Two protonation switches control rhodopsin activation in membranes

Abstract

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to Ϸ50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.FTIR spectroscopy ͉ G protein-coupled receptor ͉ ionic lock ͉ membrane protein ͉ UV-visible spectroscopy G protein-coupled receptors (GPCRs) are 7-helical transmembrane proteins that exist in conformational equilibria between inactive and active conformations modulated by the binding of ligands (1). In the case of rhodopsin as a visual pigment, the ligand is the retinal chromophore, which is covalently linked to a lysine on transmembrane helix H7 by a protonated Schiff base (PSB) (2). The 11-cis retinal chromophore of the dark state is an inactivating inverse agonist that is converted by photoisomerization to the all-trans agonist, driving the conformational transitions leading to receptor activation. Within milliseconds several inactive intermediates are formed, such as Batho, BSI, Lumi, and Meta I, that can be examined by using time-resolved (3) or cryotrapping techniques (4). The early transitions involve mainly a relaxation of the isomerized retinal chromophore (5, 6), with only minor changes to the ␣-helix bundle of the receptor protein as revealed by electron crystallography of the Meta I state (7). Only in the transition from Meta I to the active receptor conformation Meta II is a rearrangement of the helix bundle observed, involving tilt movements of H6 (8-10) and presumably also of H5 (11).Activation of the receptor is proposed to involve 2 distinct protonation switches ...

Solid-state 2 H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

“…Solid-state NMR spectroscopy (28) is a powerful adjunct to X-ray crystallography, as it gives knowledge of membrane proteins in a native-like environment where function is preserved (17,21). In the case of rhodopsin, solid-state NMR clearly demonstrates mobility of the retinylidene ligand-despite its crucial biological role as an inverse agonist-even at cryogenic temperatures, as typically used in X-ray crystallography.…”

“…By trapping the conformational states and probing their local dynamics, we explore the energy landscape for the receptor activation. According to our molecular dynamics simulations (45), helices H1-H4 make up the TM core of the receptor (4,17). The rhodopsin core is stabilized by two ionic locks involving the PSB and the E(D)RY motif with their counterions, plus three hydrogen-bonded networks and several conserved microdomains (27).…”

“…Upon light absorption, rhodopsin catalyzes the rapid and highly selective 11-cis to trans isomerization of retinal (15,16) switching it from an inverse agonist to an agonist. In the Metarhodopsin I-Metarhodopsin II equilibrium, recognition sites are exposed for a heterotrimeric G protein (transducin or G t ), initiating the amplified visual response (17,18). However, the lightactivated Meta II state is transient and crystal deformation occurs giving low-resolution data (14).…”

“…Spectroscopic approaches including spin-label EPR (23), 13 C NMR (18,(24)(25)(26), and Fourier transform infrared (FTIR) (17,27) studies are thus needed to establish the activation mechanism of the photoreceptor as it underlies the visual process. Solid-state NMR spectroscopy (28) is particularly important, as it gives knowledge of both protein structure and dynamics in a membrane lipid environment (29).…”

A Comparison between the Photoactivation Kinetics of Human and Bovine Rhodopsins

“…The helix movement also results in a pH-dependent protonation of glutamate 134. 14,18,19 Previous studies of the human rhodopsin attempted to characterize human rhodopsin intermediates using both temperature trapping and time-resolved spectroscopic measurements to determine the similarities and differences between the activation of human and bovine rhodopsins. 20,21 It was expected that differences in the sequences of human and bovine rhodopsins in the connection from transmembrane 5 and extracellular loop 2 could affect rhodopsin activation and G-protein binding.…”