Structure and function in rhodopsin: Packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled* (G-protein-coupled receptorysignal transductiony11-cis-retinalydisulfide bondymetarhodopsin II)

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UVyvisible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110OCys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain. A large number of point mutations in rhodopsin that occur in all three [intradiscal, transmembrane (TM), and cytoplasmic] domains in rhodopsin are associated with retinitis pigmentosa (RP) (2–6). Previous work with designed (7, 8) and naturally occurring RP mutants (9) in the intradiscal domain has demonstrated partial or total misfolding of the corresponding opsins upon expression of the mutant genes in COS-1 cells. Operationally, misfolding in opsin has been defined as loss of the ability to bind 11-cis-retinal (7). This in turn has been concluded to be due to the formation in the intradiscal domain of a disulfide bond other than the native Cys-110OCys-187 disulfide bond (7–10). A recent study of the RP mutation, L125R, in helix C of the TM domain and two additional mutations at this site, L125A and L125F, also demonstrated misfolding (11). These results led to the conclusion that defects in the packing of the helices are relayed to the intradiscal domain, where they also cause misfolding by inducing the formation of an abnormal disulfide bond. In undertaking the present work we argued that helix C, where the Leu-125 mutations are located, may be a special case in causing misfolding. This helix contains the conserved charge pair (Glu-134zArg-135), the counterion to the protonated Schiff base (Glu-113), and Cys-110, a participant in the native disulfide bond. Consequently, we wished to determine if coupling between folding in the two domains is a basic characteristic such that certain mutations in every one of the seven TM helices could cause misfolding. We have now studied the following RP mutations in the TM domain that, together with the previously studied mutations in helix C, represent all seven of the TM helices. The mutations studied (Fig. 1) are G51V (12), G51A (5), G89D (4, 12), A164V (13), H211P (14), P267L (14), P267R (15), and S297R (15). The mutant opsin genes corresponding to all the above mutations were expressed in COS cells and the opsins produced were purified. They all were found to be mixtures of retinal-binding and non-retinal-binding fractions as seen from the UVyvisible (UVyVis) absorption spectral characteristics. The mixtures were separated into fractions that reconstituted with 11-cis-retinal to form the mutant rhodopsins and fractions that did not bind 11-cis-retinal. The degrees of misfolding varied with the mutants, H211P and P267R forming mostly misfolded opsins. Furthermore, when the purified 500-nm chromophore-forming mutant rhodopsins were tested for their bleaching properties (metarhodopsin II formation and its decay) these were strikingly abnormal except for G51A. Thus, the results show that RP mutations in every one of the TM helices can cause misfolding of the opsin, and they substantiate the earlier conclusion that there is coupling between the packing of the helices to form the TM domain and folding in the intradiscal domain to a tertiary structure. While the correctly folded rhodopsin structure contains the disulfide bond between Cys-110 and Cys-187, the misfolded structures contain a different intradiscal disulfide bond. MATERIALS AND METHODS Materials. 11-cis-Retinal was a gift from Rosalie Crouch (University of South Carolina and the National Eye Institute, National Institutes of Health). Dodecyl maltoside (DM) was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9410571-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: TM, transmembrane; RP, retinitis pigmentosa; DM, dodecyl maltoside. Mutant rhodopsins with amino acid substitutions are designated by the one letter abbreviations for amino acids. The amino acid to the left of the residue number is the original, whereas that to the right is the substituted amino acid. *This is paper 24 in the series ‘‘Structure and function in rhodopsin.’’ Paper 23 is ref. 1. †J.H. and P.G. contributed equally to this work. ‡Present address: Departament d’Enginyeria Quimica, Escola d’Optica I Optometria, Universitat Politecnica de Catalunya, 08222 Terrassa, Catalonia, Spain. §Present address: CytoMed, Inc., 840 Memorial Drive, Cambridge, MA 02139. ¶To whom reprint requests should be addressed.