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

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.

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

Horst Vogel, Shuguang Yuan

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.

Nature Publishing Group2014

Downscaling Fourier Transform Infrared Spectroscopy to the Micrometer and Nanogram Scale: Secondary Structure of Serotonin and Acetylcholine Receptors

Rudolf Hovius, Erwin Ilegems, Horst Pick, Per Rigler, Horst Vogel

High signal-to-noise Fourier transform IR (FTIR) spectra of the 5-hydroxytryptamine (serotonin) receptor (5-HT3R) and the nicotinic acetylcholine receptor (nAChR) were obtained by microscope FTIR spectroscopy using micrometer-sized, fully hydrated protein films. Because this novel procedure requires only nanogram quantities of membrane proteins, which is 4-5 orders of magnitude less than the amt. of protein typically used for conventional FTIR spectroscopy, it opens the possibility to access the structure and dynamics of many important mammalian receptor proteins. The secondary structure of detergent-solubilized 5-HT3R detd. by curve fitting of the amide I band yielded 36% a-helix, 33% b-strand, 15% b-turn, and 16% nonregular structures, which remained unchanged upon reconstitution in lipid membranes. From hydrogen-deuterium exchange, the secondary structure of the water-accessible part of 5-HT3R was detd. as 14% a-helix, 16% b-strand, 26% b-turn, and 14% nonregular structures. Interestingly, we found that both the overall and the water-accessible nAChR secondary structures were nearly identical to those of 5-HT3R, in agreement with predicted structures of this class of receptors. This is the first time that structural investigations were obtained for two closely related ligand-gated ion channels under strictly identical exptl. conditions. [on SciFinder (R)]

2003

Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles

Olivia Baud, Horst Pick, Luc Veya, Horst Vogel, Shuguang Yuan

A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level.

Nature Publishing Group2015