Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4029-4033, April 1994 Biochemistry Structure and function in rhodopsin: Replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state (signal transduction/protein folding/G protein-coupled receptors/monodonal antibody/transducin) FLORENCE F. DAVIDSON, PETER C. LOEWENt, AND H. GOBIND KHORANA Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 Contributed by H. Gobind Khorana, December 1, 1993 ABSTRACT A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties ofthe resulting rhodopsin mutants. The mutant protein C11OA/C187A expressed in COS cells resembles wild-tpe rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chro- mophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that ofthe wild type, by a AAG* of only -2.9 kcal/mol. Further, the afi~ties of the mutant and wild-type rhodopsins to the anti- rhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin H (MU) and MIII photoin- termediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates oftransducin activation are the same for both mutant and wild-type MUI FIG. 1. A secondary structure model for bovine rhodopsin. at Shown are (i) K2%, which forms a Schiffbase with 11-cis-retinal; (ii) intermediates 4rC, at 150C the MU photointermediate in the E113, counterion to the protonated Schiff base; (iii) the 10 cysteine mutant decays more than 20 times faster than in wild type. We residues of rhodopsin, of which two, C322 and C323, are palmitoy- conclude that the disulfide bond between Cys-110 and Cys-187 lated and two, C110 and C187, form a disulfide bond with each other; is a key component in determining the stability of the MU and (iv) glycosylation sites, N2 and N15. The membrane interface structure and its coupling to transducin activation. boundaries indicated by double lines are tentative. The intradiscal domain is equivalent to the extracellular domain ofrelated G-protein Rhodopsin, the vertebrate dim light photoreceptor, is a coupled receptors. member of the superfamily of guanine nucleotide-binding extracellular cysteine pair yields receptors with some ligand- protein (G-protein)-coupled signal-transducing membrane binding capacity (15). proteins. One structural feature in rhodopsin, which is con- We report here a study of mutant rhodopsins in which one served in this class of receptors, is an intradiscal disulfide or more of the three intradiscal cysteine residues, 110, 185, bond between Cys-110 and Cys-187 (1-5) (Fig. 1). Replace- or 187 (Fig. 1), has been replaced by alanine. The mutant ment of either of these cysteine residues in rhodopsin by opsins, expressed in COS-1 cells, fold stably and bind 11- serine prevents functional receptor formation (4). Likewise, cis-retinal to form chromophores with the characteristics of rhodopsin mutants with a short deletion in the N-terminal wild-type rhodopsin. By the present criteria, the mutant sequence or in any one of the intradiscal§ loops BC, DE, or C11OA/C187A has a ground-state structure that is indistin- FG (Fig. 1) yield completely or partially nonfunctional guishable from wild-type rhodopsin, and thermal bleaching opsins, as do many point mutants in this domain (6). It has shows only a 10%6 decrease in the transition free energy. therefore been concluded that the intradiscal loops and the However, the metarhodopsin II (MII) intermediate formed N-terminal region form an integrated tertiary structure, of by the light-activated mutant is destabilized. At 40C, under which the Cys-110 to Cys-187 disulfide bond is a critical conditions where MII decay is insignificant, initial rates of component. In the present workl we have asked, is transducin (GT) activation by the mutant and wild-type rhodopsin able to fold and function correctly when the rhodopsins are identical. Yet at 15'C, the decay of mutant cysteine residues forming the disulfide bond are replaced by MII is at least 20 times faster than that ofwild type. Thus, the certain amino acids other than serine? Studies of small disulfide bond between Cys-110 and Cys-187 appears to be a soluble proteins, such as bovine pancreatic trypsin inhibitor key determinant of stability of the MII conformation and its (11-13) and lysozyme (14), have shown that alanine and coupling to GT activation. valine replacements ofthe disulfide-bonded cysteine residues in these proteins allow the formation of structures virtually Abbreviations: G protein, guanine nucleotide-binding protein; GT, identical to those of the native proteins. Moreover, in the transducin; GTP[y-35S], guanosine 5'-y[-[35S]thio]triphosphate; MR, ,B3adrenergic receptor, a member of the G protein-coupled metarhodopsin II; LM, lauryl maltoside. receptor superfamily, valine replacement of the conserved tPresent address: Department of Microbiology, University of Man- itoba, Winnipeg, Manitoba R3T 2N2 Canada. §Usually designated extracellular in other G-protein coupled recep- The publication costs of this article were defrayed in part by page charge tors. payment. This article must therefore be hereby marked "advertisement" IThis is paper 6 in the series "Structure and Function in Rhodopsin." in accordance with 18 U.S.C. §1734 solely to indicate this fact. Paper 5 is ref. 10. 4029 Downloaded by guest on October 2, 2021 4030 Biochemistry: Davidson et al. Proc. Natl. Acad Sci. USA 91 (1994) MATERIALS AND METHODS IDetermination of Thermal Stabilities. The thermal stability of ground-state rhodopsins was measured by monitoring the Expression, Purification, and Characterization of Wild- rate of loss of 500-nm absorbance and appearance of 380-nm Type and Mutant Rhodopsins. Mutations in the coding se- absorbance as a function of temperature in the dark. At quence of the synthetic bovine rhodopsin gene (16) were temperatures where decay of the chromophore was slow, made by cassette mutagenesis as described (17) using the measurements were made by repetitive scanning at 240 vector pMT4 (18). COS-1 cells were transiently transfected nm/min from 650 nm to 250 nm. At higher temperatures, with mutant and wild-type plasmids (17), harvested 60-72 h scans were taken before and after the experiment, to look for later, and resuspended in PBS buffer (1.5 mM KH2PO4/8 mM any changes in baseline, light scattering, or loss of sample, Na2HPO4/2.7 mM KCI/137 mM NaCi, pH 7.3) containing and the decay kinetics were monitored by single-wavelength benzamidine, aprotinin, leupeptin, and pepstatin A each at 25 measurements at 510 nm. The decays were fit to single pg/ml and phenylmethylsulfonyl fluoride at 0.1 mM. Ex- exponentials using the KALEIDAGRAPH program (Synergy pressed opsins were reconstituted by the addition of 11-cis- Software, Reading, PA). From these, the first-order decay retinal (5 1.M) to the cell suspension, followed by gentle rate constants were derived. The thermodynamic parameters agitation for 2 h at 40C in the dark. Reconstituted rhodopsins Ea, AGt, AHt, and ASt were calculated from the rate data were solubilized with 1% (wt/vol) lauryl maltoside (LM; according to standard theory for irreversible processes (23) Anatrace, Maumee, OH), incubated for 2 h at 40C in the dark, using values for the slopes of the Arrhenius plots which were and centrifuged for 1 h at 100,000 x g at 40C. Rhodopsins derived from least-squares analysis. were purified from the 100,000 x g supernates by immunoaf- finity chromatography using rholD4-Sepharose (17) in 0.05% Assay for GT Activation. GT activation was measured by the LM in 10 mM sodium phosphate buffer (pH 6.0) or 50 mM light-stimulated formation of the GT-guanosine 5'-[Y- Tris HCl, pH 7.0/150 mM NaCl. Purity of the immunopuri- [35S]thio]triphosphate (GTP[y-35S]) complex, as described fled rhodopsins was assessed after SDS/PAGE in 10-12% (24) with the following modifications. Assays were carried polyacrylamide gels (19) by silver staining (Bio-Rad silver out using 1-12.5 nM rhodopsin, as indicated, 250 nM GT (25), stain plus kit), by immunoreactivity against the monoclonal and 125 nM GTP[y_35S] (1.4 x 105 cpm/pmol) in 10 mM antibody rholD4, and by measurement of the absorbance Tris-HCl, pH 7.4/100 mM NaCl/0.5 mM MgCl2/1 mM di- ratios at 280 nm to 500 nm (A2o/A5oo) in the UV-visible thiothreitol/0.0125% LM. Reactions were initiated by the spectrum (20). addition of GTPty_35S] to rhodopsin/GT mixes preequili- UV-Visible Spectroscopy. All measurements were made on brated at 4°C in the dark, with and without prior illumination a Perkin-Elmer A7 UV-visible spectrometer, equipped with for 90 sec with >495-nm light. Four 25-pi aliquots were water-jacketed cuvette holders connected to a circulating removed at 15- to 30-sec intervals and filtered through water bath (model RTE 5DD, Neslab Instruments, Ports- nitrocellulose (Millipore HAWP-45 pm). The filters were mouth, NH). Temperatures at the cuvette were monitored by rinsed three times with ice-cold buffer, dried, and subjected a digital thermometer mounted on the sample cuvette holder to scintillation counting to quantitate bound GTd-GTP[-35S]. (Cole-Parmer model 8402-00). Samples were illuminated in Dark activities were subtracted from light activities for each the cuvettes with a 150-W fiber optic light (Dolan-Jenner, time point, and the data were analyzed by linear regression Woburn, MA; Fiber Lite A-200) equipped with a 495-nm to determine the rates of rhodopsin-catalyzed exchange of cutoff filter (Melles Griot, Irvine, CA).