RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5–7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6–9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution. To test for the existence of such a constraint, we asked whether it were possible to relocate the highly conserved Lys296 in the visual pigment bovine rhodopsin. We show here that the Lys can be moved to three other locations in the protein while maintaining the ability to form a pigment with 11-cis-retinal and activate the G protein transducin in a light-dependent manner. These results contradict the convergent hypothesis and support the homology of type I and type II rhodopsins by divergent evolution from a common ancestral protein.T he retinylidene proteins are integral membrane proteins that covalently bind a retinal chromophore. Amino acid sequence comparison divides these proteins into two families known as type I and type II rhodopsins (1). Type I rhodopsins, such as bacteriorhodopsin from the archaeon Halobacterium salinarum, function as light-driven ion transporters, channels, and phototaxis receptors. Type II rhodopsins, best known for the visual pigment of mammalian rod photoreceptor cells, function primarily as photosensitive receptor proteins in metazoan eyes and in certain extraocular tissues. Henceforth, we will use the term "rhodopsin" to refer to the visual pigment of bovine rod photoreceptor cells and "bacteriorhodopsin" to refer to the lightdriven proton pump of H. salinarum.Rhodopsin is a prototypical member of the large family of G protein-coupled receptors (GPCRs; specifically class A GPCRs) (2). It is composed of an apoprotein (called "opsin") and an 11-cis-retinal chromophore, resulting in a pigment with λ max = 500 nm. The GPCR fold comprises seven transmembrane α-helices oriented in a particular spatial arrangement with a specific connectivity (SCOP classification scop.b.g.c.A; ref. 3). In rhodopsin, the N terminus resides in the intradiscal (i.e., extracellular) space and the C terminus in the cytoplasm. The 11-cisretinal chromophore is covalently attached to the protein by means of a protonated Schiff base to the e-amino group of Lys296 in the seventh helix. The GPCR fold and active-site Lys are absolutely conserved among all visual pigments of higher eukaryotes.Upon absorption of light, the 11-cis-retinal isomerizes to the all-trans form. The protein responds with a conformational change leading to an enzymatic cascade that begins with activation of the G protein transducin and ends with closure of cation channels in the plasma membrane and hyperpolarization of the rod cell (4). A key intermediate in the photoactivation of rhodopsin is the species metarhodopsin II (MII) (5). MII is the only intermediate capable of activating transducin and is...

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λ = 390 nm; D380N, λ = 506 nm) and one with red-shifted (D380E, λ = 533 nm) absorption maxima relative to the wild-type protein (λ = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. While this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [Devine et al. (2013) Proc. Natl. Acad. Sci. USA 110, 13351–13355]. Since the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.