Noncatalytic cGMP-binding Sites Research Articles

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ∼ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.

Regulation of photoreceptor phosphodiesterase catalysis by its non-catalytic cGMP-binding sites

The photoreceptor 3ʹ:5ʹ-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory γ subunit (Pγ), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pγ, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic αβ dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pγ for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pγ had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pγ. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pγ peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pγ with the catalytic domain.

Regulation of photoreceptor phosphodiesterase catalysis by its non-catalytic cGMP-binding sites

The photoreceptor 3':5'-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory gamma subunit (Pgamma), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pgamma, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic alphabeta dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pgamma for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pgamma had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pgamma. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pgamma peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pgamma with the catalytic domain.

Probing Domain Functions of Chimeric PDE6α′/PDE5 cGMP-Phosphodiesterase

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6alpha') in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6alpha'/PDE5 enzyme containing the PDE6alpha' noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6alpha'. The inhibitory gamma subunit of PDE6 (Pgamma) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6alpha' by approximately 6-fold. The polycationic region of Pgamma, Pgamma-24-45, was mainly responsible for this effect, while the inhibitory domain of Pgamma, Pgamma-63-87, was ineffective. On the contrary, Pgamma failed to inhibit catalytic activity of the chimeric PDE6alpha'/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6alpha' are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 --> Ala resulted in an approximately 10-fold increase of the Km value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6alpha' may regulate catalytic properties of the enzyme.

Identification and Characterization of a Novel Family of Cyclic Nucleotide Phosphodiesterases

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a, 7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2, 1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 microM. This new phosphodiesterase is highly specific for cGMP. Its Km of approximately 0.07 microM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE. None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2'-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2' regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.

Photoreceptor Phosphodiesterase: Interaction of Inhibitory γ Subunit and Cyclic GMP with Specific Binding Sites on Catalytic Subunits

The photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the phototransduction cascade of photoreceptor cells. It is the only known PDE isoform the activity of which is regulated by interaction with a heterotrimeric G protein. The rod PDE6 holoenzyme is a tetrameric protein consisting of two large catalytic α and β subunits and two small γ subunits, which serve as potent inhibitors of PDE6. In dark-adapted photoreceptors, the γ subunits maintain PDE6 activity at a low level. When exposed to light the visual pigment rhodopsin activates the retinal G protein, transducin, leading to release of the inhibitory action of the γ subunits. In addition to the active sites where cGMP is hydrolyzed, the α and β catalytic subunits have high-affinity, noncatalytic cGMP binding sites. These noncatalytic sites do not directly regulate cGMP catalysis at the active site, but rather can modulate the affinity with which the γ subunits bind to the catalytic subunits. This article describes a number of experimental approaches that have recently been developed for studying the interactions between catalytic and inhibitory subunits of PDE6, as well as the dynamics of cGMP binding to and dissociation from the PDE6 noncatalytic sites.

Subunit Structure of Rod cGMP-Phosphodiesterase

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.

Cyclic nucleotides as regulators of light-adaptation in photoreceptors

AbstractCyclic nucleotides can regulate the sensitivity of retinal rods to light through phosducin. The phosphorylation state of phosducin determines the amount of G available for activation by Rho*. Phosducin phosphorylation is regulated by cyclic nucleotides through their activation of cAMP-dependent protein kinase. The regulation of phosphodiesterase activity by the noncatalytic cGMP binding sites as well as Ca2+/calmodulin dependent regulation of cGMP binding to the cation channel are also discussed.

CGMP binding sites on photoreceptor phosphodiesterase: role in feedback regulation of visual transduction.

A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transduction lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (Kd = 60 nM) for the nonactivated PDE, intermediate affinity (Kd approximately 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (Kd > 1 microM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites > 50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.

Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP.

THE photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase γ-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP1–4. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity5–7. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its γ-subunits, accelerates GTPase activity by several fold. Thus the γ-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase α and β-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.

Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cGMP binding sites of rod phosphodiesterase.

In illuminated rod outer segment membranes, GTP and guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) have reciprocal effects on cGMP phosphodiesterase (PDEase; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) activity and cGMP binding to noncatalytic sites on that enzyme. Two micromolar p[NH]ppG increased PDEase activity more than 2-fold while inhibiting cGMP binding more than 40%. Reduction of noncatalytic cGMP binding, which followed addition of p[NH]ppG, was not a result of PDEase activation. Both effects of p[NH]ppG were completely dependent on the presence of bleached rhodopsin. A heat-stable factor has been found to inhibit PDEase activity and also to stimulate cGMP binding to noncatalytic cGMP binding sites. Addition of p[NH]ppG reversed the effects of this factor on both PDEase activity and cGMP binding. During purification of this material, the activity peaks for both PDEase inhibition and activation of noncatalytic cGMP binding comigrated on both Blue Sepharose CL-6B column chromatography and sucrose density gradients centrifugation, suggesting that the same factor could be responsible for both inhibition of PDEase activity and enhancement of noncatalytic cGMP binding. Limited tryptic proteolysis of PDEase, which markedly reduced cGMP binding to the noncatalytic sites, and experiments using highly purified cAMP (free of cGMP) as substrate for PDEase showed that the binding of cGMP to noncatalytic sites was not required for the heat-stable inhibitory factor to inhibit PDEase activity. We discuss possible relationships between the regulation of PDEase and the binding of cGMP to noncatalytic sites.