High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Abstract

Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is a rigid-body movement of transmembrane helix 6 (TM6) at the cytoplasmic surface of the molecule. To place this result on a quantitative footing, and to identify movements of other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used to determine distances and distance changes between pairs of nitroxide side chains introduced in helices at the cytoplasmic surface of rhodopsin. Sixteen pairs were selected from a set of nine individual sites, each located on the solvent exposed surface of the protein where structural perturbation due to the presence of the label is minimized. Importantly, the EPR spectra of the labeled proteins change little or not at all upon photoactivation, suggesting that rigid-body motions of helices rather than rearrangement of the nitroxide side chains are responsible for observed distance changes. For inactive rhodopsin, it was possible to find a globally minimized arrangement of nitroxide locations that simultaneously satisfied the crystal structure of rhodopsin (Protein Data Bank entry 1GZM), the experimentally measured distance data, and the known rotamers of the nitroxide side chain. A similar analysis of the data for activated rhodopsin yielded a new geometry consistent with a 5-Å outward movement of TM6 and smaller movements involving TM1, TM7, and the C-terminal sequence following helix H8. The positions of nitroxides in other helices at the cytoplasmic surface remained largely unchanged.DEER ͉ G protein-coupled receptor ͉ photoreceptor ͉ spin labeling G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that mediate cellular responses to a variety of both physical and chemical signals.Interaction of a GPCR with a cognate signal produces a conformational change leading to an activated receptor. The activated state subsequently binds to and activates a corresponding heterotrimeric G protein, which in turn triggers events that ultimately modulate an amplified cellular response (for recent reviews, see refs. 1 and 2). The nature of the conformational switch or switches that underlie receptor activation is a subject of much interest (3, 4). Crystal structures of an inactive state of the GPCRs rhodopsin (5-9) and ␤ 2 -adrenergic receptor (10, 11) have been reported, and they provide a crucial foundation for designing and interpreting the results of spectroscopic experiments aimed at revealing conformational changes associated with activation.Site-directed spin-labeling (SDSL) studies of rhodopsin provided the first direct structural evidence for an outward tilt of the cytoplasmic end of transmembrane helix 6 (TM6) as a major event in the activation of rhodopsin, both in solutions of dodecyl maltoside (DM) and in phospholipid bilayers (12-15). Data from site-directed fluorescent labeling and chemical reactivity (16), UV spectroscopy (17), zinc cross-linking of histidines (18), and disulfide cross-linking (13) offered s...

Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH

“…To distinguish between the different submodels will require other, more structure sensitive time-resolved spectral techniques. The scheme presented here, however, is consistent with EPR studies describing the helix-6 movement involved in activation, 31 and G-protein peptide mimic binding studies. 41 Many structural studies reveal complex helix movements involving the rearrangements of H-bonding in the transmembrane region of the protein, and several states of the chromophore relaxation during activation.…”

“…As expected, the ratio of the Meta Ib 480 formation of Meta Ib 480 and the enhanced deprotonation leading to the formation of Meta IIb to the helix-6 movement observed by EPR on a similar time scale. 28,31 This helix movement is necessary for G-protein binding. It is also suggested that the proton uptake by the protein, leading to its active state and causing the anomalous pH dependence of the equilibrium between Meta I and Meta II in Scheme 1, is initiated by the helix movement.…”

An unbalanced translocation unmasks a recessive mutation in the follicle-stimulating hormone receptor (FSHR) gene and causes FSH resistance

“…31 In the activation-related scenario, TMH6 undergoes the most important movement where the kink functions as a pivot and finally leads to G-protein coupling and activation. 32,33 Substitution of the proline is expected to lead to modifications in the signaling properties by causing changes in the helix conformation, intramolecular interactions and thereby the helix arrangement. This is supported by recent findings regarding corresponding prolines in other glycoprotein hormone receptors, the thyrotropin receptor (hTSHR, Pro639), 34 and the lutropin/choriogonadotropin receptor (rLHCGR, Pro588).…”

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.…”