Microstructure at the water lipid protein interface controls conformational switching mechanisms in the conserved D(E)RY motif of G-protein coupled receptors

G-Protein coupled receptors (GPCRs) play a fundamental role in many physiological processes due to their ability to switch between different structures upon activation. The prototypical GPCR rhodopsin serves as a model to study molecular switching mechanisms. Upon photoisomerization of the chromophore retinal, protonation of a glutamic acid (Glu 134) in the highly conserved D(E)RY motif at the cytosolic end of transmembrane helix 3 (TM3) leads to breakage of an ionic lock which stabilizes the inactive state [1]. Due to the low dielectricity of the lipidic environment, side chain charges and their neutralization contribute to the energetics of conformational transitions much more than in a purely aqueous environment. Our aim is to elucidate the functional implication of lipid protein interactions and microstructure at the water lipid protein interface in controlling protein conformation. We have studied synthetic peptides derived from rhodopsin TM3 by fluorescence spectroscopy at different pH in a hydrophobic environment. In [2] pH dependency of FRET between Trp at the cytosolic side of a TM3-derived peptide and DANSYL-PE was used as a monitor for helix motion. The observed pH dependency argues for stabilization of the protonated state by lipid protein interactions. In addition, we studied a TM3-derived peptide with a Trp probe shifted into the hydrophobic region. This peptide showed a red-shifted emission maximum of Trp, indicative of water accessibility. Moreover, at low pH the red-shift was less pronounced supporting the hypothesis that the neutralized Glu134 repels water and in general provides a pH-regulated hydration site. We conclude that microstructure at the water lipid protein interface and lipid protein interaction play a key role in the switching mechanism of GPCRs. The predominance of these local interactions which are not strictly dependent on intramolecular contacts to specific amino acids reconciles the highly conserved proton uptake at the D(E)RY motif in GPCR activation on the one hand and the diverse ligand specificity of class-A GPCRs on the other hand.
References
[1] J. A. Ballesteros, A. D. Jensen, G. Liapakis, S. G.F. Rasmussen, L. Shi, U. Gether, J. A. Javitch, J. Biol. Chem. 276, 29171-29177 (2001)
[2] S. Madathil, G. Furlinski, K.Fahmy, Biopolymers 82, 329-333 (2006).