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The Molecular Switching Mechanism at the Conserved D(E)RY Motif in Class-A GPCRs

Sandoval, A and Eichler, S and Madathil, S and Reeves, PJ and Fahmy, K and Böckmann, RA (2016) 'The Molecular Switching Mechanism at the Conserved D(E)RY Motif in Class-A GPCRs.' Biophysical Journal, 111 (1). 79 - 89. ISSN 0006-3495

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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 Glu134 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 Glu134- and Gln134-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.

Item Type: Article
Subjects: Q Science > QC Physics
Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Philip Reeves
Date Deposited: 18 Aug 2016 10:06
Last Modified: 30 Jun 2021 10:15

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