Retinitis Pigmentosa Rhodopsin Mutations L125R and A164V Perturb Critical Interhelical Interactions

Abstract

L125R, a severe retinitis pigmentosa rhodopsin missense mutation, results in rhodopsin protein misfolding, retinal degeneration, and ultimately blindness. The initiating structural events leading to this protein misfolding are unknown. Through the use of compensatory mutations, in conjunction with crystal structure-based molecular analysis, we established that the larger and positively charged Arg . Combined, these localized defects in interhelical interactions cause structural changes that interfere with the ability of opsin to bind 11-cis-retinal. These distortions ultimately lead to the formation of an abnormal disulfide bond, severe protein instability, aggregation, and endoplasmic reticulum retention. In the absence of a crystal or NMR structure of each retinitis pigmentosa mutation, compensatory mutagenesis and crystal structure-based analysis are powerful tools in determining the localized molecular disturbances. A detailed understanding of the initiating local perturbations created by missense mutations such as these, not only identifies critical factors required for correct folding and stability, but additionally opens avenues for rational drug design, mimicking the compensatory mutations and stabilizing the protein.Rhodopsin, the dim light-activated photoreceptor in the retina, is considered to be a prototypical G protein-coupled receptor (1). A substantial understanding of the structure of rhodopsin has been obtained from electron cryomicroscopy (2), electron paramagnetic resonance (3, 4), and biochemical studies (5). More recently, the 2.8-Å resolution crystal structure of rhodopsin (the first solved among the G protein-coupled receptors) has provided a detailed three-dimensional model of the structure of rhodopsin (6, 7). Through the induction of structural defects, naturally occurring rhodopsin mutations result in the onset of a heterogeneous group of inherited retinal degenerative disorders collectively called retinitis pigmentosa (RP) 1 (reviewed in Ref. 8). Thus far, more than 90 different rhodopsin point mutations have been identified, affecting ϳ1 in 3500, or an estimated 2 million people worldwide (8). Although there are no current, effective therapies for retinitis pigmentosa, supplementation with vitamin A (the precursor of 11-cisretinal) has been helpful in treating a subset of RP patients (9). More detailed analyses have examined the direct effect of vitamin A, as well as other retinoids, on the protein folding and stability properties of specific retinitis pigmentosa mutants. A treatment protocol of Vitamin A plus 11-cis-retinal improved stability of the T17M mutant protein and delayed photoreceptor degradation in a transgenic mouse model expressing that retinitis pigmentosa mutant (10). Additional studies have shown that improved folding of P23H, the most common retinitis pigmentosa mutant in North America, can be achieved with both 11-cis-7-ring-retinal (11) and 9-cis-retinal (12) addition.As a representative G protein-coupled receptor, rhodopsin is an integral membrane protein compose...

Critical Role of Transmembrane Segment Zinc Binding in the Structure and Function of Rhodopsin

“…The conserved nature of this site raises the possibility that the Zn 2ϩ 3 -mediated Meta II formation may be an acquired feature that accounts for the 100-fold difference in light sensitivity between rod and cone pigments (40). The fact that Zn 2ϩ deficiency results in abnormal dark adaptation and night blindness in humans and other animals (41) (25). We now propose that the disrupted Zn 2ϩ 3 coordination site in the rhodopsin transmembrane domain directly leads to the protein misfolding and night blindness associated with these mutations.…”

“…UV-visible Absorption Spectroscopy-UV-visible absorption spectra were recorded on a PerkinElmer Life Sciences -40 UV-visible spectrophotometer at 2 or 25°C (25). Rhodopsin thermal stability was determined by monitoring the decay of the 500 nm absorbance at a constant temperature of 50°C.…”

“…ROS (rod outer segments) from bovine retinas was a kind gift from Dr. Phillip Reeves (M.I.T.). The presence of rhodopsin protein, both from COS-1 cells and from ROS, was determined through UV-visible spectroscopy (500 nm absorbance) and Western blot analysis (25).…”

“…Preparation of Rhodopsin Protein Samples-Amino acid substitutions were introduced into the synthetic rhodopsin gene through PCR mutagenesis, as described previously (25). COS-1 cells were transiently transfected with pMT4 vectors carrying the opsin genes.…”

Role of group-conserved residues in the helical core of β 2 -adrenergic receptor

“…S161V (4.53)/L115A (3.34). It was shown previously in rhodopsin that the L119A mutation rescues the chromophore-binding defect caused by the A164V mutation (14). Because Leu-119 (3.34) in rhodopsin corresponds to Leu-115 (3.34) in ␤ 2 -AR [supporting information (SI) Fig.…”