Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway

Recent crystal structures of G-protein-coupled receptors (GPCRs) have revealed ordered internal water molecules, raising questions about the functional role of those waters for receptor activation that could not be answered by the static structures. Here, we used molecular dynamics simulations to monitor-at atomic and high temporal resolution-conformational changes of central importance for the activation of three prototypical GPCRs with known crystal structures: the adenosine A(2A) receptor, the beta(2)-adrenergic receptor and rhodopsin. Our simulations reveal that a hydrophobic layer of amino acid residues next to the characteristic NPxxY motif forms a gate that opens to form a continuous water channel only upon receptor activation. The highly conserved tyrosine residue Y-7.53 undergoes transitions between three distinct conformations representative of inactive, G-protein activated and GPCR metastates. Additional analysis of the available GPCR crystal structures reveals general principles governing the functional roles of internal waters in GPCRs.

Lipid Receptor S1P(1) Activation Scheme Concluded from Microsecond All-Atom Molecular Dynamics Simulations

Shuguang Yuan

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator which activates G protein-coupled sphingosine 1-phosphate receptors and thus evokes a variety of cell and tissue responses including lymphocyte trafficking, endothelial development, integrity, and maturation. We performed five all-atom 700 ns molecular dynamics simulations of the sphingosine 1-phosphate receptor 1 (S1P(1)) based on recently released crystal structure of that receptor with an antagonist. We found that the initial movements of amino acid residues occurred in the area of highly conserved W269(6.48) in TM6 which is close to the ligand binding location. Those residues located in the central part of the receptor and adjacent to kinks of TM helices comprise of a transmission switch. Side chains movements of those residues were coupled to the movements of water molecules inside the receptor which helped in the gradual opening of intracellular part of the receptor. The most stable parts of the protein were helices TM1 and TM2, while the largest movement was observed for TM7, possibly due to the short intracellular part starting with a helix kink at P-7.50, which might be the first helix to move at the intracellular side. We show for the first time the detailed view of the concerted action of the transmission switch and Trp (W-6.48) rotamer toggle switch leading to redirection of water molecules flow in the central part of the receptor. That event is a prerequisite for subsequent changes in intracellular part of the receptor involving water influx and opening of the receptor structure.

Public Library Science2013

X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.

Nature Publishing Group2011

Probing Odorant-Mediated Receptor Activation

Olivia Baud

Molecular interactions of odorants with their olfactory receptors (ORs) are of central importance for the mammalian olfactory system to detect and discriminate a large variety of odors with a limited set of receptors. How a particular OR binds and distinguishes different odorant molecules remains unknown on a structural basis as there is no three- dimensional OR structure available. Progress in the area of ligand assignment to specific receptors with the development of functional assays allowing efficient screening of numerous odorant compound libraries is essential for the understanding of the selectivity and sensitivity of odor detection. To improve our knowledge of an OR binding site environment, the first part of this thesis focused on the characterization of the molecular interactions of the mouse eugenol receptor (mOR-EG) which has been shown to be activated by a wide variety of odorants. Computer modeling based on the resolved 3D structure of the β2-adrenergic receptor and mutational studies on mOR-EG were used to determine the key amino acid residues responsible for specific OR-ligand recognition and to determine the localization and chemical properties of the putative ligand binding site. Activation of mOR-EG point mutants was monitored using a cAMP-induced reporter gene assay in Hana3A cells. These investigations were used to improve ligand docking in the binding site and further define a more precise model of the mOR-EG ligand-binding cavity. With the development of genome sequencing, the comparison of ortholog/paralog pairs is becoming increasingly important to study the molecular basis of OR specificity. Emphasis has thus been placed on the characterization of the mOR-EG’s mouse paralog mOR-EV and on its putative human ortholog hOR5D18. These two ORs have been functionally expressed in Hana3A cells and the visualization of both their overall- and cell-surface expression have been performed using different OR fusion proteins. New specific agonists for the mOR-EV have been identified (jasmone, rose oxyde and nootkatone) and first attempts to de-orphanize hOR5D18 have been carried out, by random screening a large panel of more than 250 odorant compounds coupling the OR activation to cAMP-induced reporter gene expression. Based on our knowledge of the mOR-EG ligand binding site, three important positions for ligand recognition that diverge between mOR-EG and mOR-EV were mutated in both receptors to increase their sequence identity. Comparison of their respective ligand repertoires has provided novel insight into the molecular determinants for odor detection and discrimination. Given the importance to assign ligands to their cognate receptors, we established a novel functional assay based on electrical impedance measurements. The detection of ligand-induced cell morphological changes during and after receptor stimulation has been used to quantify the activation of different GPCRs including mOR-EG with their respective ligands. This label-free technique is applicable for many different cell types as well as for endogenously expressed receptors and is suitable for screening large libraries of ORs and compounds. Odorants are generally small hydrophobic cell-membrane-permeable organic molecules with highly variable chemical structures and properties. Due to their large chemical diversity, it is likely that some of them might trigger other cellular processes apart from their "conventional" role in olfaction. The dual role of some odorants acting on the mOR-EG and/or the nuclear estrogen receptor α (ERα) has therefore been explored. Estrogenic effect of these odorants has been measured on ER endogenously expressing MCF7 cells, which represent a sensitive and well-established system for the detection of estrogenic activities, using a Luciferase reporter gene assay and different cell proliferation measuring methods. Finally, computational modeling and ligand docking have provided informations on the molecular recognition principles of odorants on two functionally non-related receptors and will therefore help for the evaluation of the potential risk of chemical compounds used in perfumery.

EPFL2012

Probing Odorant-Mediated Receptor Activation

Olivia Baud

EPFL2012