Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors |
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Authors: | Vogel Reiner Mahalingam Mohana Lüdeke Steffen Huber Thomas Siebert Friedrich Sakmar Thomas P |
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Affiliation: | 1 Biophysics Section, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 9, D-79104 Freiburg, Germany 2 Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY 10065, USA |
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Abstract: | Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic “ionic lock.” Glu1343.49 of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg1353.50 in the dark-state crystal structure of rhodopsin. We examined the roles of Glu1343.49 and Arg1353.50 on H3 and Glu2476.30 and Glu2496.32 on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu1343.49 of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg1353.50 similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu2476.30 and Glu2496.32 on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu2476.30 and Glu2496.32 on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu1343.49 and Arg1353.50 is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the β2-adrenergic receptor. |
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Keywords: | FTIR, Fourier transform infrared GPCR, G-protein-coupled receptor H, transmembrane helix β2AR, β2-adrenergic receptor PC, phosphatidyl choline DDM, n-dodecyl-β- smallcaps" >d-maltoside OG, n-octyl β- smallcaps" >d-glucoside H, transmembrane helix MES, 2-(N-morpholino)ethanesulfonic acid |
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