Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II

Abstract

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y(6.40) constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223(5.58) in helix 5, Y306(7.53) of the NPxxY motif and R135(3.50) of the E(D)RY motif, highly conserved residues of the G protein binding site.

Fig. 1.

Biochemical assessment of M257Y^6.40 in the context of the N2C/D282C background. (A) Assessment of G protein activation (pmol) by bovine opsin (open symbols and dashed regression lines) or 11-cis-retinal reconstituted rhodopsin (solid symbols and regression lines) for the N2C/D282C (blue circles) and N2C/M257Y/D282C (red triangles) mutants. The shaded area represents dim red light conditions that preserve the inactive ground state of rhodopsin. At time 5 min 30 s, samples were subjected to bright illumination and the rest of the data were collected. (B) Ability of opsin to covalently bind all-trans-retinal. Absorbance spectra of purified N2C/M257Y/D282C reconstituted with all-trans-retinal were recorded at pH 7.4 (solid line) and pH 3.5 (dotted line). At pH 7.4, the pigment absorbs maximally at 380 nm, which is characteristic of metarhodopsin-II and free retinal. Acidification causes a red-shift to 415 nm, resulting from the protonation of the Schiff base formed between opsin and retinal. The inset shows that reconstitution of N2C/M257Y/D282C with all-trans-retinal (squares, dotted regression line, slope = 1.37 pmol/ min) increases G protein activation levels to those of fully light activated metarhodopsin-II that had been reconstituted with 11-cis-retinal (red triangles, solid regression line, slope = 1.35 pmol/ min). Error bars in (A) and (B) represent standard error, n = 3.

Fig. 2.

Effect of the M257Y^6.40 mutation on the ionic lock region. The 2F_o-F_c density map (A, blue mesh, contoured at 1.5σ) indicates a high degree of order around the NPxxY (dark blue) and E(D)RY motifs (salmon) that are close to the binding site for the G protein peptide GαCT (orange). The bulky side chains of Y223^5.58 and Y306^7.53, as well as the mutated side chain of Y257^6.40 can be accurately positioned within their respective densities. In the ground state of wild-type rhodopsin (B), M257^6.40 is part of the hydrophobic barrier (green) that separates the NPxxY motif from the ionic lock region including the E(D)RY motif. The M257Y^6.40 mutation (C) interferes with this packing and lowers the energy barrier for reorganizations of TM6 that characterize the active conformation. In the structure of the constitutively active E113Q^3.28 structure (D) the hydrophobic barrier has opened and Y223^5.58 in TM5 and Y306^7.53 of the NPxxY motif in TM7 swing into the created cavity, stabilized by a water-mediated H-bond network (9). In both active state structures (C + D), the ionic lock interactions between R135^3.50 and E134^3.49 of the E(D)RY motif in TM3 and E247^6.30 in TM6 are opened allowing R135^3.50 to adopt an elongated rotamer that forms part of the GαCT peptide binding site. The constitutively active M257Y^6.40 mutant (C) introduces only minor changes to the overall structure of the ionic lock region, however the introduced tyrosine forms an edge-face interaction with Y223^5.58, a parallel-displaced π-π stacking interaction to Y306^7.53, and hydrophilic interactions to R135^3.50, three interactions that stabilize the open G protein binding site.

Fig. 3.

Conformation of all-trans-retinal in the M257Y/GαCT structure. The 2F_o-F_c density map contoured at 1σ (A, blue mesh) shows clear uninterrupted density for all-trans-retinal including the covalent bond to K296^7.43 that is characteristic for the active metarhodopsin-II state. The retinal binding pocket (B, C, defined as residues with interatomic distances to the retinal ≤ 4 Å) is dominated by hydrophobic van der Waals contacts between the β-ionone ring and four amino acids in TM5 (green), three amino acids in TM6 (blue), and one in EL2 (violet). The polyene part of the retinal is covalently bound to K296^7.43 in TM7 (red) and stacked against Y268^6.51 in TM6 (blue), while the two amino acids in TM3 (orange) directly interact with the C19 and C20 methyl groups.