Structural and functional dynamics of human cone cGMP-phosphodiesterase important for photopic vision

Abstract

Cone cGMP-phosphodiesterase (PDE6) is the key effector enzyme for daylight vision, and its properties are critical for shaping distinct physiology of cone photoreceptors. We determined the structures of human cone PDE6C in various liganded states by single-particle cryo-EM that reveal essential functional dynamics and adaptations of the enzyme. Our analysis exposed the dynamic nature of PDE6C association with its regulatory γ-subunit (Pγ) which allows openings of the catalytic pocket in the absence of phototransduction signaling, thereby controlling photoreceptor noise and sensitivity. We demonstrate evolutionarily recent adaptations of PDE6C stemming from residue substitutions in the Pγ subunit and the noncatalytic cGMP binding site and influencing the Pγ dynamics in holoPDE6C. Thus, our structural analysis sheds light on the previously unrecognized molecular evolution of the effector enzyme in cones that advances adaptation for photopic vision.

Keywords: cryo-EM; phosphodiesterase 6; photoreceptor; phototransduction.

Fig. 1.

Cryo-EM structures of the complexes of PDE6C with Pγ_r and Pγ_c in the presence of cGMP. (A) The final cryo-EM map for the (PDE6C)₂•(Pγ_r)₂ complex. The densities corresponding to the PDE6C subunits are colored in salmon and blue, whereas the densities for the Pγ_r subunits are colored in green and wheat. Pt–pony-tail motif. (B) The structure of the (PDE6C)₂•(Pγ_r)₂ complex modeled into cryo-EM map is shown in cartoon_. Dashed lines delimit Pt-motif, the GAF-A, GAF-B, and catalytic domains (PDB 9CXH). The linker helices connecting GAF-A with the GAF-B domains (LH1) and GAF-B with the catalytic domains (LH2) are indicated by arrows. cGMP bound to the GAF-A domains and 5′-GMP bound to the catalytic pockets are shown as spheres colored by element. Zn²⁺ and Mg²⁺ are shown as gray and magenta spheres, respectively. (C and D) The final cryo-EM map (C) for the (PDE6C)₂•(Pγ_c)₂ complex and the structure modeled into cryo-EM map shown in cartoon (D) (PDB 9CXG). The color scheme as in (A) and (B). (E) Superimposition of the structures of the (PDE6C)₂•(Pγ_r)₂ (9CXH) and (PDE6C)₂•(Pγ_c)₂ (9CXG) complexes. PDE6C chains are colored green and cyan (9CXH) and wheat and gray (9CXG). Pγ_r chains are colored orange and pink, Pγ_c chains are colored magenta and hot pink. cCMP and 5′-GMP are shown as blue spheres.

Fig. 2.

(A) Deficient interaction of the Pγ subunit with the GAF-A of PDE6C. The GAF-A/GAF-B domains and the Pγ subunits (magenta and yellow) are from the structure of the (PDE6C)₂•(Pγ_c)₂ complex (PDB 9CXG). The first modeled residue of Pγ_c K21 points away from the GAF-B and GAF-A domains. Pγ_r assumes similar conformation in the structure of the (PDE6C)₂•(Pγ_r)₂ complex (PDB 9CXH). Pγ_r from the structure of rod PDE6 (blue) is superimposed from PDB 6MZB. Residue Pγ_rR15 (sticks) essential for Pγ_r binding the GAF-A domains of PDE6AB, is not conserved in Pγ_c. Pγ would clash with PDE6C K132 and not be able to interact with the PDE6C GAF-A domain as it does with the GAF-A domain of rod PDE6. (B) Key contacts of 5′-GMP in the catalytic pocket (PDB 9CXH). The guanine ring makes hydrogen bonds with the side chain of conserved Q776, which serves as a key selectivity determinant. The two divalent ions in the active site critical for cGMP hydrolysis, Zn²⁺ and Mg²⁺, are coordinated by conserved H and D residues. The electron density for ligand is shown from a 2.8 Å map after homogeneous refinement with C2 symmetry (SI Appendix, Fig. S2).

Fig. 3.

Disorganization of the PDE6C GAF-A domains in the absence of noncatalytic cGMP. (A and B) Maps and structures of the (PDE6C)₂•(Pγ_r)₂ complex were obtained based on two major particle subsets. (A) The first map based on 83% of particles features all three structural domains: GAF-A, GAF-B, and the catalytic domain. Approximately 50 N-terminal residues of the Pt motif are not modeled due to poor density. cGMP bound to the GAF-A domains is shown as spheres. Zn²⁺ and Mg²⁺ in the catalytic pocket are shown as gray and magenta spheres, respectively. There is no ligand in the catalytic pocket. (B) The second map based on 17% of particles lacks the density for the GAF-A (230 N-terminal residues). The coloring scheme is as in Fig. 1.

Fig. 4.

Conformational heterogeneity and dynamics of Pγ in the complex with PDE6C. (A) Map for the (PDE6C)₂•(Pγ_r)₂ complex in the presence of cGMP with a single open catalytic site. The map reconstruction is based on the ~20% particles used in the processing of the final map and selected from the 3DVA. (B) Map for the (PDE6C)₂•(Pγ_c)₂ complex in the presence of cGMP with a single open catalytic site. The map reconstruction is based on the ~28% particles used in the processing of the final map and selected from the 3DVA. (A and B) The densities corresponding to the PDE6C subunits are colored in salmon and blue, whereas the densities for the Pγ_r and Pγ_c subunits are colored in green and wheat. The Pγ subunits are superimposed from the final models of the complexes. The sites with the Pγ C-terminus shown in the green cartoon lack corresponding density. (C) Clustal Omega multiple sequence alignment of the C termini of Pγ subunits. Cone Pγ subunits: cLam, lamprey A3RLS1; cD.r., Danio rerio NP_957079; cHum, human Q13956; cBov, bovine P22571; cMus, murine NP_076387; cGal.g, chicken AAO67734; cXen.l, Xenopus laevis AAH74358. Rod Pγ subunits: rLam, lamprey A4LAP0; rD.r., Danio rerio NP_997964; rHum, human P18545; rBov, bovine P04972; rMus, murine P09174; rGal.g, chicken NP_989776; rXen.l, Xenopus laevis AAH74367. The Y/F substitution in cone Pγ subunits (except in fish) indicated by red arrow. (D) The Y/F substitution disrupts hydrogen bonding between Y84 hydroxyl group and backbone atoms of PDE6C. Interactions between rod PγY84 and backbone atoms of PDE6C (PDB 9CXH). Similar interactions present between PγY84 and rod PDE6AB (PDB 6MZB). Distances between the hydroxyl group of Y84, and backbone oxygen of G778, backbone nitrogen of F779 and backbone oxygen of L775 are 3.3 Å each. F779 side chain is involved in π–π stacking (4 Å) with the guanosine group of 5′-GMP. For clarity, only the side chain of F779 is shown. The backbone of PDE6C residues 774 to 782 is shown as sticks, and the rest of the PDE6C molecule is shown as cartoon with transparency set to 0.7. (E) Inhibition of PDE6 by the C-terminal peptides of rod and cone Pγ. Activity of tPDE6 from mouse retina was measured in the presence of increasing concentrations of Pγ_r -63-87 or Pγ_c-59-83. Representative experiment is shown. From three similar experiments (Mean ± SD), K_i = 2.5 ± 0.4 μM for Pγ_r-63-87, maximal inhibition 100%; K_i = 8.2 ± 2.4 μM for Pγ_c-59-83, maximal inhibition 95%. t test P = 0.015.

Fig. 5.

cGMP-binding by PDE6C GAF-A domain. (A) In PDE6C proteins of higher vertebrates, L (human PDE6C-L115), is localized at the opening of the GAF-A cGMP-binding pocket (PDB 9CXG). Rod PDE6 isoforms contain F at this position. The bulky side chain of PDE6A-F113 points into the binding pocket making π–π stacking interaction with the guanine ring. The side chain of F113 is superimposed from PDB 6MZB. (B) The rate of dissociation of cGMP bound to chicken PDE6C-42-458 was measured following the protein preincubation with [³H]cGMP and cold chase with an excess of unlabeled cGMP by the filter binding assay. For the WT+Pγ_c samples, 50 µM Pγ_c was added prior to the addition of unlabeled cGMP. The k_off values from the exponential decay fits to the data are 0.015 ± 0.001 min⁻¹ for PDE6C-42-458 and 0.002 ± 0.0003 min⁻¹ for the L/F mutant, 0.012 ± 0.002 min⁻¹ for PDE6C-42-458 plus 50 µM Pγ_c (Mean ± SD, n = 3). t test: WT vs. L/F P < 0.0001; WT vs. WT+ Pγ_c P = 0.017. (C) The steady state binding curves as determined using BLI for MBP-Pγ_c binding to PDE6C-42-458 in the presence or absence of cGMP. The K_D values (Mean ± SD, n = 3): K_D 36.1 ± 8.3 µM; +cGMP, K_D 10.0 ± 1.9 µM. t test P = 0.007.