The molecular switching mechanism at the conserved D(E)RY motif in class-A GPCRs

The molecular switching mechanism at the conserved D(E)RY motif in class-A GPCRs

Sandoval, A.; Eichler, S.; Madathil, S.; Reeves, P. J.; Fahmy, K.; Boeckmann, R. A.


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 photoreceptor rhodopsin, this is accompanied by proton uptake at Glu134 in the class-conserved E(D)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 here by molecular dynamics (MD) simulations on TM3 model peptides in a DOPC bilayer. We show that protonation of the conserved glutamic acid alters its side chain rotamer preferences and stabilizes the C-terminal helical structure. Both 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 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 opsin mutants and demonstrates that the E(D)RY motif is a hydrated microdomain. 25 The function of the E(D)RY motif as a proton switch is suggested to be based on the reorganization of the H-bond network at the membrane interface.

Keywords: infrared; fluorescence; membrane protein; hydration