Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta

Abstract

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 k_cat. A truncation consisting solely of the phosphodiesterase domain is also active with a k_cat 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.

Figure 1

Predicted domain structure and transmembrane topology for RhoPDE. Domain nomenclature: N-term, N-terminal domain; Rho, microbial type I rhodopsin domain; linker, sequence connecting the Rho and PDE domains; PDE, phosphodiesterase domain; and C-term, short C-terminal domain. Transmembrane helices were predicted and drawn with Protter (wlab.ethz.ch/protter/start). Amino acids are colored: green, Lys296 (numbering of the native protein), the presumed covalent attachment site for the retinal chromophore; blue, the C8 epitope tag; and orange, the 1D4 epitope tag.

Figure 2

SDS–PAGE and Western blot analysis of fractions from immunoaffinity purification of RhoPDE from HEK293 cells. (A) Coomassie-stained gel and (B) C8-probed Western blot of C8-purified RhoPDE: lane M, molecular mass markers in kilodaltons; lane L, load, postnuclear supernatant fraction; lane FT, flow-through, not bound to the C8 column; lane W, final wash before the first elution with the C8 peptide; lane E1, first elution with the C8 peptide; lane E2, second elution with the C8 peptide. (C) C8-probed Western blot of tandem C8- and 1D4-purified RhoPDE. Lanes from left to right: lane M, molecular mass markers; lane L, load, postnuclear supernatant fraction; lane FT, flow-through, not bound to the C8 column; lane W, final wash before the first elution with the C8 peptide; lane E1, first elution with the C8 peptide; lane E2, second elution with the C8 peptide; lane E3, third elution with the C8 peptide; lane FT, flow-through, nonbound fraction from E1–E3 from the C8 column applied to the 1D4 column; lane W, final wash before the first elution with the 1D4 peptide; lane E1, first elution with the 1D4 peptide; lane E2, second elution with the 1D4 peptide. Note that these experiments were optimized to yield a high concentration of RhoPDE in the final eluate and not for a high yield of protein from the HEK293 cells. Thus, the C8 column was overloaded in these examples where excess RhoPDE can be observed in Western blots of the FT lanes. In addition, it must be kept in mind that the Western blots in this figure are significantly overloaded with protein levels well outside of the linear detection range (the same amount of material was loaded on the Western blots as was loaded on the Coomassie-stained gel). This can be seen most readily from the truncated Rho domain (migrating at approximately 35–40 kDa) observed in both Western blots of panels B and C, but not visible in the Coomassie-stained gel in panel A.

Figure 3

UV–vis absorption spectra of purified RhoPDE from HEK293 cells. (A) Spectrum of the C8-purified pigment with ATR in the dark state. (B) Expanded visible region of the dark state spectrum in panel A. (C) Visible region spectrum of the 1D4-purified pigment with ATR in the dark state. (D) Spectrum of the tandem C8- and 1D4-purified pigment with ATR in the dark state. (E) Visible region of the spectrum in panel A before (dotted line) and after (solid line) a 30 s exposure to room lights.

Figure 4

Determination of the RhoPDE transmembrane topology and orientation. Immobilized COS-7 cells were transfected with the dual (C8 and 1D4) epitope-tagged RhoPDE on glass coverslips and incubated with the antibodies under permeabilized or unpermeabilized conditions for immunofluorescent staining, as indicated in the figure and described in Experimental Procedures. Panels A and B show intact and detergent-permeabilized cells, respectively, probed with the 1D4 antibody. Panels C and D show intact and detergent-permeabilized cells, respectively, probed with the C8 antibody. Fluorescence from either probe is observed only in permeabilized cells, indicating both epitopes are intracellular. The COS-7 cells in panels B and D show a diffuse staining pattern typical of proteins localized to the plasma membrane.

Figure 5

Phosphodiesterase activity of RhoPDE from transiently transfected HEK293-GnT1⁻ cells. The reaction mixture contained RhoPDE in membranes isolated from transfected cells or in liposomes reconstituted with protein that had been purified from transfected cells. The RhoPDE used in this figure contained only the C-terminal 1D4 tag (i.e., no C8 tag). The formation of 5′-GMP from cGMP (or 5′-AMP from cAMP) was followed by HPLC: (●) reaction in the dark and (○) reaction in the light. Integrated peaks corresponding to GMP (or AMP) at each time point were converted into concentrations with a standard curve and plotted vs time. Panels A and B show two representative reactions with RhoPDE (50 nM) in HEK293 membranes reconstituted with ATR. (A) k_cat = 19.6 ± 0.2 s⁻¹; (B) k_cat = 25.4 ± 0.2 s⁻¹. (C) Reaction with RhoPDE (50 nM) in HEK293 membranes not reconstituted with ATR. This reaction was performed in the absence of light from the 300 W tungsten source. k_cat = 21.3 ± 0.2 s⁻¹. (D) cGMP phosphodiesterase actvity of RhoPDE (50 nM) in reconstituted liposomes. k_cat = 27.7 ± 0.2 s⁻¹. (E) cAMP phosphodiesterase actvity of RhoPDE (100 nM) in reconstituted liposomes. k_cat = 3.7 ± 0.1 s⁻¹.

Figure 6

Purification and assay of RhoPDE from HEK293 cells grown in the presence of ATR. (A) Absorption spectra of RhoPDE after purification (C8 antibody column) from HEK293 membranes. The figure compares the yield of RhoPDE from cells grown in the presence and absence of ATR: yellow, cells grown in the presence of ATR; and blue, cells grown in the absence of ATR. Membranes from cells grown in the absence of ATR were treated with retinal before detergent solubilization. No ATR was added to membranes from cells grown in the presence of ATR. (B) cGMP phosphodiesterase activity of RhoPDE in membranes isolated from cells grown in the presence of ATR. No additional ATR was added. The assay buffer for this reaction consisted of 50 mM HEPES (pH 6.5), 50 mM NaCl, 10 mM MgCl₂, and 0.5 mM EDTA.

Figure 7

Phosphodiesterase activity of the isolated PDE domain purified from transformed E. coli T7 Express cells. (A) Coomassie-stained SDS–PAGE gel showing fractions from purification of the isolated PDE domain from transformed E. coli T7 Express cells using a six-His tag with a Ni affinity column: lane M, molecular mass markers in kilodaltons; lane P, pellet from the cell extract; lane S, soluble fraction from the cell extract; lane FT, flow-through, nonbound fraction from the Ni affinity column; lane W, last wash before elution with an imidazole gradient; and lanes E1–E3, three representative fractions from the imidazole gradient eluate. (B) AKTA FPLC profile for size exclusion chromatography of the isolated PDE domain on a Superdex-200 10/300 GL column. Molecular mass standards: (1) blue dextran, 2000 kDa, 8.5 mL (void volume); (2) aldolase, 158 kDa, 13.15 mL; (3) albumin, 67 kDa, 14.57 mL; (4) ovalbumin, 43 kDa, 15.41 mL; and (5) chymotrypsinogen, 25 kDa, 17.35 mL. The isolated PDE domain elutes at 14.5 mL, giving a mass of 74 kDa, consistent with a homodimer. (C) HPLC assay of the cGMP phosphodiesterase activity of the isolated PDE domain. The concentration of PDE in this assay was 100 nM, which gives an apparent k_cat of 3.13 ± 0.12 s⁻¹.

Figure 8

Structure of the PDE domain homodimer. The figure shows a ribbon diagram of molecules A and B of the dimer, colored green and cyan, respectively. Zn²⁺ and Mg²⁺ ions are modeled as purple and green spheres, respectively. N- and C-termini are marked.

Figure 9

Active site of PDE monomer A, showing metal ion coordination, water molecules, and hydrogen bonds to active site residues. The figure shows an omit map (F_o − F_c = 3σ) with electron density for the Zn²⁺ and Mg²⁺ ions in the active site of monomer A. The water molecules and Zn²⁺ and Mg²⁺ ions are modeled as red, purple, and green spheres, respectively.

Figure 10

Superposition of PDE and PDE9 (PDB entry 2HD1) dimers using monomer A as a reference molecule. Monomers A and B of PDE are colored green and cyan, respectively. Both monomers of PDE9 are colored salmon. Zn²⁺ (purple) and Mg²⁺ (green) ions are modeled as spheres. Helices of monomer A are labeled according to the PDE9 model.