptor superfamily and include three subtypes: FPR, FPR-like-1 and FPR-like-2. They have been involved in the control of␣many inflammatory processes promoting the recruitment and infiltration of leukocytes in regions of inflammation through the molecular recognition of chemotactic factors. A large number of structurally diverse chemotypes modulate the activity of FPRs. Newly identified antagonists include bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The molecular recognition of these compounds at FPR receptor was computationally investigated using both ligand- and structure-based approaches. Our findings suggest that all antagonists bind at the first third of the seven helical bundles. A closer inspection of bile acid interaction reveals a number of unexploited anchor points in the binding site that may be used to aid the design of new potent and selective bile acids derivatives at FPR.     

Brownian dynamics simulations of the β2-adrenergic receptor extracellular loops: evidence for helix movement in ligand binding?

Brownian dynamics simulations of the gating mechanism of the extracellular loops of the β₂-adrenergic receptor suggest that loop gating alone will not permit the larger ligands to bind and hence suggests that lateral helix movement is also required in the binding process.