Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation

Abstract

The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation.

Evolution of nonspectral rhodopsin function at high altitudes

“…The Astroblepus L59Q substitution replaces a hydrophobic residue with a polar residue into a membrane-facing site, and thus may be indirectly perturbing the geometry of these nearby HBNs, resulting in our observed increases in dark-state and light-activated rhodopsin decay rates. Our findings that L59Q and M288L both significantly increase the decay-rates of light-activated rhodopsin, while also decreasing the stability of dark-state rhodopsin against thermal activation, are consistent with the emerging theory that distinct hubs of the rhodopsin HBN have interconnected roles in modulating the kinetic properties of rhodopsin and other GPCRs (47,49,65).…”

“…This finding is consistent with the structural locations of both substitutions (Fig. 3), where both are proximal to the same intramolecular HBN controlling rhodopsin kinetics, but only site 288 is involved in the section surrounding the RBP (23,49,66,67) (Fig. 3A).…”

“…1A). These positions are, to our knowledge, completely conserved in all known vertebrate rhodopsins, consistent with their proximity to the retinal binding pocket (RBP; site 288) and the NPxxY motif (site 59): two distinct hubs of the same intramolecular HBN stabilizing inactive and active rhodopsin (27,(47)(48)(49). To better understand what ecological factors may be driving substitutions at these highly conserved positions, we investigated the geographical distribution of natural variation at these positions across the Andean collection sites.…”

“…Through interactions with water molecules, RBP HBN-participating residues stabilize both the dark-state and lightactivated (MII) forms of rhodopsin, likely through interactions with the Schiff-base and counter ion (E113), where mutations to RBP HBN residues accelerate kinetic rates and tend also to blue-shift peak absorbance (23,62,63,65,66). Although previous evidence suggested the proximity of site 288 to functional RBP water molecules involved in photoactivation (68,69), the exact functional role of site 288 in the RBP HBN and rhodopsin activation had not been directly investigated until recently (49). Our M288L rhodopsin functional results lend further support to the involvement of site 288 in the RBP HBN and steric organization.…”

Soft Matter in Lipid–Protein Interactions

“…The FSM explains functional influences of membrane lipid-protein interactions by elastic deformation of the lipid film (15,19,125). If a change in protein shape occurs (36,83,149,154), then the free energy of the entire system (comprising protein, lipids, and water) is affected by the membrane bilayer (13). At this level, the contribution to the free energy change is described by two quantities: the bending modulus κ and the monolayer H 0 spontaneous curvature.…”