Crystal structure of a thermally stable rhodopsin mutant

Abstract

We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.

Figure 1. Western blot of rhodopsin mutants and three-dimensional crystals of recombinant rhodopsins

A: The proteins were expressed and purified from transfected COS cells. Primary antibody used in the blot was 1D4. Lane 1: The N2C/D282C mutant has lost the carbohydrate chain on residue 2 but retains that attached to residue 15. Lane 2: The fully deglycosylated mutant N2C/N15D/D282C shows a single homogeneous band. Mutations in position 15 are known to cause autosomal dominant retinitis pigmentosa in humans. Photos from crystals of recombinant rhodopsins were taken under infrared illumination (>850nm). B: Crystals of recombinant wild type rhodopsin. C: Crystals of thermally stabilized rhodopsin N2C/D282C. D: Crystals of stabilized fully deglycosylated rhodopsin N2C/N15D/D282C.

Figure 2. Thermal stability of N2C/D282C rhodopsin in the detergent C₈E₄ used for crystallization

The N2C/D282C mutant was purified by 1D4 immunoaffinity chromatography. Rhodopsin solubilized from retinas (native) was purified by Con A affinity chromatography. In both cases a Sephadex G50 column was used to exchange the detergent from 0.02% (w/v) DDM to 0.2% (w/v) C₈E₄ immediately before the experiment. The protein solutions were incubated for 30 minutes at various temperatures before an absorption spectrum has been recorded. The amount of bound retinal was determined by the absorption at 500 nm, corrected by the protein absorption at 280 nm. To investigate the effect of reduced disulfide bonds the same experiment was performed in the presence of 50 mM β-mercaptoethanol (β-me). Using the program Prism sigmoidal curves were fitted to the individual data points. The T₅₀ values obtained from these curves (wt: 37.3°C., wt+β-me: 37.5°C., N2C/D282C: 47.2°C., N2C/D282C+β-me: 41.3°C.) show a ∼10°C. increase in stability of the mutant which is mostly lost in the presence of β-mercaptoethanol.

Figure 3. Data collection on a recombinant microcrystal

A: Frozen microcrystal used for data collection. The arrow represents the axis along which the data was collected on 15 distinct positions. B: Diffraction pattern obtained after 15 s exposure and 1° rotation. 1: The first diffraction pattern collected at a new position on the needle. 2: The eighth consecutive frame collected at the same position. A strong reduction in diffraction intensity caused by radiation damage is observed. 3: Fully restored diffraction intensity is obtained after the beam had been translated along the needle axis. C: Diffraction intensity represented by the number of strong spots (NSTRONG, XDS) on each frame of the 13 indexed wedges. At each minimum in the number of strong spots the beam was translated to a new position on the needle axis. The positions of diffraction patterns shown in B are marked.

Figure 4. Electron density maps of N2C/D282C rhodopsin

A: Retinal Omit electron density map (0.75 sigma) obtained after molecular replacement and density modification. The retinal (red) was not included but appears clearly in the electron density. B: Disulfide bond and N15 glycosylation. Sigma-A weighted 2F_o-F_c Electron density contoured at sigma 0.9. The disulfide bond between the mutated residues C2 and C282 is well resolved, as is the first residue of the N-glycosylation linked to N15. No density is visible adjacent to the mutated residue 2, which would have been glycosylated in wild-type rhodopsin.

Figure 5. Comparison of recombinant and native rhodopsin

A: Superposition emphasizing overall similarity of the individual monomers and their different dimer packing along helix 5. Blue: Monomer A, Cyan: Monomer B, Grey: native rhodopsin monomers A and B obtained from 1GZM coordinates, Yellow: Designed disulfide bond between C2 and C282, Red:retinal, Green: N15 linked glycosylation B: Enlargement of the extracellular region with the introduced disulfide bond. C: Coordinate differences between monomers A of both structures plotted as the root mean square deviation (rmsd) of Cα atoms against sequence position (blue). Poorly ordered regions with a B-factor higher than 100 are marked red. The designed disulfide includ ing two amino acids on each side is labeled yellow. The largest differences are found in loops C2 and C3 and are due to the inherent flexibility and poor order of these loops. The end of helix 5 before the C3 loop is shifted about 2 Å due to the different dimer packing. The disulfide is introducing small but significant changes in the backbone of the E3 loop and the N-terminus.

Figure 6. The N-terminal Cap domain

Contacts between the N-terminal cap (blue) and the rest of the receptor (cyan). Atoms of the receptor with a minimum distance of 5 Å to residues 1-33 of the N-terminus are colored beige. The covalently bound retinal (red) is separated from the N-terminal cap by the lid formed from the E2 loop. The designed disulfide between C2 and C282 connecting the N-terminal cap with loop E3 is shown as yellow sticks. The molecule is viewed with the extracellular site facing up.