D(E)RY Motif- A Conserved Proton Dependent Modular Switch In Class-1 GPCRs

Activation of G protein-coupled receptors (GPCRs) orginates in ligand-induced structural changes that are transmitted across the plasma membrane to the cytosolic receptor surface. In rhodopsin-like class-1GPCRs, protonation of a carboxylic acid (Glu134 in rhodopsin) in the conserved cytosolic D(E)RY motif in transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the proton-sensitivity of the structure of synthetic peptides consisting of 30amino acids derived from TM3 of bovine rhodopsin (rho) and the chemokine receptor CCR3. ATR- Fourier-transform-infrared (FTIR) spectroscopy reveals a helical structure of the rho-TM3 peptide in PC vesicles and detergent micelles, where Glu134 exchanges protons with the solvent at a pKa of 5.5-6.0 as determined from the pH sensitivity of the COO- stretching mode. The amide I modes reveal a proton-induced helical turn formation in rho-TM3 and CCR3-TM3. The conformational change in the ERY motif is affects also the Tyr136 fluorescence which exhibits a strong pH sensitivity (pKa of 6) which is abolished upon replacement of Glu134 by Gln in rho-TM3. Förster-Resonance-Energy-transfer from a C-terminal Trp to DANSYL-labelled lipids in mixed micelles and lipid vesicles evidences that the D(E)RY motif constitutes an autonomous proton-driven conformational switch which couples protonation to helical turn formation and probably helix insertion depth. We propose that the protonation-controlled hydrophobicity profile at the cytosolic end of TM3 provides a pH-sensitive module that couples helix packing in the active receptor conformation of class I GPCRs to proton exchange reactions with the cytosol.