Retinal Ligand Mobility Explains Internal Hydration and Reconciles Active Rhodopsin Structures
ABSTRACT Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors-a pharmaceutically important class of membrane proteins that has garnered much attention due to the recent availability of structural information. Yet, the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state 2H NMR spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states; retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent active-like crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and to a concerted transition in the highly conserved Trp2656.48 residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.
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ABSTRACT: Cell-permeable orthosteric ligands can assist folding of G protein coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore, if endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected HEK293 cells but rapidly reached the plasma membrane in cells incubated with an A1-antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound an A1-selective antagonist with an affinity indistinguishable form the wild-type A1-receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2--vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression A1-receptor-Y(288)A within 1 h. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1-receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1-receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.Molecular pharmacology 10/2014; 87(1). DOI:10.1124/mol.114.094045 · 4.12 Impact Factor
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ABSTRACT: Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that β2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.Nature Communications 10/2014; 5:5115. DOI:10.1038/ncomms6115 · 10.74 Impact Factor