Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

Abstract

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

Fig. 1. The cryo-EM structure of PDE6αβ2γ ternary complex at 3.4-Å resolution.

(A) Overall cryo-EM 3D reconstruction of the PDE6αβ2γ complex displaying the 34-Å-long N-terminal pony-tail domain (Pt motif) (top) and the local resolution estimation map (bottom). Resolution keys are labeled from 3.2 (blue) to 8.0 Å (red). (B) Four orthogonal views of the cryo-EM structure of PDE6αβ2γ complex displaying PDE6α (purple), PDE6β (green cyan), and two molecules of PDE6γ (red) subunits. (C) Structures of PDE6α (top, 10 to 822) and PDE6β (bottom, 11 to 822) in two orthogonal views shown in cartoon representation. Scale bar, 30 Å.

Fig. 2. Structural features of the PDE6αβ2γ complex.

(A) Structure of the PDE6αβ heterodimer showing the domain distribution within PDE6α (purple) and PDE6β (green cyan) subunits where regulatory GAF domains are trailed by the phosphohydrolase catalytic domain in a trilobed organization. One molecule of PDE6γ (red ribbon) wraps around PDE6α and PDE6β individually for a tighter regulation of PDE6 activity. One molecule of cGMP (red spheres) is bound to each of the GAF-A domains of the PDE6αβ heterodimer. (B) Zoomed-in view of the GAF-A cGMP-binding pocket showing the orientation of the cGMP molecule (red sticks) with respect to the surrounding secondary structures. The electron density corresponding to the cGMP molecule was calculated after the final refinement and is displayed as gray mesh. (C) The cGMP-bound GAF-A domains of both PDE6αβ2γ complex (left) and PDE6C (gray, right) feature a salt bridge between the partial negative charge of Asn¹¹⁴ carbonyl oxygen and the guanidine group of Arg⁹³. Amino acid residue Asn¹¹⁴ provides important cGMP-specific protein-ligand hydrogen bonds that stabilize cGMP binding. (D) Topology diagram showing the secondary structure elements of the Pt motif (gray), GAF-A (red), GAF-B (purple), and catalytic domain (green). (E) Zoomed-in view of the cGMP-binding pocket (red mesh) of PDE6α GAF-A domain. The N terminus of PDE6γ (red cartoon) forms a lid over the buried cGMP molecule and thereby prevents its release from the GAF-A domain until a threshold concentration gradient of cGMP has been reached in the cell. The electron density of the N terminus of PDE6γ after the final refinement is displayed as gray mesh. (F) Interaction interface between the GAF-A β1-β2 loop and the GAF-B domain of PDE6α. PDE6γ (red) staples the GAF-A β1-β2 loop interaction to the GAF-B domain and likely senses changes associated with cGMP binding in the GAF-A domain. The refined electron density around the GAF-A β1-β2 loop is displayed as gray mesh. (G) The interface between GAF-B β1-β2 loop of PDE6α and the catalytic domain of PDE6β features a short two-turn helical structure that forms hydrophobic interactions with α6, α7, α8, and α9 helices of the catalytic domain. The refined electron density corresponding to the key amino acid residues is displayed as gray mesh.

Fig. 3. The PDE6γ subunit stabilizes the open-state conformation of PDE6αβ heterodimer.

(A) Cryo-EM density (left, red surface) and partial de novo model (right, red ribbon) of PDE6γ around the PDE6αβ heterodimer (PDE6α, purple; PDE6β, green cyan). One molecule of PDE6γ wraps around PDE6α and PDE6β individually for a tighter regulation of PDE6 activity. (B) Local resolution estimation map (left) and partial de novo model (right) of PDE6γ showing a good correlation between the 3D map and the refined PDE6γ model. The density of PDE6γ after the final refinement is displayed as gray mesh. (C) Comparison between the catalytic domains of PDE6α (purple), closed-state PDE2A (pink, PDBID: 3IBJ), and inhibitor-bound PDE2A (orange, PDB ID: 4D08) displaying different orientations of the H-loop (606 to 629, left) and M-loop (745 to 767, right) regions. The catalytic domain of closed-state PDE2A shows the H-loop folded into the catalytic site close to the substrate-binding pocket (black mesh). In contrast, the catalytic domain of PDE6α features an open H-loop similar to the inhibitor-bound PDE2A crystal structure. The M-loop region of PDE6α (purple) shows a similar conformation as the inhibitor-bound PDE2A structure (orange). Notably, residues 840 to 850 of the M-loop of the closed-state PDE2A crystal structure (pink) are disordered. (D) The C terminus of PDE6γ (red cartoon) binds near the substrate-binding pocket of the PDE6 catalytic domain. PDE6γ binding to the catalytic domain of PDE6 mimics a substrate/inhibitor-bound form where the H-loop displays an open conformation and the substrate-binding pocket is occluded by the C terminus of PDE6γ. Structurally conserved regions of the catalytic domain (RMSD ≤ 0.2 Å between PDE6α and PDE2A) are shown in gray.

Fig. 4. The β1-β2 loop of regulatory GAF domains as potential allosteric triggers to PDE allosteric regulation.

(A) Comparison between overall structures of the closed-state PDE2A homodimer and the PDE6αβ heterodimer. M-loop regions of the closed-state PDE2A and the PDE6αβ heterodimer are shown in gold. (B) Schematic representation showing the extent of potential conformational changes that occur during PDE activation. These conformational changes include a downward movement of the GAF-A β1-β2 loop, a 10° inward twist of the LH1 coiled coil, an 80° outward twist of the GAF-B β1-β2 loop, and an 80° rotation of the catalytic domains. M-loop regions are shown in gold. (C) A comparison between the 2D class averages of the untreated PDE6αβ2γ complex and PDE6αβ2γ treated with sildenafil recapitulates some of the key structural differences illustrated in (B). The face views of PDE6αβ2γ treated with sildenafil feature changes in the interaction profile of the GAF-B β1-β2 loop with the catalytic domain of the PDE6αβ heterodimeric core. The GAF-B β1-β2 loops are denoted by asterisks. Scale bar, 60 Å.