Structural insights into the mechanism of rhodopsin phosphodiesterase

Abstract

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Fig. 1. Overall structure.

a Ribbon diagrams viewed from the membrane plane (left), the extracellular side (upper right) and the intracellular side (lower right). One protomer is colored rainbow and the other is colored orange. The retinal chromophores are shown as orange stick models. b Schematic representation of the ribbon diagram. c Monoolein molecules and residues at the dimer interface, viewed from the membrane plane (left) and the extracellular side (right). Monoolein molecules are shown as spheres with gray carbon atoms and red oxygen atoms. d Close-up view of the residues on the intracellular side.

Fig. 2. Structural comparisons with BR and ChR.

a, b Rh-PDE structure superimpositions on the BR structure (a PDB ID: 1C3W) and the ChR2 structure (b PDB ID: 6EID). Rh-PDE, BR, and ChR2 are colored orange, blue and green, respectively. c–e Residues around the RSBs of BR (c), Rh-PDE (d), and ChR2 (e). Dashed lines represent hydrogen bonds. f–h Structures of the retinal binding pockets of BR (f), Rh-PDE (g), and ChR2 (h). i Schematic diagram of the ground and excited states. j Residues contributing to proton transfer in BR (left) and corresponding residues in Rh-PDE (right).

Fig. 3. Structure and function of TM0.

a Residues at the interface between TM0 and the rhodopsin domain. b Absorption spectrum with hydroxylamine bleach of the N-terminal truncated mutants. c Representative immunoblots indicating expression levels of bovine Rh (~35 kDa), Rh-PDE wt (~75 kDa), Rh-PDE Δ32 (~72 kDa), Rh-PDE Δ76 (~67 kDa), and Rh-PDE Δ88 (~65 kDa) in detergent solubilized fraction isolated from HEK293T cells and detected with the anti-Rho C-terminal 1D4-tag antibody. Source data are provided as a Source Data file. d Band intensities in (c) were quantified and calculated the relative expression level against to bovine Rh obtained with imageJ software (n = 1). e, f Photoactivities of truncated mutants with cAMP (e) and cGMP (f). Data are presented as mean values ± standard deviation (SD) (n = 6 independent experiments).

Fig. 4. Structures with the linker region.

a Overall structure of Rh-PDE TMD–Linker. The linker region is colored gray. b Overall structure of Rh-PDE Linker–PDE. The newly observed region, as compared to the previous structure (PDB ID: 5VYD), is shown as gray bold coils. Zinc and magnesium ions are colored gray and green, respectively. c Glu478 and the backbone of the newly observed region are shown as stick models. Dashed lines show hydrogen bonds. d Ribbon diagrams of the linker structure of Rh-PDE TMD–Linker. e Ribbon diagrams of the linker region structure, modeled with Rosetta. f Ribbon diagrams of the final structure from the aMDdual simulation.

Fig. 5. Photoactivation mechanism of Rh-PDE.

a HS-AFM images of the full-length Rh-PDE. b Ribbon diagrams of the full-length Rh-PDE model. One protomer is colored rainbow and the other is colored light orange. c Bulky residues between the intracellular side of TM7 and the retinal chromophore. d Relative activities of mutants, measured with a GloSensor assay. Data are presented as mean values ± standard deviation (SD) (n = 5 for I302A; n = 6 for the other, independent experiments). e, f Molecular surfaces of BR (e) and Rh-PDE (f), drawn without TM7. g Residues of ICL1 and ICL3 in Rh-PDE. h Photoactivities of the ICL3 mutant with cAMP (left) and cGMP (right). Data are presented as mean values ± standard deviation (SD) (n = 6 independent experiments).