Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC

Abstract

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCC_Rho) and without (GC_Rho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GC_Rho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl₂, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GC_Rho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Keywords: Blastocladiella emersonii; class III nucleotidyl cyclase; crystal structure; cyclic GMP (cGMP); disulfide; guanylate cyclase (guanylyl cyclase); optogenetics; rhodopsin.

Figure 1.

Model for the transmembrane topology and orientation of RhoGC. Domains are: N-term, N-terminal domain; Rho, microbial type I rhodopsin domain; CC, coiled-coil domain; and GC, guanylyl cyclase domain. Transmembrane helices were predicted and drawn by Protter (wlab.ethz.ch/protter/start (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site)) (34). Cyan, N-term; green, Rho; purple, CC; yellow, GC; blue square, Lys-384, presumed site of covalent attachment to the chromophore; and red circles, Glu-497 and Cys-566, residues controlling specificity for the GTP substrate.

Figure 2.

Purification and initial characterization of the isolated GCwCC_Rho and GC_Rho domains. A, purification of GCwCC_Rho and GC_Rho. Left panel, Coomassie-stained SDS-polyacrylamide gel showing fractions for purification of the isolated GCwCC_Rho domain from transformed E. coli BL21(DE3)-pGro7 cells (co-expressing GroEL/ES) using nickel-affinity chromatography. Right panel, Coomassie-stained SDS-polyacrylamide gel showing fractions from purification of GC_Rho. The last lane contains purified GCwCC_Rho for size comparison. B, initial rate data for guanylyl cyclase activity from GC_Rho and GCwCC_Rho in the presence of MnCl₂ (left panel) or MgCl₂ (right panel). Reactions were performed as described under “Experimental procedures” and contained 10 μm enzyme, 5 mm GTP, and 10 mm divalent cation. C, Äkta FPLC profiles for size-exclusion chromatography of GC_Rho (solid line) and GCwCC_Rho (dashed line) on a Superdex-200 10/300 GL column in 25 mm HEPES buffer, pH 7.0, containing 100 mm NaCl. Both proteins were loaded onto the column at a concentration of 200 μm. 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; 5, chymotrypsinogen-A, 25 kDa, 16.8 ml; and 6, ribonuclease-A, 14 kDa, 18 ml. Both GC_Rho and GCwCC_Rho elute as monomers under these conditions.

Figure 3.

Overall structure of the GC_Rho monomer. Secondary structure elements are labeled according to conventional nomenclature and are color-coded: red, α-helix; yellow, β-strand; and green, loop.

Figure 4.

Formation of GC_Rho dimers. A, normalized c(s) distribution plot for AUC of GC_Rho in the absence (black) and presence (red) of 1 mm MnCl₂ and 60 μm ddGTP (ddGTP equimolar with GC_Rho). B, SEC profiles for GC_Rho in the absence (solid line) and presence (dotted line) of MnCl₂ and ddGTP. Solid line, data for GC_Rho in the absence of MnCl₂ and ddGTP are from Fig. 2C. Dotted line, data from protein that was subjected to the same conditions as for the AUC sample in A containing MnCl₂ and ddGTP. Superdex-200 column was run in 25 mm HEPES, pH 7.0, containing 100 mm NaCl at 4 °C with a flow rate of 0.5 ml/min.

Figure 5.

Structure of the GC_Rho homodimer. A, overall structure of the GC_Rho homodimer. Schematic representations of molecule A and B are colored green and gold, respectively. Selected secondary structure elements are labeled according to convention. Tartrate is modeled as sticks and shown in yellow. Manganese ions are shown as green spheres. Cysteine residues are shown as sticks and colored according to the respective main chain coloring. B, structural superposition of the GC homodimer with the “active conformation” of the human AC heterodimer (PDB entry 1CJU). Schematic representations are color-coded as follows: GC_Rho molecule A, green; GC_Rho molecule B, gold; and human AC molecules A and B, blue. N- and C-terminal amino acids are marked and color-coded according to the coloring of the respective molecule. C, disulfide cross-link at the dimer interface. F_o − F_c omit map is contoured at 3 σ cutoff. Parts of the protein backbone have been omitted for clarity. D, tartrate-binding site at the dimer interface. F_o − F_c omit map is contoured at 4 σ cutoff. Metal ion coordination and hydrogen bonding are indicated with dotted lines. Parts of the protein backbone and additional solvent molecules have been omitted for clarity.

Figure 6.

Analysis of disulfide cross-linking by SDS-PAGE. Protein samples (GC_Rho or GCwCC_Rho) were incubated for 20 h under the conditions indicated in the figure before being loaded onto 10% polyacrylamide gels so as to simulate the AUC conditions in Fig. 4A. All samples were prepared in 25 mm HEPES, pH 7.0, 100 mm NaCl using either 1 μm GC_Rho or GCwCC_Rho. Top panels, GC_Rho; bottom panels, GCwCC_Rho; left panels, without DTT; and right panels, with 5 mm DTT. Other conditions are as indicated in the figure: Mn, 2 mm MnCl₂; Hi Mn, 20 mm MnCl₂; Mg, 10 mm MgCl_2; cGMP, 1 mm cGMP; GTP, 1 mm GTP; and PPi, 1 mm pyrophosphate.

Figure 7.

GC_AC activity data and omit map. A, initial rate data for catalytic activity of GC_AC when provided ATP (black circles) or GTP (red circles) as a substrate. The reactions contained 5 mm substrate (GTP or ATP) and 10 mm MnCl₂. B, F_o − F_c omit map contoured at 3σ cutoff shows electron density for the two mutated active site residues Lys-497 and Asp-566 in GC_AC. Secondary structure elements are colored green and labeled according to convention. Amino acid side chains are shown as sticks. Hydrogen bonding is indicated by the dotted line. Part of the protein backbone has been removed for clarity.

Figure 8.

Guanylyl cyclase activity as a function of enzyme concentration. A, rate of cGMP formation as determined by the HPLC assay is plotted as a function of total enzyme concentration showing a non-linear dependence that is well fit by Equation 1 under “Experimental procedures” relating initial rate to a monomer/dimer equilibrium model in which only the dimer is active. The construct and condition for each set of data are as indicated in the figure. B, data for GC_Rho with MgCl₂ are re-plotted from A on a more sensitive scale to show clearly the non-linear dependence on enzyme concentration. All reactions contained 10 mm GTP and 20 mm metal ion (Mn²⁺ or Mg²⁺). The dotted curves are fits of Equation 1 to the data using the parameters of K_D and k_cat listed in Table 1. The enzyme concentration is total enzyme added to the reaction, expressed in terms of the micromolar concentration of monomer. An expanded view of the data for GCwCC_Rho-Mn is shown in Fig. S3A. A control experiment showing that the non-linear behavior is not a result of non-specific protein interactions at low concentrations is shown in Fig. S3B.