Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz, Jay M.; Fay, Jonathan F.; Farrens, David

doi:10.1074/jbc.m210567200

Cited by 78 publications

(95 citation statements)

References 78 publications

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“…A number of factors are shown to affect the thermal stability of dark-state rhodopsin, including retinal disease-causing mutations (44) or zinc binding (45). Other factors mediating the stability of dark-state rhodopsin are proposed, like a conserved ion-pair interaction in the intradiscal loop E-2 of rhodopsin (46). A destabilizing effect is also observed for the active intermediate Meta II of the G51 mutants formed upon photobleaching.…”

Section: Discussionmentioning

confidence: 93%

Structural and Functional Role of Helices I and II in Rhodopsin

Bosch

Ramon

Valle

et al. 2003

Journal of Biological Chemistry

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The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.

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Section: Discussionmentioning

confidence: 93%

Structural and Functional Role of Helices I and II in Rhodopsin

Bosch

Ramon

Valle

et al. 2003

Journal of Biological Chemistry

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“…Janz et al (51) In addition, congenital night blindness mutants that are characterized by an open conformation of the chromophorebinding site for nucleophiles have been identified. For example, T94I opsin folds properly and binds 11-cis-retinal to form pigment, but T94I Rho displays reduced thermal stability, has a long lived Meta II photostate, and shows highly increased reactivity toward NH 2 OH in the dark compared with WT Rho (54,55).…”

Section: Discussionmentioning

confidence: 99%

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

Zhu

Jang

Jastrzębska

et al. 2004

Journal of Biological Chemistry

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Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO T4R/T4R dog retina, we found that the mutation abolished glycosylation at Asn 2 , whereas glycosylation at Asn 15 was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho* lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G t ). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

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“…Also the cytoplasmic loops (C1 -C3) of opsin and rhodopsin have significantly higher temperature factors (Bfactor) than the TM segments. On the other hand, the extracellular loops and the N-terminus form a compact extracellular domain, the so-called "retinal plug" [49,113]. The "retinal plug"…”

Section: Overall Structure Of Opsinmentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

858

925

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Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x) 5,6 F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

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Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Cited by 78 publications

References 78 publications

Structural and Functional Role of Helices I and II in Rhodopsin

Structural and Functional Role of Helices I and II in Rhodopsin

A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor

Crystal structure of the ligand-free G-protein-coupled receptor opsin

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