title: The Molecular Switching Mechanism at the Conserved D(E)RY Motif in Class-A GPCRs.
abstract: The disruption of ionic and H-bond interactions between the cytosolic ends of transmembrane helices TM3 and TM6 of class-A (rhodopsin-like) G protein-coupled receptors (GPCRs) is a hallmark for their activation by chemical or physical stimuli. In the bovine photoreceptor rhodopsin, this is accompanied by proton uptake at Glu(134) in the class-conserved D(E)RY motif. Studies on TM3 model peptides proposed a crucial role of the lipid bilayer in linking protonation to stabilization of an active state-like conformation. However, the molecular details of this linkage could not be resolved and have been addressed in this study by molecular dynamics (MD) simulations on TM3 model peptides in a bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We show that protonation of the conserved glutamic acid alters the peptide insertion depth in the membrane, its side-chain rotamer preferences, and stabilizes the C-terminal helical structure. These factors contribute to the rise of the side-chain pKa (> 6) and to reduced polarity around the TM3 C terminus as confirmed by fluorescence spectroscopy. Helix stabilization requires the protonated carboxyl group; unexpectedly, this stabilization could not be evoked with an amide in MD simulations. Additionally, time-resolved Fourier transform infrared (FTIR) spectroscopy of TM3 model peptides revealed a different kinetics for lipid ester carbonyl hydration, suggesting that the carboxyl is linked to more extended H-bond clusters than an amide. Remarkably, this was seen as well in DOPC-reconstituted Glu(134)- and Gln(134)-containing bovine opsin mutants and demonstrates that the D(E)RY motif is a hydrated microdomain. The function of the D(E)RY motif as a proton switch is suggested to be based on the reorganization of the H-bond network at the membrane interface.
authors.affiliation: Computational Biology, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Angelica
authors.initials: A
authors.lastname: Sandoval
authors.affiliation: Helmholtz-Zentrum Dresden - Rossendorf, Institute of Resource Ecology, and Technische Universität Dresden, Dresden, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Eichler
authors.affiliation: Department of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors.forename: Sineej
authors.initials: S
authors.lastname: Madathil
authors.affiliation: School of Biological Sciences, University of Essex, Colchester, United Kingdom.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.affiliation: Helmholtz-Zentrum Dresden - Rossendorf, Institute of Resource Ecology, and Technische Universität Dresden, Dresden, Germany. Electronic address: k.fahmy@hzdr.de.
authors.forename: Karim
authors.initials: K
authors.lastname: Fahmy
authors.affiliation: Computational Biology, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany. Electronic address: rainer.boeckmann@fau.de.
authors.forename: Rainer A
authors.initials: RA
authors.lastname: Böckmann
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D006860:Hydrogen Bonding; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D056004:Molecular Dynamics Simulation; D011522:Protons; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D011522:Protons; D043562:Receptors, G-Protein-Coupled