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

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

Free backbone carbonyls mediate rhodopsin activation

Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins

Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor. © 2009 Nature America, Inc. All rights reserved

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer.

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins

Capping of Aβ42 Oligomers by Small Molecule Inhibitors

Aβ42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer’s disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aβ42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1–2 nm and high MW oligomers with heights of 3–5 nm. In both cases, the oligomers are disc-shaped with diameters of ∼10–15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aβ42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5–20) of Aβ42 monomers and cap the height of the oligomers that are formed at 1–2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aβ42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aβ oligomers provides insights into the mechanism of inhibition and the pathway of Aβ aggregation

Location of the Retinal Chromophore in the Activated State of Rhodopsin

Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific 13C-labeled sites located on the β-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly188 (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the β-ionone ring moves to a position between Met207 and Phe208 on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the C∈ carbons of Lys296 (H7) and Met44 (H1) and between Gly121 (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu122 (H3) and His211 (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc