Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Abstract

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

A cholesterol switch controls phospholipid scrambling by G protein–coupled receptors

“…4 A , (P(mock)), indicating that β1AR is less efficiently reconstituted. This may be because the protein multimerizes as detergent is withdrawn during the reconstitution process (see related analyses of opsin in references ( 7 , 8 , 50 )), resulting in fewer vesicles being populated. However, as seen for opsin, the fluorescence decay trace required a double-exponential fit, with the slow second phase providing a measure of the scrambling rate.…”

Phospholipid Scrambling by G Protein–Coupled Receptors

“…Opsin self-associates to form dimers and higher-order multimers in the membrane (22,44,53) but purifies as a monomer when solubilized in n-dodecyl-β-D-maltoside (DDM) (29,45,53). However, the protein reversibly self-associates when the concentration of DDM is lowered (45,53) and reconstitutes into vesicles as a dimer or higher-order multimer, as explained below.…”

“…Opsin self-associates to form dimers and higher-order multimers in the membrane (22,44,53) but purifies as a monomer when solubilized in n-dodecyl-β-D-maltoside (DDM) (29,45,53). However, the protein reversibly self-associates when the concentration of DDM is lowered (45,53) and reconstitutes into vesicles as a dimer or higher-order multimer, as explained below. This behavior makes it difficult to determine the functional oligomeric state of the protein and, more specifically, to address the question of whether an opsin monomer is sufficient to facilitate scrambling.…”

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors

“…4A, (P(mock)), indicating that β1AR is less efficiently reconstituted. This may be because the protein multimerizes as detergent is withdrawn during the reconstitution process (see related analyses of opsin in references (7, 8, 50)), resulting in fewer vesicles being populated. However, as seen for opsin, the fluorescence decay trace required a double-exponential fit, with the slow second phase providing a measure of the scrambling rate.…”