A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin

Despite high-resolution crystal structures of both inactive and active G protein-coupled receptors (GPCRs), it is still not known how ligands trigger the large structural change on the intracellular side of the receptor since the conformational changes that occur within the extracellular ligand-binding region upon activation are subtle. Here, we use solid-state NMR and Fourier transform infrared spectroscopy on rhodopsin to show that Trp2656.48 within the CWxP motif on transmembrane helix H6 constrains a proline hinge in the inactive state, suggesting that activation results in unraveling of the H6 backbone within this motif, a local change in dynamics that allows helix H6 to swing outward. Notably, Tyr3017.48 within activation switch 2 appears to mimic the negative allosteric sodium ion found in other family A GPCRs, a finding that is broadly relevant to the mechanism of receptor activation. [Display omitted] •Rhodopsin activation results in unraveling of the H6 backbone within the CWxP motif•Tyr3017.48 appears to mimic the negative allosteric sodium ion found in other GPCRs•The A295V7.42 constitutively active CSNB mutation unravels the H6 backbone•Switch 2 is likely the primary activation switch in rhodopsin and other class A GPCRs The hallmark of GPCR activation is a large outward motion of helix H6. NMR spectroscopy was used to determine how interactions in the prototypical GPCR rhodopsin regulate the dynamics of the conserved proline kink within this helix. These findings establish the mechanism for coupling of retinal isomerization to receptor activation.