Journal of Molecular Biology
Structural Characterization of the Rod cGMP Phosphodiesterase 6
Introduction
Rod cGMP phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate visual signal transduction. PDE6 belongs to the phosphodiesterase (PDE) superfamily, whose members (PDE1–11) regulate the cellular concentrations of the second messengers cAMP and cyclic guanosine 3′,5′-monophosphate (cGMP).1, 2 PDEs exist as homodimeric or heterodimeric proteins that comprise a highly conserved C-terminal catalytic domain and a regulatory N-terminus, often consisting of an N-terminal GAF-A domain and an intermediate GAF-B domain.1, 2, 3, 4 In contrast to other PDE family members, PDE6 utilizes an additional small regulatory protein and is thus made of three subunits—PDE6α (∼ 99 kDa), PDE6β (∼ 99 kDa), and PDE6γ (∼ 10 kDa)—that form a heterotetramer in a molar ratio of 1:1:2.5, 6, 7 Although PDE6γ inhibits the catalytic domains of both PDE6α and PDE6β,8, 9 PDEαβγ2 still has basal cGMP hydrolytic activity.6
Both catalytic subunits PDE6α and PDE6β carry a CAAX motif at their C-termini for posttranslational isoprenylation and carboxymethylation; PDE6α is farnesylated, whereas PDE6β is geranylgeranylated.10 These modifications serve as membrane anchors for attachment of PDE6 to rod outer segment (ROS) membranes.5, 10, 11 The 17-kDa prenyl-binding protein δ (PrBP/δ)12, 13, 14 is able to extract PDE6 from ROS membranes by binding to the isoprenylated carboxyl-terminus of the catalytic subunits.15 Using fluorescence resonance energy transfer techniques, Zhang et al. showed that PrBP/δ specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 μM and 0.70 μM, respectively.13 PrBP/δ is evolutionarily highly conserved and expressed throughout the animal kingdom. The homology among bovine, human, and mouse PrBP/δ polypeptides is ∼ 99% and falls to 69% between Caenorhabditis elegans and humans.16, 17 In mammals, PrBP/δ is primarily localized to the retina, but is also present in nonretinal tissues.12 Deletion of PrBP/δ in mouse has profound effects on the trafficking of farnesylated GRK1 and PDE6 to the outer segments,18 suggesting that PrBP/δ functions as a chaperone in the transport of a subset of prenylated proteins.
A wealth of structural information on PDEs exists; the recently determined nearly full-length structure of PDE2 provides the first structure of an intact PDE and insights into its regulation, domain arrangement, and function.19 The structure of the cone GAF-A domain of chicken PDE6 has been solved.20 NMR measurements indicate conformational change upon cGMP binding, and unfolding experiments revealed that that cGMP-free GAF-A is thermodynamically less stable than the cGMP-bound form.20 Neither the structure of the PDE6α or PDE6β GAF-B domains nor their functional implications are known, but the structure of the catalytic domain of a PDE5/6 chimeric protein complexed with the inhibitory PDE6γ peptide has been elucidated, giving insight into the regulatory mechanism of PDE6γ.21 A cocrystal of the transducin α subunit (Gtα) complexed with the core of PDE6γ specifies the region of interaction between these entities.22 Moreover, the crystal structure of PrBP/δ revealed that it has an immunoglobulin-like β-sandwich fold that forms a hydrophobic isoprenyl-binding pocket.18 Finally, three-dimensional (3D) electron microscopy of negatively stained samples unveiled the overall architecture of PDE6 at 30 Å resolution.23, 24
Herein we describe a novel method for purification of PDE6 in complex with PrBP/δ. We present the biochemical characterizations of both PDE6αβγ2 and PDE6αβγ2–PrBP/δ. By comparing the projection structures of negatively stained PDE6αβγ2 and PDE6αβγ2–PrBP/δ samples, we identified the PrBP/δ binding site/isoprenylation site. In addition, we present the 3D structure of the PDE6 complex at 18 Å resolution generated from single-particle images by weighted backprojection.25 Model building has been performed to fully interpret this structure.
Section snippets
Purification of PDE6 and PDE6–PrBP/δ
PDE6 complexes were purified as described in Materials and Methods. PDE6 was extracted from ROS membranes with a hypotonic buffer and further purified by propyl-agarose affinity chromatography. Fractions containing PDE6 were pooled, concentrated, and subjected to gel-filtration chromatography using a Superdex 200 column (Fig. 1a). PDE6–PrBP/δ-GST (PrBP/δ and GST fusion protein) was isolated by extraction of PDE6 from ROS membranes with recombinant PrBP/δ-GST and purified on a GSTrap column.
Discussion
In this study, we present the purification of free PDE6 and the complexes formed by PDE6 and PrBP/δ in the form of PDE6–PrBP/δ-GST and PDE6–PrBP/δ. We compare the biochemical properties of PDE6 and PDE6–PrBP/δ and elucidate their structure by electron microscopy and single-particle processing of negatively stained PDE6 samples.
Expression and purification of PrBP/δ proteins
PrBP/δ-GST plasmid in pGex-2T expression vector was used, and purification was performed as previously described.13 The PrBP/δ protein was purified from an overnight digest of PrBP/δ-GST with biotinylated thrombin, followed by 30 min of incubation with streptavidin agarose to remove thrombin. PrBP/δ was then separated from GST by Superdex 200 10/300 gel-filtration chromatography.
Purification of PDE6
Bovine ROS membranes were prepared from 100 frozen retinas (W. L. Lawson Co., Lincoln, NE) under dim red light in
Acknowledgements
This research was supported by National Institutes of Health grants EY008061, EY019478, EY019298, T32EY007157, GM079191, and P30EY11373; the Foundation Fighting Blindness; the Swiss National Science Foundation; the Swiss National Center of Competence in Structural Biology; and the Maurice E. Müller Foundation, Switzerland. K.P. is a Senior Fellow of the American Asthma and Sandler Program for Asthma Research.
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Cited by (22)
New focus on regulation of the rod photoreceptor phosphodiesterase
2021, Current Opinion in Structural BiologyCitation Excerpt :Previous studies used negatively stained transmission electron microscopy and cryo-electron microscopy (cryo-EM) to study the structure of the PDE6αγβγ complex at a low resolution [8,52,53].
The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa
2019, Computational and Structural Biotechnology JournalDomain organization and conformational plasticity of the G protein effector, PDE6
2015, Journal of Biological ChemistryCitation Excerpt :A final map was generated from ∼9200 particles (Fig. 1C), and comparison of class averages and corresponding model projections (Fig. 1B) indicates good agreement of the model with the data. The overall appearance of the map is similar to that of previously reported models of PDE6 imaged in negative stain (12–14), exhibiting a flattened and extended shape with two prominent cavities. Most of the structure has an approximate 2-fold symmetry as a consequence of the 84% sequence similarity between the PDE6α and PDE6β subunits.
Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling
2014, Journal of Molecular BiologyCitation Excerpt :Rod PDE6 is the only enzyme of the 11-member phosphodiesterase superfamily that consists of two different catalytic subunits forming a heterodimer, Pαβ, which is regulated by two identical inhibitory Pγ subunits. Tangible progress has been made in determining atomic structures of several individual domains and low-resolution topological organization of the PDE6 holoenzyme [11–13,17]. However, a more detailed structure of the entire PDE6 remains lacking.
Structural approaches to understanding retinal proteins needed for vision
2014, Current Opinion in Cell Biology
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A.G., M.C., and D.T.L. contributed equally to this work.