SCIENCE ADVANCES | RESEARCH ARTICLE
BIOCHEMISTRY Copyright © 2019 The Authors, some rights reserved; Cryo-EM structure of phosphodiesterase 6 reveals exclusive licensee American Association insights into the allosteric regulation of for the Advancement of Science. No claim to type I phosphodiesterases original U.S. Government Sahil Gulati1,2,3, Krzysztof Palczewski1,2,3*, Andreas Engel4, Works. Distributed 4 4 under a Creative Henning Stahlberg *, Lubomir Kovacik Commons Attribution NonCommercial Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate License 4.0 (CC BY-NC). 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 PDE62 complex. The final density map resolved at 3.4 Å reveals several previously unseen Downloaded from structural features, including a coiled N-terminal domain and the interface of PDE6 subunits with the PDE6 heterodimer. Comparison of the PDE62 complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.
INTRODUCTION
vation by transducin (12), PDE6 is crucial for the visual phototrans- http://advances.sciencemag.org/ The phosphodiesterase (PDE) family displays a conserved catalytic duction. Rod photoreceptor PDE6 is an atypical phosphohydrolase phosphohydrolase domain, whose activity is controlled by diverse that consists of a heterodimeric catalytic core composed of PDE6 domain structures and regulatory mechanisms (1, 2) (figs. S1 and S2). and PDE6 subunits (PDE6 heterodimer) and two inhibitory Because of their association with various pathologies and distinct PDE6 subunits (11). All other members of type I PDE harbor cellular and subcellular distributions, PDEs are targets of several a homodimeric catalytic core, including PDE6 from cone photo- widely used drugs and remain a major target for drug development receptors that is composed of two ′ subunits (13). All PDE6 (3). Most structure-activity relationship studies have so far focused catalytic subunits contain two regulatory N-terminal GAF domains on their conserved catalytic domains that share 25 to 52% sequence (GAF-A and GAF-B) and a C-terminal catalytic domain (14). GAF homology among type I PDE family members. As a result, most domains and the tightly bound inhibitory PDE6 subunits regulate PDE inhibitors display a substantial degree of cross-reactivity within the activity of catalytic domain allosterically (15). Binding of the PDE family (4, 5) and other related enzymes (6). In particular, cGMP to the GAF-A domain of PDE6 increases affinity for the on February 13, 2020 inhibitors of PDE5, including sildenafil and vardenafil, are widely PDE6 subunit that inhibits the catalytic activity of the PDE6 used for the treatment of erectile dysfunction and pulmonary hyper- heterodimer when bound. Reciprocally, the binding of PDE6 to tension (7). However, PDE5 inhibitors have been associated with the PDE6 heterodimer catalytic subunits enhances the affinity of several ocular side effects, including blurred vision, changes in color cGMP for noncatalytic sites, which, in turn, increases the affinity of vision, transient alterations in the electroretinogram, conjunctival the catalytic domains for PDE6 (15–17). During phototransduction, hyperemia, ocular pain, photophobia, and, in extreme cases, dam- activated transducin [Gt-GTP (guanosine 5′-triphosphate)] re- age to the optic nerve (8). These secondary effects are mediated duces the inhibitory constraint of the PDE6 C-terminal region by the binding of PDE5 inhibitors to a closely related phospho- on the catalytic site of PDE6 (16), resulting in a reduced binding hydrolase, PDE6 (9), with about 10% of the inhibitory effect as affinity of cGMP to one of the two GAF-A domains and the release of compared to PDE5 (10). PDE6 is an essential component of vi- cGMP. Gt-GTP has been previously shown to bind amino acid sual phototransduction that catalyzes the hydrolysis of guanosine residues 24 to 45 (18–21), 54 to 55 (22), and 55 to 62 (22) and the 3′,5′-monophosphate (cGMP) to GMP in response to light (9, 11). C terminus of PDE6 (23). Among these interaction sites, the The reduction in cGMP concentration results in the closure of Na+ glycine-rich region of PDE6 (residues 55 to 62) has been implicated 2+ and Ca ion channels in the photoreceptor plasma membrane, in facilitating the interaction of Gt-GTP with the N-terminal leading to their hyperpolarization. Because of its catalytic efficiency polycationic region of PDE6 (18–22). operating close to the diffusion limit of cGMP and a ~100-fold acti- The absence of a detailed understanding of the allosteric modu- lation of PDE isoenzymes has limited the commercial success of PDE inhibitors due to cross-reactivity within the PDE family. Sev- 1 Gavin Herbert Eye Institute and the Department of Ophthalmology, University of eral high-resolution structures of individual domains of PDE isoen- California, Irvine, 829 Health Sciences Road, Irvine, CA 92617, USA. 2Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid zymes, including PDE1, PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, Avenue, Cleveland, OH 44106, USA. 3Cleveland Center for Membrane and Structural PDE8, PDE9, and PDE10, are available (fig. S1). A crystal structure of Biology, Case Western Reserve University, 1819 East 101st Street, Cleveland, OH 4 near full-length PDE2A that includes both GAF and catalytic domains 44106, USA. Center for Cellular Imaging and NanoAnalytics, Biozentrum, University was reported previously (24). In addition, several low-resolution of Basel, Mattenstrasse 26, 4058 Basel, Switzerland. *Corresponding author. Email: [email protected] (K.P.); [email protected] cryo–electron microscopy (cryo-EM) structures of PDE6 that de- (H.S.) scribe the overall architecture of the isoenzyme have been reported
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 1 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
(14, 25, 26). Despite the wealth of knowledge that these partial interface as well as 3 and 4 helices on the distal side (Fig. 2A and structures have provided, the structural relationships between the fig. S4A). In comparison to previously determined structures of regulatory domains and the catalytic domain of PDE family mem- GAF-A domains, a substantial structural change is observed in bers remain elusive. the 14–amino acid–long loop that connects the 1 with the 2 strand (1-2 loop) of the GAF-A six-stranded -sheet cluster. The GAF-A 1-2 loop directly interacts with the 4 strand and the 6-5 loop RESULTS of the GAF-B regulatory domain, suggesting a potential allosteric To gain structural insights into this allosteric mechanism, we puri- signal transduction route from the GAF-A to GAF-B domain upon fied native bovine PDE6 from rod outer segments (ROS) and used cGMP binding (Fig. 2F). cryo-EM to determine the structure of the PDE62 complex. The The refined cryo-EM map shows one molecule of cGMP bound structure of the PDE62 complex resolved at 3.4-Å resolution to the GAF-A domain of both PDE6 and PDE6 (Figs. 2, A and B, shows a linear organization of the three domains (GAF-A, GAF-B, and 3A). The 3′,5′-cyclic phosphate group of cGMP points toward and the catalytic phosphohydrolase domain, respectively) connected the 3 and 4 helices, whereas its guanine ring makes several well- by long helices in both PDE6 and PDE6 subunits (Fig. 1). The defined contacts with the 1 and 2 strands (Fig. 2B and fig. S4C). overall structure of PDE6 shows a trilobed architecture with each lobe The buried cGMP molecule makes several electrostatic contacts with corresponding to the GAF-A, GAF-B, and phosphohydrolase cata- the side chains of Ser95, Asn114, Val166, and Thr174 in addition to lytic domains of the PDE6 heterodimer (Figs. 1 and 2, A and D). hydrophobic contacts with Phe113, Phe134, Val140, Tyr172, and M193 Downloaded from The PDE6 heterodimer attains a pseudo-twofold symmetry where that determine its orientation in the cGMP binding pocket (Fig. 2B the three domains of PDE6 and PDE6 are organized in a head-to- and fig. S4C). As observed in the crystal structure of cGMP-bound head arrangement with a dimension of 154 Å by 115 Å by 74 Å. The PDE6C GAF-A domain (28), the GAF-A domain of PDE62 fea- heterodimeric interface of the PDE6 heterodimer (~5036 Å2) ex- tures Asn114 locked into position by a 3.0-Å salt bridge between the tends over the entire length of the molecule. Major interfaces lay in partial negative charge of its carbonyl oxygen and the guanidine the N-terminal pony-tail helical structure (Pt motif, ~895 Å2) and group of Arg93 (Fig. 2C). The N-terminal polycationic region of http://advances.sciencemag.org/ extended linker helices that connect GAF-A to GAF-B (LH1; ~510 Å2) PDE6 binds close to the GAF-A cGMP-binding site, suggesting its and GAF-B to the catalytic domain (LH2; ~685 Å2) of PDE6 and potential role in preventing the release of bound cGMP unless a PDE6. Local resolution analysis of the PDE6 heterodimer shows certain threshold of cGMP gradient has been established in the cell a resolution better than 3.4 Å in the core and 5 to 7 Å in peripheral and thereby lowering the dissociation constant (KD) of cGMP for the regions (Fig. 1A, bottom). The Pt motif is among the most flexible GAF-A domain (Figs. 2E and 3A). In addition, PDE6 staples the regions within the PDE62 complex with a local resolution of GAF-A 1-2 loop interaction site to GAF-B domain and likely 7 to 8 Å (Fig. 1A, bottom), and therefore has uncertainty in the de senses changes upon cGMP binding (Figs. 2, A and F, and 3A). One novo model. The final model is composed of amino acid residues molecule of PDE6 interacts exclusively with GAF-A and the catalytic 10 to 822 of PDE6 and 11 to 822 of PDE6 (Fig. 1C). The amino domain of the same subunit in the PDE6 heterodimer, suggestive acid residues 2 to 9 and 823 to 856 of PDE6 as well as 2 to 10 and of a tighter allosteric regulation (Figs. 1B, 2A, and 3A). PDE6 sub- 823 to 850 of PDE6 could not be modeled because of their flexibility. units display a resolution range of 3.2 to 9 Å with the highest reso- on February 13, 2020 This flexibility is exemplified by the two-dimensional (2D) class lution achieved in the N- and C-terminal ends due to their direct averages that show blurring of the flexible C-terminal ends of both interaction with GAF-A and catalytic domains, respectively (Figs. 2, A, PDE6 and PDE6 (fig. S3D, arrows). These long C-terminal helices E, and F, and 3, A and B). Amino acid residues 30 to 70 of PDE6 extend in opposite directions and serve as sites for lipid modifica- were omitted from the final de novo model of the PDE62 complex tions that anchor PDE62 in the ROS disk membranes. Although because of poor cryo-EM density near that region (Fig. 3, A and B). the distal C-terminal helices of the PDE6 heterodimer were aver- This could be explained by the flexible nature of the polypeptide seg- aged out during the final 3D cryo-EM reconstruction, this is the ment and the lack of direct interface contacts with the PDE6 het- first instance where 2D snapshots of these long C-terminal helices erodimer. Overall, these findings suggest a direct communication have been visualized. between the cGMP-bound GAF-A and GAF-B regulatory domains The N-terminal region of the PDE6 heterodimer displays an in PDE62 complex through the 1-2 loop of GAF-A. asymmetric double-stranded Pt motif that extends to more than The GAF-A C-terminal 5 helix (206 to 232) is linked to the 34 Å in length (Fig. 1). The Pt motif likely provides structural stability N-terminal 1 helix of GAF-B (254 to 280) by LH1 (Figs. 1C and to the PDE6 heterodimer by providing an interaction interface of 2D). Although the GAF-B domain maintains a familiar topology to ~895 Å2 between PDE6 and PDE6 subunits. The asymmetry of those of previously published GAF-B domain structures of PDE2A the Pt motif can be attributed to the low sequence identity between (RMSD = 1.120 Å between 59 pruned atom pairs) (24) and PDE5 the N-terminal regions of the two subunits of PDE6 heterodimer, (RMSD = 1.06 Å between 56 pruned atom pairs) (27), substantial with 27% identity in the first 70 amino acids as compared to 77.5% structural differences were observed in the orientation of LH1 (~20° tilt), identity in the rest of the sequence (fig. S2). The GAF-A domain of 2 (~10° tilt), and LH2 (~30° tilt). A major structural difference was the PDE6 heterodimer shows a high structural similarity to pre- found in the 34–amino acid–long 1-2 loop (280 to 313) that con- viously published GAF-A domains of PDE2A (24), PDE5 (27), and nects 1 with 2 in the six-stranded -sheet cluster of the GAF-B PDE6C (28) with root mean square deviations (RMSDs) of 1.34, 1.17, domain (Fig. 2D and fig. S4B). The GAF-B 1-2 loop of one sub- and 1.12 Å, respectively. The conserved structural features of the unit of the PDE6 heterodimer directly interacts with the catalytic GAF-A domain include an antiparallel -sheet cluster with strand domain of the other subunit (Fig. 2G). This cross-talk is mediated order 3-2-1-6-5-4 (Fig. 2D and fig. S4A). This -sheet cluster is sand- by the intertwined architecture of the PDE6 heterodimer, where wiched by 1, 2, and 5 helices that lay toward the heterodimer LH2 helices cross over the pseudo-twofold axis such that the catalytic
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 2 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
A N-ter B 115 Å 74 Å C α10/β11 822 Pt motif N C Nα Nα Nα 34 Å Nβ Pt motif
GAF-A
90° 90° LH1 LH1 154 Å PDEγ GAF-B
PDEγ LH2 LH2 Cα Catalytic Cα core Cβ
Nβ Nβ 3.2 Å 90° 90° Pt motif Downloaded from PDEγ Cα GAF-A Cβ
LH1 90° LH1 180° GAF-B 8.0 Å
LH2 http://advances.sciencemag.org/ Cα LH2
Cβ Catalytic Cβ core Cβ
Fig. 1. The cryo-EM structure of PDE62 ternary complex at 3.4-Å resolution. (A) Overall cryo-EM 3D reconstruction of the PDE62 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 PDE62 complex displaying PDE6 (purple), PDE6 (green cyan), and two molecules of PDE6 (red) subunits. (C) Structures of PDE6 (top, 10 to 822) and PDE6 (bottom, 11 to 822) in two orthogonal views shown in cartoon representation. Scale bar, 30 Å.
domain of one subunit lies directly below the GAF-B domain of the strate or an inhibitor to the PDE catalytic domain (Fig. 3C) (24). on February 13, 2020 other subunit of the heterodimer (Figs. 1, B and C, and 2A). The Whereas during PDE2A inactivation, the H-loop occludes the interacting region of the GAF-B 1-2 loop adopts a short two-turn substrate-binding pocket, which results in deactivation of the iso- helical structure that forms hydrophobic interactions with 6, 7, enzyme (Fig. 3C) (24). Unlike the structure of closed-state PDE2A, 8, and 9 helices of the catalytic domain (Fig. 2G). The direct in- the H-loop in the PDE62 complex features an open conforma- teraction suggests a potential allosteric signal transduction route tion that is reminiscent of the substrate/inhibitor-bound state of from the cGMP-bound GAF-A to the catalytic domain via GAF-B. PDE2A and PDE5 catalytic domains (Fig. 3, C and D, and fig. S5). This feature makes the PDE62 complex the only PDE isoenzyme This open H-loop conformation is stabilized by the C terminus showing the second direct association between the GAF-B domain of PDE6 that binds near the substrate-binding site and occludes and the catalytic phophohydrolase domain in addition to the LH2 substrate binding (Fig. 3D). helix that links the two domains together. This finding suggests a In contrast to the closed-state structure of PDE2A homodimer, role of the GAF-B 1-2 loop in the direct regulation of PDE6 cata- the M-loop region (745 to 767) that connects 14 and 15 of the lytic domains transducing the allosteric signal from cGMP-bound catalytic domain does not participate in heterodimer formation in GAF-A domains to the catalytic domains. the PDE62 complex (Fig. 4A). In light of the direct interaction of The GAF-B C-terminal 5 helix (414 to 441) is linked to the GAF 1-2 loops connecting the regulatory cGMP-bound GAF-A to N-terminal 1 helix of the catalytic domain (558 to 577) through the catalytic domain through the GAF-B domain of the PDE62 com- the LH2 helix and a helix-loop-helix region comprising a four-helix plex and the comparison with the closed state of PDE2A (Fig. 4A) (24), bundle (Figs. 1C and 2, A and D). The all–-helical structure of the a model for allosteric activation of PDE isoenzymes can be deduced catalytic domain in PDE6 (PDE6, 558 to 799; PDE6, 556 to 796) to reveal the representative conformational changes (Fig. 4B). is structurally conserved with previously published crystal structures These conformational changes include a downward movement of of the catalytic domains of several PDE isoenzymes (24, 29). How- the GAF-A 1-2 loop, a 10° inward twist of LH1, an 80° outward ever, a substantial conformational change was seen in the H-loop twist of the GAF-B 1-2 loop, and an 80° rotation of the catalytic region (606 to 629) that flanks the substrate-binding site (Fig. 3C). domains (Fig. 4A). However, the rearrangement of catalytic domains is The H-loop is a characteristic loop that changes its conformation upon unlikely to occur during the allosteric activation of PDE6 because of ligand binding. During PDE2A activation, the H-loop swings out and its association with PDE6. In an attempt to visualize some of these attains an open-loop conformation that allows the binding of a sub- structural changes, we performed cryo-EM on the PDE62 complex
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 3 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
A Nα Nα B C
R93 R93 β2 β1 cGMP cGMP GAF-A β3 β3
cGMP β3 N114 N114
GAF-B β1 β1
α4 Catalylic α3 core β2 β2
Membrane
D E GAF-A cGMP pocket Catalytic core α 5 β1 N α α6
C Downloaded from 5 α4 α5 α3 α4 GAF-B α2 L β2 β β β H α α α α β β β α4 α 4 5 6 1 1 α 7 10 α 12 Pt 1 2 3 2 L α 2 11 α4 motif H 1 α β β β β β β 2 http://advances.sciencemag.org/ GAF-A 1 α4 α3 4 5 6 1 2 3 α8 α9 LH1 α3 PDE6γ F G M98 I105 GAF-A M98 I105 A299 M282 M282 A299 β1-β2 loop GAF-B β1-β2 loop
α5 α5 GAF-B β1-β2 loop 45° 90°
α7 on February 13, 2020 α6 α6 α8 PDE6γ β5 β5 β4 β4 α8 α7 α9 β6 α9 β6 GAF-B GAF-B PDE6β catalytic core PDE6β catalytic core
Fig. 2. Structural features of the PDE62 complex. (A) Structure of the PDE6 heterodimer showing the domain distribution within PDE6 (purple) and PDE6 (green cyan) subunits where regulatory GAF domains are trailed by the phosphohydrolase catalytic domain in a trilobed organization. One molecule of PDE6 (red ribbon) wraps around PDE6 and PDE6 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 PDE6 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 PDE62 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 PDE6 GAF-A domain. The N terminus of PDE6 (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 PDE6 after the final refinement is displayed as gray mesh. (F) Interaction interface between the GAF-A 1-2 loop and the GAF-B domain of PDE6. PDE6 (red) staples the GAF-A 1-2 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 1-2 loop is displayed as gray mesh. (G) The interface between GAF-B 1-2 loop of PDE6 and the catalytic domain of PDE6 features a short two-turn helical structure that forms hydrophobic interactions with 6, 7, 8, and 9 helices of the catalytic domain. The refined electron density corresponding to the key amino acid residues is displayed as gray mesh. in the presence of fivefold molar excess of sildenafil. High-resolution of sildenafil in the catalytic site of PDE62 complex due to a com- 2D class averages of PDE62 complex treated with sildenafil dis- petition with the inhibitory PDE6 subunit for the same binding site plays a reduced density of the GAF-B 1-2 loop associated with its (30). Notably, the catalytic domains did not show notable structural increased flexibility (Fig. 4C; compare panels marked with asterisks). changes likely because of the partially bound PDE6 that stabilizes The observed structural changes are consistent with partial occupancy the PDE62 complex in its open state. Overall, these results further
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 4 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
A Nα Nα B Nγ Nγ 3.2 Å
PDE6γ cryo-EM density
PDE6γ de novo model 9.0 Å
Unmodeled density
Cγ Cγ Membrane
C H-loop Substrate-binding D M-loop Downloaded from pocket H-loop M-loop
PDE6γ
30° http://advances.sciencemag.org/
Cα Cα Cα
PDE6 Closed-state PDE2A Substrate/inhibitor-bound PDE2A
Fig. 3. The PDE6 subunit stabilizes the open-state conformation of PDE6 heterodimer. (A) Cryo-EM density (left, red surface) and partial de novo model (right, red ribbon) of PDE6 around the PDE6 heterodimer (PDE6, purple; PDE6, green cyan). One molecule of PDE6 wraps around PDE6 and PDE6 individually for a tighter regulation of PDE6 activity. (B) Local resolution estimation map (left) and partial de novo model (right) of PDE6 showing a good correlation between the 3D map and the refined PDE6 model. The density of PDE6 after the final refinement is displayed as gray mesh. (C) Comparison between the catalytic domains of PDE6 (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 PDE6 features an open H-loop similar to the inhibitor-bound PDE2A crystal structure. The M-loop region of PDE6 (purple) shows a on February 13, 2020 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 PDE6 (red cartoon) binds near the substrate-binding pocket of the PDE6 catalytic domain. PDE6 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 PDE6. Structurally conserved regions of the catalytic domain (RMSD ≤ 0.2 Å between PDE6 and PDE2A) are shown in gray. support the role of GAF 1-2 loops in the allosteric regulation of N-terminal GAF domains (fig. S1). Notably, PDE2A activation is PDE catalytic activity. mediated by binding of cGMP to the GAF-B domain, whereas cGMP- induced activation of PDE5 is mediated by cGMP binding to the GAF-A domain. On the other hand, the activation of PDE6 is more DISCUSSION complex and involves the binding of Gt-GTP, which releases the This study provides structural insights into the allosteric regula- inhibitory constraint of PDE6 on the catalytic site of PDE6 (16) tion of PDE6 catalytic activity by both N-terminal GAF domains and reduces the binding affinity of cGMP to one of the two GAF-A and the PDE6 subunit. A comparison of the PDE62 complex domains. Therefore, the proposed model displaying the representative structure with the closed-state structure of PDE2A shows reorgani- structural changes associated with PDE allosteric regulation (Fig. 4B) zation of the GAF 1-2 loop regions that form potential allosteric is likely limited to the closely related structural homologs of PDE6. signal transduction routes from GAF-A to the catalytic domain The structure of PDE62 complex displays several features through the GAF-B domain upon cGMP binding (Fig. 4). These al- that are in agreement with previously published chemical cross-linking losteric signals are then translated into the outward movement of the and mass spectrometric data (31). These features include a tandem or- H-loop region that allows substrate binding and hydrolysis (Figs. 3 ganization of GAF domains, parallel organization of the two catalytic and 4). The N-terminal regulatory domains are crucial for tightly subunits, and presence of juxtaposed -helical segments in the PDE6 regulating the cyclic nucleotide signaling by PDE isoenzymes. The heterodimer (31). PDE6 is the only type I PDE family member that type I PDE family features a variety of N-terminal domains includ- has inhibitory subunits bound to the catalytic dimer (32). Previous ing calcium/calmodulin-binding domains, protein kinase–regulated cross-linking studies have indicated the ability of PDE6 to bind the upstream conserved regions, and GAF domains. However, the al- catalytic domains of the PDE6 heterodimer, whereby its C-terminal losteric regulation of the majority of PDEs is governed through residues block the substrate-binding pocket (30). PDE6 binding has
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 5 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
A PDE2A PDE6 B PDE PDE Closed-state Open-state Closed-state Open-state
Missing N-ter Nα Nβ
10° Ligand 80° GAF-A loop 80°
M-loop GAF-B Membrane loop c Sildenafil-treated PDE6αβ2γ PDE6αβ2γ M-loop M-loop
* ** Downloaded from Cβ C M-loop C Cα Membrane Increased flexibility of GAF-B β1-β2 loops
Fig. 4. The 1-2 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 PDE6 heterodimer. M-loop regions of the closed-state PDE2A and the PDE6 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 1-2 loop, a 10° inward twist of the LH1 coiled coil, an 80° outward twist of the GAF-B 1-2 loop, and an 80° rotation of the catalytic domains. M-loop re- http://advances.sciencemag.org/ gions are shown in gold. (C) A comparison between the 2D class averages of the untreated PDE62 complex and PDE62 treated with sildenafil recapitulates some of the key structural differences illustrated in (B). The face views of PDE62 treated with sildenafil feature changes in the interaction profile of the GAF-B 1-2 loop with the catalytic domain of the PDE6 heterodimeric core. The GAF-B 1-2 loops are denoted by asterisks. Scale bar, 60 Å. also been proposed to modulate cGMP binding at the regulatory 30 min at 4°C to completely remove any residual ROS pellet. The GAF-A domain (33). This unusual interdomain allosteric control clear supernatant was dialyzed against buffer containing 10 mM Hepes for a 9.7-kDa PDE6 polypeptide has been debated often because of (pH 7.5), 6 mM MgCl2, and 1 mM DTT for 3 hours at 4°C. The hypo- the lack of structural evidence. This study provides the direct struc- tonic solution was supplemented with ROS membranes (25 M tural confirmation that PDE6 adopts an extended and mostly un- rhodopsin) and 250 M GTPS (Sigma-Aldrich), followed by light structured conformation to enable its interaction with both the GAF-A illumination for 30 min with a 150-W fiber light (NCL-150, Volpi, on February 13, 2020 cGMP-binding pocket and the catalytic domain of the PDE6 het- USA) delivered through a 480- to 520-nm band-pass filter (Chroma erodimer. In addition, the interaction of PDE6 C terminus with Technology Corporation, USA). The resuspension was then centri- the catalytic subunits stabilizes the open-state conformation of the fuged multiple times at 40,000g for 30 min at 4°C to completely re- PDE62 complex. move any residual ROS pellet. The supernatant was loaded onto a Overall, the high-resolution structure of the PDE62 complex C10/10 column (GE Healthcare) with 6 ml of propyl-agarose resin and its comparison with the closed-state PDE2A structure provide pre-equilibrated with 10 mM Hepes (pH 7.5), 2 mM MgCl2, and a solid structural framework for rational design of candidate mole- 1 mM DTT. Next, the column was washed with 30 resin volumes of cules that are selective to specific PDE isoenzymes to alleviate side the equilibration buffer followed by 2 resin volumes of buffer con- effects induced by conventional therapeutic approaches. taining 10 mM Hepes (pH 7.5), 2 mM MgCl2, 1 mM DTT, and 50 mM NaCl. Bound proteins were eluted with 30 ml of equilibra- tion buffer containing 0.4 M NaCl. The eluate was then dialyzed MATERIALS AND METHODS against buffer containing 10 mM Hepes (pH 7.5), 6 mM MgCl2, Purification of PDE6 1 mM EDTA, and 1 mM DTT. All experimental procedures were carried out in a darkroom under dim The dialyzed eluate was loaded onto a C10/20 column (GE red light (>670 nm). Bovine ROS were prepared as described else- Healthcare) with 15 ml of Blue Sepharose CL-6B resin (Sigma-Aldrich) where (34). ROS were diluted in isotonic buffer containing 20 mM Hepes pre-equilibrated with 10 mM Hepes (pH 7.5), 6 mM MgCl2, 1 mM (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 100 mM NaCl. EDTA, and 1 mM DTT. The flow-through was supplemented with The suspension was then centrifuged at 30,000g at 4°C for 25 min a nanobody that specifically binds to G11 (37) to accomplish to remove soluble and some membrane-associated proteins (35, 36). its removal from the sample (fig. S3). After 30 min of incubation, The pellet was resuspended in hypotonic buffer containing 5 mM Ni2+–nitrilotriacetic acid resin pre-equilibrated with 10 mM Hepes Hepes (pH 7.5), 0.1 mM EDTA, and 1 mM DTT and gently (pH 7.5), 6 mM MgCl2, 1 mM EDTA, and 1 mM DTT was added. homogenized three times by manually passing the suspension through Following 30 min of incubation, the resin bound with G11 was re- a glass-to-glass homogenizer. The homogenate was centrifuged at moved by passing the resuspension through a Pierce disposable column 40,000g for 30 min at 4°C. Supernatants from the two hypotonic (Thermo Fisher Scientific). The flow-through containing Gt and washes were pooled and centrifuged multiple times at 40,000g for PDE6 obtained from the immobilized-Ni2+ affinity chromatography
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 6 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE was then concentrated and loaded onto a Superdex 200 10/300 GL compute 30 reprojections covering views from all directions to serve column equilibrated with buffer containing 10 mM Hepes (pH 7.5), as templates for the final round of Gautomatch particle selection. 2 mM MgCl2, 1 mM DTT, and 100 mM NaCl (fig. S3, A and B). The final dataset comprising 199,658 particles was processed by Fractions containing PDE6 were combined, concentrated to about Relion 2.1. Five rounds of 2D classification reduced the dataset to 0.7 mg ml−1, and used for cryo-EM analyses. The functional charac- 82,558 particles, which were subjected to three rounds of 3D classi- terization of PDE6 has been described previously (26, 38). fication (with seven, five, and five 3D classes, respectively) with the rotationally symmetric initial 3D model as a reference (fig. S6A). Cryo-EM specimen preparation, data acquisition, and Two identical 3D classes displaying a resolution of 8.3 Å and com- movie processing prising a total of 43,597 particles were combined into a single data- Three microliters of the purified PDE62 or PDE62 with 5 M set. The combined dataset was then refined against one of the two excess of sildenafil at a concentration of 0.7 mg ml−1 were applied to a 8.3-Å 3D classes, which was low-pass–filtered to 35 Å. Upon mask- Quantifoil R2/2 400 mesh grid (Electron Microscopy Sciences) with- ing and modulation transfer function correction, the reconstructions out a prior glow discharge. The grids were plunge-frozen in liquid reached a resolution of 4.1 Å, as measured by Fourier shell correla- ethane with a FEI Vitrobot Mark IV (Thermo Fisher Scientific) under tion (FSC) of a refinement in which two halves of the dataset were these conditions: temperature, 4°C; humidity, 100%; blotting time, refined separately and combined only when building the final map 2 s; and blotting force set to −10. Frozen grids were imaged in a FEI (fig. S6A). The same dataset was then reprocessed with the cisTEM Titan Krios (300 kV, Thermo Fisher Scientific) equipped with a Gatan program (47) that uses per-particle CTF refinement and B-factor Downloaded from Quantum-LS energy filter (20-eV zero-loss filtering) connected to a particle weighting. The final 3D reconstruction reached a resolution Gatan K2 Summit detector operating in super-resolution counting of 3.4 Å at an FSC of 0.143 (fig. S6B) (48). Local resolution distribu- mode. Super-resolution movies of 50 frames were acquired at a tion of the final map was determined by ResMap (49). The data ac- magnification of ×47,259 in the nanoprobe mode using the SerialEM quisition and processing of PDE62 treated with sildenafil was software (39). A total dose of 80 e− Å−2 and a pixel size of 0.529 Å for done under identical conditions as the untreated PDE62 holo- the super-resolution pixels were used during data collection (fig. S3C). enzyme. In total, 4780 images were acquired, from which 223,656 http://advances.sciencemag.org/ The acquired movies were processed during the imaging session particles were isolated and subjected to 2D classification. with the Focus program (40), which included (i) gain reference application and binning 2× by the clip and resample_mp.exe Model building and refinement programs from the IMOD (41) and Frealign (42) packages, respec- The cryo-EM map was put into an artificial crystal lattice to calcu- tively; (ii) motion correction and dose weighting by MotionCor2 late its structure factor using the phenix.map_to_structure_factors (43); and (iii) contrast transfer function (CTF) estimation by script in the PHENIX program (50). The 3D density map was sharp- CTFFIND4 (44). A total of 3134 aligned movies were used for ened by applying a negative B-factor of −148 Å2 as determined with further single-particle processing. Images displaying a resolution the phenix.auto_sharpen script (50) and by manual intervention. lower than 7 Å during CTF correction or average drifts higher than De novo modeling of PDE6, PDE6, and PDE6 subunits was per- 2 Å per frame were excluded from the analysis. formed in Coot 0.8.8 (51) using secondary structure predictions calculated by PSIPRED (52). The densities of bulky side chains were on February 13, 2020 Image processing used as references during the backbone tracing. A partial PDE6 Particles in micrographs were selected with the EMAN2 boxer (45) model (PDE6, 10 to 822; PDE6, 11 to 822; and PDE6 1 to 30 and and Gautomatch (www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/) 70 to 87) was used for rigid body fitting into the 3D density map programs. During the initial processing, 2176 particles were auto- with Chimera (53). The model was then refined by rigid body re- matically selected from 100 preselected images with reference-free finement of individual chains in the PHENIX program (50), where Gautomatch localization. The selected particles were inspected with the amplitudes and phases of the structural factors were used as the e2boxer.py script and 2D classified in Relion 2.1 (46). A few pseudo-experimental diffraction data for model refinement. Initial high-resolution 2D classes were low-pass–filtered to 30 Å and used models were improved by multiple rounds of PHENIX real-space as templates for Gautomatch particle selection on all aligned images. refinement (50) and REFMAC version 5.8 (54) refinement against the This larger dataset of particles was 2D classified in Relion 2.1, and 30 overall map at a resolution of 3.4 Å. Each round of refinement was high-resolution class averages covering a broader range of views followed by manual model building and adjustments with Coot 0.8.8 were selected as templates for another round of Gautomatch particle (51). Simultaneous optimization of the stereochemical and molecular selection. The final particle selection resulted in a total of 197,171 clashes was performed during the refinements, resulting in a final particles, which were reprocessed with Relion 2.1. After seven rounds model to map a cross-correlation coefficient of 0.79 (fig. S6C). of 2D classification, a 2D class average representing the face-view of The stereochemical quality of the final PDE62 complex model PDE6 (fig. S3D) was used to create a rotationally symmetric starting was assessed with the Molprobity server (55). The final PDE62 model for 3D processing. The Fourier transform of the selected pro- model was cross-validated with previously available crystal structures jection was low-pass–filtered to 60 Å and rotated around the long of GAF-A (27, 28), GAF-B (27, 56), and catalytic (29, 30) domains axis of the class average. The created 3D Fourier volume was back- of PDE family members. Details of the cryo-EM data collection and transformed to obtain the initial real-space 3D model. structural refinement statistics are provided in table S1. Protein co- The 2D class averages comprising 85,929 particles were subjected ordinates and the EM map were deposited in the Protein Data Bank to three rounds of 3D classification with the 60-Å rotationally sym- (PDB) (PDB accession number: 6MZB and Electron Microscopy metric model as a reference (fig. S6A). The resulting five 3D classes Data Bank ID: EMD-9297). The raw image data have been deposited were used to generate the initial map of PDE6 at a resolution of in the Electron Microscopy Public Image Archive (EMPIAR) database 8.2 Å, which was subjected to the e2project.py script of EMAN2 to (www.ebi.ac.uk/pdbe/emdb/empiar/; access number: EMPIAR-10228).
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 7 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
All structural and density representations were generated using 19. N. O. Artemyev, H. M. Rarick, J. S. Mills, N. P. Skiba, H. E. Hamm, Sites of interaction either Chimera (53) or Pymol (www.pymol.org). between rod G-protein -subunit and cGMP-phosphodiesterase -subunit. Implications for the phosphodiesterase activation mechanism. J. Biol. Chem. 267, 25067–25072 (1992). SUPPLEMENTARY MATERIALS 20. N. O. Artemyev, J. S. Mills, K. R. Thornburg, D. R. Knapp, K. L. Schey, H. E. Hamm, A site on Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ transducin -subunit of interaction with the polycationic region of cGMP content/full/5/2/eaav4322/DC1 phosphodiesterase inhibitory subunit. J. Biol. Chem. 268, 23611–23615 (1993). Fig. S1. Domain organization in the type I phosphodiesterase superfamily. 21. N. O. Artemyev, Binding of transducin to light-activated rhodopsin prevents transducin Fig. S2. Domain organization and primary sequence comparison between interaction with the rod cGMP phosphodiesterase -subunit. Biochemistry 36, PDE6 and PDE6 subunits. 4188–4193 (1997). Fig. S3. Biochemical characterization and cryo-EM of the PD62 complex. 22. X. J. Zhang, X. Z. Gao, W. Yao, R. H. Cote, Functional mapping of interacting regions of the Fig. S4. Structural features of regulatory GAF domains of the PDE62 complex. photoreceptor phosphodiesterase (PDE6) -Subunit with PDE6 catalytic dimer, Fig. S5. Comparison between the H-loop orientations of PDE6 and PDE5. transducin, and regulator of G-protein signaling9-1 (RGS9-1). J. Biol. Chem. 287, Fig. S6. Cryo-EM reconstruction and data fitting of the PDE62 complex. 26312–26320 (2012). Table S1. Cryo-EM data collection, refinement, and validation statistics for the PDE62 23. K. C. Slep, M. A. Kercher, W. He, C. W. Cowan, T. G. Wensel, P. B. Sigler, Structural complex. determinants for regulation of phosphodiesterase by a G protein at 2.0 Å. Nature 409, 1071–1077 (2001). 24. J. Pandit, M. D. Forman, K. F. Fennell, K. S. Dillman, F. S. Menniti, Mechanism for the REFERENCES AND NOTES allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near 1. M. Conti, J. Beavo, Biochemistry and physiology of cyclic nucleotide phosphodiesterases: full-length construct. Proc. Natl. Acad. Sci. U.S.A. 106, 18225–18230 (2009). Annu. Rev. Biochem. 76 Essential components in cyclic nucleotide signaling. , 481–511 25. B. M. Qureshi, E. Behrmann, J. Schöneberg, J. Loerke, J. Bürger, T. Mielke, Downloaded from (2007). J. Giesebrecht, F. Noé, T. D. Lamb, K. P. Hofmann, C. M. T. Spahn, M. Heck, It takes two 2. D. M. G. Halpin, ABCD of the phosphodiesterase family: Interaction and differential transducins to activate the cGMP-phosphodiesterase 6 in retinal rods. Open Biol. 8, Int. J. Chron. Obstruct. Pulmon. Dis. 3 activity in COPD. , 543–561 (2008). 180075 (2018). 3. M. P. DeNinno, Future directions in phosphodiesterase drug discovery. Bioorg. Med. 26. A. Goc, M. Chami, D. T. Lodowski, P. Bosshart, V. Moiseenkova-Bell, W. Baehr, A. Engel, Chem. Lett. 22, 6794–6800 (2012). K. Palczewski, Structural characterization of the rod cGMP phosphodiesterase 6. 4. X. Zhang, Q. Feng, R. H. Cote, Efficacy and selectivity of phosphodiesterase-targeted J. Mol. Biol. 401, 363–373 (2010). drugs in inhibiting photoreceptor phosphodiesterase (PDE6) in retinal photoreceptors. 27. H. Wang, H. Robinson, H. Ke, Conformation changes, N-terminal involvement, and cGMP http://advances.sciencemag.org/ Invest. Ophthalmol. Vis. Sci. 46, 3060–3066 (2005). signal relay in the phosphodiesterase-5 GAF domain. J. Biol. Chem. 285, 38149–38156 5. E. Bischoff, Potency, selectivity, and consequences of nonselectivity of PDE inhibition. (2010). Int. J. Impot. Res. 16 (Suppl. 1), S11–S14 (2004). 28. S. E. Martinez, C. C. Heikaus, R. E. Klevit, J. A. Beavo, The structure of the GAF A domain 6. F. G. Boess, M. Hendrix, F. J. van der Staay, C. Erb, R. Schreiber, W. van Staveren, from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding J. de Vente, J. Prickaerts, A. Blokland, G. Koenig, Inhibition of phosphodiesterase 2 surface, and a large cGMP-dependent conformational change. J. Biol. Chem. 283, increases neuronal cGMP, synaptic plasticity and memory performance. 25913–25919 (2008). Neuropharmacology 47, 1081–1092 (2004). 29. K. Y. Zhang, G. L. Card, Y. Suzuki, D. R. Artis, D. Fong, S. Gillette, D. Hsieh, J. Neiman, 7. D. H. Maurice, H. Ke, F. Ahmad, Y. Wang, J. Chung, V. C. Manganiello, Advances in B. L. West, C. Zhang, M. V. Milburn, S. H. Kim, J. Schlessinger, G. Bollag, A glutamine switch targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov. 13, 290–314 mechanism for nucleotide selectivity by phosphodiesterases. Mol. Cell 15, 279–286 (2014). (2004). 8. M. M. Moschos, E. Nitoda, Pathophysiology of visual disorders induced by 30. B. Barren, L. Gakhar, H. Muradov, K. K. Boyd, S. Ramaswamy, N. O. Artemyev, Structural phosphodiesterase inhibitors in the treatment of erectile dysfunction. Drug Des. Devel. Ther. basis of phosphodiesterase 6 inhibition by the C-terminal region of the -subunit. 8, 3407–3413 (2016). EMBO J. 28, 3613–3622 (2009). on February 13, 2020 9. J. A. Beavo, Cyclic nucleotide phosphodiesterases: Functional implications of multiple 31. X. Zeng-Elmore, X.-Z. Gao, R. Pellarin, D. Schneidman-Duhovny, X.-J. Zhang, K. A. Kozacka, isoforms. Physiol. Rev. 75, 725–748 (1995). Y. Tang, A. Sali, R. J. Chalkley, R. H. Cote, F. Chu, Molecular architecture of photoreceptor 10. S. A. Ballard, C. J. Gingell, K. Tang, L. A. Turner, M. E. Price, A. M. Naylor, Effects of sildenafil phosphodiesterase elucidated by chemical cross-linking and integrative modeling. on the relaxation of human corpus cavernosum tissue in vitro and on the activities of J. Mol. Biol. 426, 3713–3728 (2014). cyclic nucleotide phosphodiesterase isozymes. J. Urol. 159, 2164–2171 (1998). 32. P. Deterre, J. Bigay, F. Forquet, M. Robert, M. Chabre, cGMP phosphodiesterase of retinal 11. W. Baehr, M. J. Devlin, M. L. Applebury, Isolation and characterization of cGMP rods is regulated by two inhibitory subunits. Proc. Natl. Acad. Sci. U.S.A. 85, 2424–2428 phosphodiesterase from bovine rod outer segments. J. Biol. Chem. 254, 11669–11677 (1988). (1979). 33. V. Y. Arshavsky, C. L. Dumke, M. D. Bownds, Noncatalytic cGMP-binding sites of 12. T. D. Lamb, M. Heck, T. W. Kraft, Implications of dimeric activation of PDE6 for rod amphibian rod cGMP phosphodiesterase control interaction with its inhibitory phototransduction. Open Biol. 8, 180076 (2018). -subunits. A putative regulatory mechanism of the rod photoresponse. J. Biol. Chem. 13. S. Kolandaivelu, B. Chang, V. Ramamurthy, Rod phosphodiesterase-6 (PDE6) catalytic 267, 24501–24507 (1992). subunits restore cone function in a mouse model lacking cone PDE6 catalytic subunit. 34. D. S. Papermaster, Preparation of retinal rod outer segments. Methods Enzymol. 81, 48–52 J. Biol. Chem. 286, 33252–33259 (2011). (1982). 14. Z. Zhang, F. He, R. Constantine, M. L. Baker, W. Baehr, M. F. Schmid, T. G. Wensel, 35. S. Gulati, B. Jastrzebska, S. Banerjee, Á. L. Placeres, P. Miszta, S. Gao, K. Gunderson, M. A. Agosto, Domain organization and conformational plasticity of the G protein G. P. Tochtrop, S. Filipek, K. Katayama, P. D. Kiser, M. Mogi, P. L. Stewart, K. Palczewski, effector, PDE6. J. Biol. Chem. 290, 17131–17132 (2015). Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism. 15. H. Mou, R. H. Cote, The catalytic and GAF domains of the rod cGMP phosphodiesterase Proc. Natl. Acad. Sci. U.S.A. 114, E2608–E2615 (2017). (PDE6) heterodimer are regulated by distinct regions of its inhibitory subunit. 36. B. Y. Baker, S. Gulati, W. Shi, B. Wang, P. L. Stewart, K. Palczewski, Crystallization of J. Biol. Chem. 276, 27527–27534 (2001). proteins from crude bovine rod outer segments. Methods Enzymol. 557, 439–458 16. A. W. Norton, M. R. D’Amours, H. J. Grazio, T. L. Hebert, R. H. Cote, Mechanism of (2015). transducin activation of frog rod photoreceptor phosphodiesterase. Allosteric 37. S. Gulati, H. Jin, I. Masuho, T. Orban, Y. Cai, E. Pardon, K. A. Martemyanov, P. D. Kiser, interactiona between the inhibitory subunit and the noncatalytic cGMP-binding sites. P. L. Stewart, C. P. Ford, J. Steyaert, K. Palczewski, Targeting G protein-coupled receptor J. Biol. Chem. 275, 38611–38619 (2000). signaling at the G protein level with a selective nanobody inhibitor. Nat. Commun. 9, 17. M. Yamazaki, N. Li, V. A. Bondarenko, R. K. Yamazaki, W. Baehr, A. Yamazaki, Binding of 1996 (2018). cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). 38. B. Y. Baker, K. Palczewski, Detergents stabilize the conformation of phosphodiesterase 6. Identification of GAF domains in PDE subunits and distinct domains in the PDE Biochemistry 50, 9520–9531 (2011). subunit involved in stimulation of cGMP binding to GAF domains. J. Biol. Chem. 277, 39. D. N. Mastronarde, Automated electron microscope tomography using robust prediction 40675–40686 (2002). of specimen movements. J. Struct. Biol. 152, 36–51 (2005). 18. D. F. Morrison, J. M. Cunnick, B. Oppert, D. J. Takemoto, Interaction of the -subunit of 40. N. Biyani, R. D. Righetto, R. McLeod, D. Caujolle-Bert, D. Castano-Diez, K. N. Goldie, retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides H. Stahlberg, Focus: The interface between data collection and data processing in as functional probes. J. Biol. Chem. 264, 11671–11681 (1989). cryo-EM. J. Struct. Biol. 198, 124–133 (2017).
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 8 of 9 SCIENCE ADVANCES | RESEARCH ARTICLE
41. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, Computer visualization of contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996). W375–W383 (2007). 42. N. Grigorieff, FREALIGN: High-resolution refinement of single particle structures. 56. M. Russwurm, C. Schlicker, M. Weyand, D. Koesling, C. Steegborn, Crystal structure of the J. Struct. Biol. 157, 117–125 (2007). GAF-B domain from human phosphodiesterase 5. Proteins 79, 1682–1687 (2011). 43. S. Q. Zheng, E. Palovcak, J.-P. Armache, K. A. Verba, Y. Cheng, D. A. Agard, MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Acknowledgments: We thank A. Sears, R. Zimmerman, and other members of the Nat. Methods 14 , 331–332 (2017). Palczewski laboratory for their helpful comments regarding this manuscript and 44. A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron J. Thorne-Wallis from C-CINA for assistance with the preparation of cryo-EM grids. K.P. is the J. Struct. Biol. 192 micrographs. , 216–221 (2015). Leopold Chair of Ophthalmology. Funding: This research was supported in part by grants 45. G. Tang, L. Peng, P. R. Baldwin, D. S. Mann, W. Jiang, I. Rees, S. J. Ludtke, EMAN2: from the National Institutes of Health (NIH) (R24EY024864 and R01EY027283 to K.P.) and J. Struct. Biol. 157 An extensible image processing suite for electron microscopy. , Research to Prevent Blindness (RPB) to The Department of Ophthalmology at UCI, the 38–46 (2007). Canadian Institute for Advanced Research (CIFAR), Alcon Research Institute (ARI), and the 46. S. H. W. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure Swiss Commission for Technology, and Innovation (CTI) grant 18272.1 PFLS-LS (H.S.). J. Struct. Biol. 180 determination. , 519–530 (2012). Author contributions: S.G. and K.P. designed the research and wrote the paper. S.G. cis 47. T. Grant, A. Rohou, N. Grigorieff, TEM, user-friendly software for single-particle image performed the protein purifications and optimizations. S.G. prepared PDE6 samples for eLife 7 processing. , e35383 (2018). cryo-EM specimen preparation. S.G., L.K., and H.S. performed the cryo-EM work. L.K. did the 48. S. H. W. Scheres, S. Chen, Prevention of overfitting in cryo-EM structure determination. cryo-EM data processing and contributed to the written paper. S.G. carried out de novo Nat. Methods 9 , 853–854 (2012). model building and refinement and analyzed the structure of the PDE62 complex. A.E. 49. A. Kucukelbir, F. J. Sigworth, H. D. Tagare, Quantifying the local resolution of cryo-EM made intellectual contributions to the study and contributed to the written paper. All density maps. Nat. Methods 11, 63–65 (2014). authors reviewed and edited the manuscript. Competing interests: The authors declare
50. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, that they have no competing interests. Data and materials availability: All data supporting Downloaded from L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, the findings of this study are available within this paper. Additional data supporting the R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: findings of this manuscript are available from the corresponding authors upon reasonable A comprehensive Python-based system for macromolecular structure solution. request. The cryo-EM map of PDE62 complex has been deposited in the Electron Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). Microscopy Data Bank under accession code EMD-9297. The modeled structure of the 51. P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr. PDE62 complex has been deposited at the PDB under accession code 6MZB. The cryo-EM D Biol. Crystallogr. 60, 2126–2132 (2004). movie files have been deposited at the EMPIAR under accession code EMPIAR-10228. 52. D. W. Buchan, F. Minneci, T. C. Nugent, K. Bryson, D. T. Jones, Scalable web services for http://advances.sciencemag.org/ the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013). Submitted 14 September 2018 53. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, Accepted 14 January 2019 T. E. Ferrin, UCSF Chimera—A visualization system for exploratory research and analysis. Published 27 February 2019 J. Comput. Chem. 25, 1605–1612 (2004). 10.1126/sciadv.aav4322 54. Collaborative Computational Project, Number 4, The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994). Citation: S. Gulati, K. Palczewski, A. Engel, H. Stahlberg, L. Kovacik, Cryo-EM structure of 55. I. W. Davis, A. Leaver-Fay, V. B. Chen, J. N. Block, G. J. Kapral, X. Wang, L. W. Murray, phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases. W. B. Arendall III, J. Snoeyink, J. S. Richardson, D. C. Richardson, MolProbity: All-atom Sci. Adv. 5, eaav4322 (2019). on February 13, 2020
Gulati et al., Sci. Adv. 2019; 5 : eaav4322 27 February 2019 9 of 9 Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases Sahil Gulati, Krzysztof Palczewski, Andreas Engel, Henning Stahlberg and Lubomir Kovacik
Sci Adv 5 (2), eaav4322. DOI: 10.1126/sciadv.aav4322 Downloaded from
ARTICLE TOOLS http://advances.sciencemag.org/content/5/2/eaav4322
SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/02/25/5.2.eaav4322.DC1 MATERIALS http://advances.sciencemag.org/
REFERENCES This article cites 56 articles, 18 of which you can access for free http://advances.sciencemag.org/content/5/2/eaav4322#BIBL
PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions on February 13, 2020
Use of this article is subject to the Terms of Service
Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).