Free backbone carbonyls mediate rhodopsin activation

Abstract

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.

Figure 1

Crystal structure of the visual receptor rhodopsin (PDB ID 1U19 ) showing the positions of Pro215^5.50, Pro267^6.50, Pro291^7.38 and Pro303^7.50. These prolines are located on helices H5, H6 and H7. The lack of an NH group results in a carbonyl group at the i-4 position from these prolines that is free to form interhelical hydrogen-bonds. The residues with the free carbonyl (His211^5.46, Ile263^6.46, Phe287^7.34, and Ala299^7.46) are shown in grey. The three free C=O groups that are conserved across the family A GPCRs (His211^5.46, Ile263^6.46, and Ala299^7.46) lie within or near the TM core of the receptor.

Figure 2

REDOR NMR as a probe of hydrogen bonding changes of carbonyl residues at the i-4 positions of Pro215^5.50, Pro267^6.50, Pro291^7.38 and Pro303^7.50. (a, c, e, g) One-dimensional MAS NMR difference spectra between rhodopsin and Meta II are shown in the top panel. REDOR filtered spectra of rhodopsin and Meta II are shown in the middle and lower panels, respectively. The spectra highlight the i-4 for carbonyls associated with His211^5.46 using rhodopsin labeled with 1-¹³C His, ¹⁵N Phe (a), Ile263^6.46 using rhodopsin labeled with 1-¹³C Ile, ¹⁵N Cys (c), Ser299^7.46 using rhodopsin labeled with 1-¹³C Ser, ¹⁵N Val (e), and Phe287^7.34 using rhodopsin labeled with 1-¹³C Phe, ¹⁵N Met (g). (b, d, f, h) Crystal structures of rhodopsin (gray, PDB ID 1U19) and Meta II (cyan, PDB ID 3PQR) are shown in the region of interest.

Figure 3

¹³C…¹⁵N REDOR NMR experiments of Meta II in the presence and absence of the Gα peptide of transducin. REDOR filtered spectra are shown for His211^5.46 (a), Ile263^6.46 (b) and Ser299^7.46 (c). The experiments on rhodopsin without (red) and with (blue) added Gα peptide were carried out in DDM micelles and mixed DDM/DOPS micelles, respectively.

Figure 4

Receptor activation leads to repacking of helices H5–H7 on the TM core of rhodopsin. (a) Inactive structure of rhodopsin. Helices H1–H4 form a scaffold onto which helices H5–H7 pack. The key packing contacts on H5 and H7 are associated with Pro215^5.50 and Pro303^7.50 and their corresponding i-4 carbonyls. Retinal isomerization disrupts both interactions. For H5, the β-ionone ring has a steric clash at the position of the His211^5.46-Glu122^3.37 hydrogen bond upon conversion the all-trans configuration. For H7, the retinal is covalently attached to Lys296^7.43. Upon isomerization, Trp265^6.48 on H6 rotates away from H7 and disrupts a water mediated hydrogen bond with Asn302^7.49, which is part of a hydrogen bonding network stretching from Asn55^1.50 and Asp83^2.50 to the Ala299^7.46 C=O. (b) Overlap of the crystal structures of rhodopsin (1GZM, purple) and Meta II (3PQR, light purple) showing the positions of the TM helices. The disruption of the interactions of the His211^5.46 C=O and Ala299^7.46 C=O with the H1–H4 scaffold allows helices H5–H7 to reorient. (c,d) Schematic of the hydrogen bonding changes occurring between inactive rhodopsin and active Meta II rhodopsin.