Structural Characterization of the Rod cGMP Phosphodiesterase 6

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Abstract

Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6α, PDE6β, and two regulatory PDE6γ subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6α and PDE6β and can form a complex with prenyl-binding protein δ (PrBP/δ), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6–PrBP/δ binding and the mechanism by which the PDE6–PrBP/δ complex assembles have not been fully characterized, and the location of regulatory PDE6γ subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6–PrBP/δ from bovine rod outer segments utilizing recombinant PrBP/δ. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/δ and, thus, where the carboxyl-termini of PDE6α and PDE6β must be located. The three-dimensional structure of the PDE6αβγ complex was determined up to 18 Å resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6γ subunits, and catalytic sites.

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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    A.G., M.C., and D.T.L. contributed equally to this work.

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