Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors

Abstract

Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.

PAR4 activation involves extracellular loop 3 and transmembrane residue Thr153

“…Amide H/D exchange was performed and analyzed as described previously. 20,25 A detailed H/D exchange procedure, software list, and analysis pipeline are described in the supplemental Data. The H/D exchange was normalized to the maximal deuterium uptake at 10 minutes and the PAR4 homology model is color-coded as indicated in the legends for Figures 2 and 3.…”

“…The computational model of PAR4 in its thrombin-activated state was generated by using the crystal structure of rhodopsin at 2.2 Å resolution (Protein Data Bank entry 1U19) as described for green cone opsin. 20 The 2 sequences were aligned using the EMBOSS Needle Pairwise Sequence Alignment tool, and the initial model was generated using the MEDELLER server. 26,27 This initial model was imported into the ROSETTA protein structure prediction suite, and the loop building protocol was applied to determine the model with the lowest free energy.…”

“…Amide hydrogen/deuterium exchange (H/D exchange) mass spectrometry (MS) is a structural approach that monitors the deuterium uptake at amide NH groups along the backbone of the protein by using MS. 17,18 The power of H/D exchange comes from comparing the deuterium uptake between 2 states to uncover the dynamics of ligand binding or receptor activation. [17][18][19][20][21][22][23] The distinct advantage of H/D exchange for G protein-coupled receptors (GPCRs) is that it overcomes the need for a stabilized receptor and allows us to examine the conformational dynamics of the tethered ligand mechanism.…”

Disease mechanisms of X‐linked cone dystrophy caused by missense mutations in the red and green cone opsins

“…AAV-mediated expression of N94K and W177R was barely detectible, whereas P307L, R330Q, and G338E showed expression levels higher than that of endogenous M-opsin in wild-type mice by an antired/green opsin antibody raised against the C terminus of human L/M-opsins 46 (Figure S2). Since human cone opsins have been shown to be N-glycosylated at Nterminal residue Asn-34, 47,48 we investigated whether N-glycosylation of these mutants was affected. Protein extracts were treated with PNGase F to remove N-linked glycosaccharides.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…8) [190]. The conformational changes in green cone opsin upon light activation have also been studied [196], as well as the alternating access mechanism of the Na + /H + antiporter NhaA [191].…”