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Partial Reconstitution of Photoreceptor cGMP Phosphodiesterase Characteristics in cGMP Phosphodiesterase-5

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100626200

ISSN

1083-351X

Autores

Alexey E. Granovsky, Nikolai O. Artemyev,

Tópico(s)

Cholinesterase and Neurodegenerative Diseases

Resumo

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory γ-subunits (Pγ). The Pγ block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pγ and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pγ and has a more than 100-fold lower k cat for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6α′ enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6α′) with Pγ is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met758, Phe777, and Phe781. The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6α′-specific glycine residues for the corresponding PDE5 alanine residues, Ala608 and Ala612. The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis. Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory γ-subunits (Pγ). The Pγ block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pγ and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pγ and has a more than 100-fold lower k cat for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6α′ enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6α′) with Pγ is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met758, Phe777, and Phe781. The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6α′-specific glycine residues for the corresponding PDE5 alanine residues, Ala608 and Ala612. The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis. cGMP phosphodiesterase γ-subunit of PDE6 α′-subunit of cone PDE6 cGMP binding, cGMP-specific PDE (PDE5 family) polymerase chain reaction high performance liquid chromatography cGMP phosphodiesterases (PDE6)1 play the role of effector enzymes in the vertebrate visual transduction cascade. In retinal rod cells, photoexcited rhodopsin induces GDP/GTP exchange on the visual G protein, transducin (Gt), and liberated GtαGTP activates PDE6. A homologous cascade operates in cone photoreceptors. cGMP hydrolysis by active PDE6 results in the closure of cGMP-gated channels in the plasma membrane (1Chabre M. Deterre P. Eur. J. Biochem. 1989; 179: 255-266Crossref PubMed Scopus (219) Google Scholar, 2Yarfitz S. Hurley J.B. J. Biol. Chem. 1994; 269: 14329-14332Abstract Full Text PDF PubMed Google Scholar). The key attributes of the visual cascade, low noise and high gain signal amplification, place specific requirements on PDE6. The enzyme must have a very low basal cGMP hydrolytic rate in the dark-adapted photoreceptors and a very high catalytic rate in the transducin-activated state. This is achieved through two unique features of PDE6: the inhibitory interaction of the catalytic subunits with the γ-subunit and an exceptionally highk cat value for cGMP hydrolysis when the inhibition is turned off. The lack of a practical expression system for PDE6 (3Piriev N.I. Yamashita C. Samuel G. Farber D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9340-9344Crossref PubMed Scopus (35) Google Scholar, 4Qin N. Baehr W. J. Biol. Chem. 1994; 269: 3265-3271Abstract Full Text PDF PubMed Google Scholar, 5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) has stalled the progress in determining the structural basis of PDE6 function. We have begun to study the structure and function relationship of PDE6 by constructing chimeras between cone PDE6α′ and cGMP binding cGMP-specific PDE (PDE5 family) (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). PDE5 and PDE6 display a high degree of identity (45–48%) between the catalytic domains, a strong substrate selectivity for cGMP, and similar sensitivity to a common set of competitive inhibitors (7McAllister-Lucas L.M. Sonnenburg W.K. Kadlecek A. Seger D. Trong H.L. Colbran J.L. Thomas M.K. Walsh K.A. Francis S.H. Corbin J.D. Beavo J.A. J. Biol. Chem. 1993; 268: 22863-22873Abstract Full Text PDF PubMed Google Scholar, 8Gillespie P.G. Beavo J.A. Mol. Pharmacol. 1989; 36: 773-781PubMed Google Scholar, 9Turko I.V. Ballard S.A. Francis S.H. Corbin J.D. Mol. Pharmacol. 1999; 56: 124-130Crossref PubMed Scopus (165) Google Scholar). Yet, the reported maximal rate of cGMP hydrolysis by PDE5 catalytic dimers is only ∼10 moles of cGMP per mole of PDE·sec, which is ∼400–550-fold lower than thek cat estimates for PDE6 (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14964-14970Abstract Full Text PDF PubMed Google Scholar, 11Turko I.V. Francis S.H. Corbin J.D. J. Biol. Chem. 1998; 273: 6460-6466Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 12Mou H. Grazio III, H.J. Cook T.A. Beavo J.A. Cote R.H. J. Biol. Chem. 1999; 274: 18813-18820Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Leskov I.B. Klenchin V.A. Handy J.W. Whitlock G.G. Govardovskii V.I. Bownds M.D. Lamb T.D. Pugh E.N. Arshavsky V.Y. Neuron. 2000; 27: 525-537Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar, 15Dumke C.L. Arshavsky V.Y. Calvert P.D. Bownds M.D. Pugh Jr., E.N. J. Gen. Physiol. 1994; 103: 1071-1098Crossref PubMed Scopus (50) Google Scholar). Furthermore, the activity of PDE5 is unaffected by the PDE6 γ-subunit (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This, and a robust functional expression of PDE5 using the baculovirus/insect cell system (16Turko I.V. Haik T.L. McAllister-Lucas L.M. Burns F. Francis S.H. Corbin J.D. J. Biol. Chem. 1996; 271: 22240-22244Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), makes PDE5 a valuable tool for “gain of PDE6 function” experiments. Recently, we have shown that a substitution of the segment PDE5-(773–820) by the corresponding PDE6α′-(737–784) sequence in the wild-type PDE5 or in a PDE5/PDE6α′ chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interaction with Pγ (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Alanine-scanning mutational analysis of the previously identified Pγ cross-linking site, PDE6α′-(750–760) (17Artemyev N.O. Natochin M. Busman M. Schey K.L. Hamm H.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5407-5412Crossref PubMed Scopus (53) Google Scholar), revealed a critical Pγ-interacting residue, Met758 (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In a model of the PDE6α′ catalytic domain, Met758 faces the opening of the catalytic cavity (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We then hypothesized that Pγ may interact with additional nonconserved residues located at the perimeter of the cavity, thus allowing Pγ to serve as a lid on the catalytic pocket. In this study, we mutated three candidate Pγ contact residues identified from the model of PDE6α′ and examined these mutants for inhibition by Pγ. The rationale for our search of the catalytic determinants of PDE6 was based on biochemical evidence and the crystal structure of the PDE4 catalytic domain (18Francis S.H. Turko I.V. Grimes K.A. Corbin J.D. Biochemistry. 2000; 39: 9591-9596Crossref PubMed Scopus (19) Google Scholar, 19He F. Seryshev A.B. Cowan C.W. Wensel T.G. J. Biol. Chem. 2000; 275: 20572-20577Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar), which suggests the critical role of the two highly conserved metal binding motifs, His-Asn-X-X-His (I) and His-Asp-X-X-His (II), in the hydrolysis of cyclic nucleotides. We replaced PDE6α′ domains containing motifs I and II into PDE5. Resulting chimeric PDEs and corresponding mutants have been analyzed to test our hypothesis. cGMP was obtained from Roche Molecular Biochemicals. [3H]cGMP was a product of Amersham Pharmacia Biotech. All restriction enzymes were purchased from New England Biolabs. AmpliTaq® DNA polymerase was a product of PerkinElmer Life Sciences, and Pfu DNA polymerase was a product of Stratagene. Rabbit polyclonal His-probe (H-15) antibodies were purchased from Santa Cruz Biotechnology. Zaprinast and all other reagents were purchased from Sigma. Pγ mutants were generated based on the pET11a-Pγ expression vector (21Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Artemyev N.O. Arshavsky V.Y. Cote R.H. Methods. 1998; 14: 93-104Crossref PubMed Scopus (30) Google Scholar). Residues Ile86 and Ile87 were substituted for alanine using PCR-directed mutagenesis. PCR products were obtained using a forward primer containing a NdeI site and a reverse primer containing the mutations and a BamHI site. The fragments were digested with NdeI/BamHI and subcloned into the pET11a-Pγ digested with the same enzymes. The Pγ-subunit and its mutants were expressed in Escherichia coli and purified on a SP-Sepharose fast flow column and on a C4 HPLC column (Microsorb-MW, Rainin) as described (22Artemyev N.O. Arshavsky V.Y. Cote R.H. Methods. 1998; 14: 93-104Crossref PubMed Scopus (30) Google Scholar). Purified proteins were lyophilized, dissolved in 20 mm HEPES buffer, pH 7.5 and stored at −80 °C until use. The constructs for expression of PDE5/PDE6α′ chimeras were obtained based on pFastBacHTb-PDE5 vector (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To obtain Chi20 and Chi21, original restriction sites in pFastBacHTb-PDE5, SpeI and SphI, were eliminated and re-introduced at desired positions to allow a site-directed cloning of PDE6α′ fragments into PDE5. To eliminate two SpeI restriction sites located within the 3′-untranslated region of PDE5 cDNA and the unique SphI site from the multiple cloning sequence of the vector, pFastBacHTb-PDE5 was digested withSpeI/SphI and treated with mung bean nuclease. New SpeI and SphI restriction sites (PDE5 codons for Arg606-His607-Ala608 and Ala618-Leu619-Lys620, respectively) were introduced into the vector using a QuikChangeTM kit (Stratagene). To obtain Chi21 (Fig. 1), a synthetic olygonucleotide duplex, encoding for PDE6α′-(561–574), was ligated into the modified pFastBacHTb-PDE5 vector digested with SpeI andSphI. To generate Chi20, a PCR fragment, encoding for PDE6α′-(575–617), was digested with SpeI/BlpI and subcloned into the modified pFastBacHTb-PDE5 vector digested withSpeI/BlpI(partial). The resulting construct was digested with SpeI/SphI and ligated to the synthetic oligonucleotide duplex encoding for PDE6α′-(561–574). Site-directed mutagenesis of PDE5 was performed using a QuikChangeTM kit. A pair of complementary oligonucleotides encoding for the Ala608→Gly and Ala612→Gly substitutions (PDE5A608G/A612G) was used to PCR-amplify the pFastBacHTb-PDE5 vector. The PCR product was treated with DpnI to eliminate the template and was transformed into E. coli DH5α. Chi16 mutants with single substitutions of residues Lys769, Phe777, and Phe781 by Ala were constructed using PCR-directed mutagenesis. A unique NheI site (PDE5 codons for Pro661-Leu662) was introduced into Chi16 using a QuikChangeTM kit. The 5′-primer sequence included the NheI recognition site. Reverse primers contained a desired mutation and the StuI site. The PCR products were digested with NheI/StuI and subcloned into the modified Chi16 vector cut with the same enzymes. Sequences of all mutants were verified by automated DNA sequencing at the University of Iowa DNA Core Facility. Sf9 cells were harvested at 60 h after infection, washed with 20 mm Tris-HCl buffer, pH 7.8 containing 50 mm NaCl, and resuspended in the same buffer containing a protease inhibitor mixture (10 μg/ml pepstatin, 5 μg/ml leupeptin, and 0.2 mm phenylmethylsulfonyl fluoride). The cell suspensions were sonicated using 30-s pulses for a total duration of 3 min. The supernatants (100,000 ×g, 45 min) were loaded onto a column with a His-Bind resin (Novagen) equilibrated with 20 mm Tris-HCl buffer, pH 7.8, containing 10 mm imidazole. The resin was washed with a 5× volume of the buffer containing 500 mm NaCl and 25 mm imidazole. Proteins were eluted with the buffer containing 250 mm imidazole. β-mercaptoethanol (2 mm) was added to the eluate. PDE5, Chi20, Chi21, and PDE5A608G/A612G were additionally purified using ion-exchange chromatography on a Mono Q® HR 5/5 column (Amersham Pharmacia Biotech). Purified proteins were dialyzed against 40% glycerol and stored at −20 °C. PDE activity was measured using [3H]cGMP as described (23Thompson W.J. Appleman M.M. Biochemistry. 1971; 10: 311-316Crossref PubMed Scopus (47) Google Scholar, 24Natochin M. Artemyev N.O. Methods Enzymol. 2000; 315: 539-554Crossref PubMed Google Scholar). Less than 15% of cGMP was hydrolyzed during these reactions. The K i values for inhibition of PDE activity by Pγ and zaprinast were measured using 0.5 μm cGMP (i.e. <35% of theK m value for chimeric and mutant PDEs). Protein concentrations were determined by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar) using IgG as a standard or by using calculated extinction coefficients at 280 nm. The molar concentrations of Chi20, Chi21, and mutatnt PDEs, [PDE], were calculated based on the fraction of PDE protein in preparations, and the molecular mass of 93.0 kDa. The fractional concentrations of PDE were determined from analysis of the Coomassie Blue-stained SDS gels using a HP ScanJet II CX/T scanner and Scion Image Beta 4.02 software. A typical fraction of Chi16 mutants in partially purified preparations was 10–15%. A typical fraction of purified Chi20, Chi21, and PDE5A608G/A612G was 65–70%. The k catvalues for cGMP hydrolysis were calculated asV max/[PDE]. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar) in 10–12% acrylamide gels. For Western immunoblotting, proteins were transferred to nitrocellulose (0.1 μm, Schleicher & Schuell) and analyzed using rabbit His-probe (H-15) or sheep anti-PDE6α′ antibodies (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 27Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44938) Google Scholar). The antibody-antigen complexes were detected using anti-rabbit or anti-goat/sheep IgG conjugated to horseradish peroxidase and ECL reagent (Amersham Pharmacia Biotech.). Fitting the experimental data to equations was performed with nonlinear least squares criteria using GraphPad Prizm Software. The K i ,K m , and IC50 values are expressed as mean ± S.E. for three independent measurements. Previously, we demonstrated that PDE5/PDE6α′ chimeras containing a PDE6α′ sequence, PDE6α′-(737–784), are effectively inhibited by Pγ, and two residues, Met758 and Gln752, participate in the inhibitory interaction (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Based on the model structure of PDE6α′ (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), three solvent-exposed nonconserved PDE6α′ residues, Lys769, Phe777, and Phe781, were chosen for further mutational analysis of the Pγ binding region (Fig.1 A). A PDE5/PDE6α′ chimera, Chi16 (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), served as a template for single substitutions of these residues by Ala. The Chi16 mutants were expressed in Sf9 insect cells and partially purified. Expression of the K769A, F777A, and F781A mutants have yielded similar amounts of soluble protein (50–100 μg/100 ml of culture). Neither of these mutations has significantly affected the catalytic properties of chimeric PDE. TheK m and k cat values for cGMP hydrolysis for all three mutants were in the 3–10 μmrange, and the 5–10 s−1 range, respectively (TableI). As an additional control for the structural integrity of the catalytic site, mutants of Chi16 were tested for the PDE activity inhibition by zaprinast, a specific competitive inhibitor of PDE5 and PDE6. The largest change, a 2-fold increase in the IC50 value, was caused by the F781A substitution (Table I). Nonetheless, such a change represents an insignificant loss of affinity to zaprinast.Table IFunctional properties of PDE5/PDE6α′ chimerasPDEK mk catIC50for zaprinastK i for PγK i for PγI86AK i for PγI87Aμms −1μmnm(max. effect, %)nm(max. effect, %)nm (max. effect, %)PDE6α′23 ± 21-aThe data are from Ref. 5.35001-aThe data are from Ref. 5.0.28 ± 0.051-aThe data are from Ref. 5.0.17 ± 0.02 (100)1-aThe data are from Ref. 5.0.75 ± 0.08 (95)0.65 ± 0.04 (100)Chi162.8 ± 0.51-bThe data are from Ref. 6.9.01-bThe data are from Ref. 6.0.12 ± 0.011-bThe data are from Ref. 6.3.6 ± 0.4 (90)1-bThe data are from Ref. 6.13 ± 1 (65)6.6 ± 1.0 (70)K769A2.2 ± 0.28.90.16 ± 0.012.9 ± 0.4 (90)F777A4.8 ± 0.77.20.19 ± 0.0119 ± 2 (45)96 ± 13 (45)64 ± 8 (25)F781A6.1 ± 0.77.50.28 ± 0.0231 ± 5 (65)49 ± 8 (40)32 ± 2 (55)M758A9.5 ± 0.91-bThe data are from Ref. 6.8.91-bThe data are from Ref. 6.0.26 ± 0.011-bThe data are from Ref. 6.97 ± 10 (75)1-bThe data are from Ref. 6.N/A (<20)N/A (<20)PDE53.3 ± 0.41-aThe data are from Ref. 5.9.61-aThe data are from Ref. 5.0.54 ± 0.02A608G/A612G14 ± 11050.30 ± 0.03Chi2012 ± 11160.35 ± 0.05Chi2117 ± 21100.39 ± 0.05PDE activity was measured using [3H]cGMP (24Natochin M. Artemyev N.O. Methods Enzymol. 2000; 315: 539-554Crossref PubMed Google Scholar). TheK m values of PDE6α′ or PDE5 and PDE5/PDE6α′ chimeras were determined in the presence of 0.1 μCi [3H]cGMP and 0.1–500 μm of unlabeled cGMP. TheK i and IC50 values for inhibition of PDE activity by Pγ and zaprinast were measured using 0.5 μmcGMP. The results are presented as the mean ± S.E. for three independent measurements.1-a The data are from Ref. 5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar.1-b The data are from Ref. 6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar. Open table in a new tab PDE activity was measured using [3H]cGMP (24Natochin M. Artemyev N.O. Methods Enzymol. 2000; 315: 539-554Crossref PubMed Google Scholar). TheK m values of PDE6α′ or PDE5 and PDE5/PDE6α′ chimeras were determined in the presence of 0.1 μCi [3H]cGMP and 0.1–500 μm of unlabeled cGMP. TheK i and IC50 values for inhibition of PDE activity by Pγ and zaprinast were measured using 0.5 μmcGMP. The results are presented as the mean ± S.E. for three independent measurements. The test of the ability of Chi16 mutants to be inhibited by Pγ showed that the K769A mutation had no effect on the inhibitory interaction with Pγ (K i 2.9 nm) (Table I). Two other mutants, F777A and F781A, displayed significant impairments in the inhibition by Pγ. The F777A substitution reduced both the maximal inhibition of PDE activity by Pγ (∼45%) and theK i value (K i of 19 nm). The inhibition of F781A mutant by Pγ also was incomplete (∼65%) and associated with an increase in theK i value (K i of 31 nm) (Fig. 2 A and Table I). C-terminal Pγ mutants were designed based on the evidence for the critical role of the Pγ C terminus in PDE6 inhibition (21Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar). The two extreme C-terminal Pγ residues, Ile86 and Ile87, were replaced by Ala to obtain the PγI86A and PγI87A mutants, respectively. The Pγ mutants were analyzed for their ability to inhibit trypsin-activated PDE6α′ (tPDE), Chi16, and the M758A, F777A, and F781A mutants (Fig. 2; TableI). PγI86A and PγI87A fully inhibited tPDE activity. However, the potency of the inhibition was reduced ∼4–5-fold (K i of 0.75 nm for PγI86A andK i of 0.65 nm for PγI87A, compared with Ki of 0.15–0.2 nm for Pγ). A similar increase in the K i values was observed from the inhibition of Chi16 activity by PγI86A (K i of 13 nm) and PγI87A (K i of 7 nm) (Fig. 2, B and C; Table I). Yet, PγI86A and PγI87A did not fully inhibit Chi16, maximal inhibition was 65 and 70%, respectively. (Fig. 2, B and C; Table I). No appreciable inhibition of M758A by either Pγ mutant was seen even at inhibitor concentrations as high as 5 μm. The inhibition of F777A by PγI86A was partial (45%) with theK i value of 96 nm, whereas PγI87A inhibited this Chi16 mutant with an even smaller maximal effect (25%,K i of 64 nm). The F781A mutant was inhibited by PγI86A and PγI87A with K i values of 49 and 32 nm and maximal effects of 40 and 55%, respectively (Fig. 2, B and C; Table I). Two conserved metal binding motifs found in all PDEs are absolutely critical for cyclic nucleotide hydrolytic activity (18Francis S.H. Turko I.V. Grimes K.A. Corbin J.D. Biochemistry. 2000; 39: 9591-9596Crossref PubMed Scopus (19) Google Scholar, 19He F. Seryshev A.B. Cowan C.W. Wensel T.G. J. Biol. Chem. 2000; 275: 20572-20577Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar). To identify the structural elements responsible for the unique catalytic properties of PDE6, chimeric PDE5/PDE6α′ have been generated by introduction into PDE5 of PDE6α′ domains containing metal binding motifs, I and II. A replacement of the PDE6α′-(562–617) segment into PDE5 yields a chimeric PDE5/PDE6α′, Chi20, that incorporates both PDE6α′ metal binding sites and the connecting sequence (Fig. 1 A). Chi20 was expressed in Sf9 cells as a functional enzyme at ∼400 μg/100 ml and purified to ∼ 65–70% purity (Fig. 1 B). The catalytic characteristics of Chi20 were examined in comparison to those of PDE5 and native PDE6α′. PDE6α′ has reportedK m (17–25 μm) andk cat (3500–4500 moles of cGMP per mole of PDE·s) values for cGMP hydrolysis that are ∼5 and ∼400-fold higher than the respective constants for PDE5 (5Granovsky A.E. Natochin M. McEntaffer R.L. Haik T.L. Francis S.H. Corbin J.D Artemyev N.O. J. Biol. Chem. 1998; 273: 24485-24490Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14964-14970Abstract Full Text PDF PubMed Google Scholar, 11Turko I.V. Francis S.H. Corbin J.D. J. Biol. Chem. 1998; 273: 6460-6466Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar). The catalytic parameters of Chi20 were significantly different from those of PDE5. Chi20 hydrolyzed cGMP with the K m value of 12 μm, which is ∼4-fold higher than theK m value for PDE5 but similar to that of PDE6α′ (Table I). The maximal activity of 116 moles of cGMP per mole of PDE·s for Chi20 is ∼10-fold higher than that of PDE5. Chi20 was inhibited by zaprinast with the IC50 value of 0.35 μm, which is comparable with that of PDE5 (Table I). To determine the role of individual metal binding motifs and their adjacent regions in cGMP hydrolysis by PDE6, we inserted a PDE6α′ fragment corresponding to the helix-α6 (20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar), PDE6α′-(562–574), into PDE5 (Chi21) (Fig. 1). The catalytic properties of Chi21 and the inhibition by zaprinast (K m of 17 μm,k cat of 110 moles of cGMP per mole of PDE·s, and IC50 0.39 μm) were similar to those of Chi20. The alignment of sequences from different PDE families corresponding to the α6 helix shows a glycine residue, PDE6α′Gly562, conserved only in photoreceptor PDEs (Fig.3 A). A second Gly residue, PDE6α′Gly566, is conserved in PDE6α′ and PDE6α, but substituted by Ala in PDE6β and PDE5 (Fig. 3 A). To test the hypothesis that Gly562 and Gly566 of PDE6α′ are responsible for the differences in catalytic properties of Chi21 and PDE5, a doubly substituted mutant of PDE5, A608G and A612G, was expressed and purified from Sf9 cells. Similar to Chi20 and Chi21, PDE5A608G/A612G hydrolyzed cGMP with a K m value of 14 μm and a k cat value of 105 moles of cGMP per mole of PDE·s (Table I). An interaction between PDE6 catalytic and inhibitory Pγ-subunits keeps the visual effector enzyme inhibited in the dark. Previous biochemical studies have established that the γ-subunit of photoreceptor PDE inhibits the enzyme activity by blocking its catalytic site (29Granovsky A.E. Natochin M. Artemyev N.O. J. Biol. Chem. 1997; 272: 11686-11689Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The major inhibitory domain has been localized to the Pγ C terminus (21Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Brown R.L. Biochemistry. 1992; 31: 5918-5925Crossref PubMed Scopus (61) Google Scholar). Recently, we have demonstrated that Pγ inhibits the activity of PDE5/PDE6α′ chimera, Chi 16, containing residues PDE6α′-(737–784) (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Essential Pγ binding residues, Gln752 and Met758, of PDEα′ have been identified via mutagenesis of Chi16 (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). A model of the PDE6α′ catalytic domain places Met758 at the opening of the catalytic pocket (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Hypothetically, to ensure an effective catalytic block, the Pγ C terminus may lie over or might be inserted into the catalytic cavity. The former appears more likely because the catalytic pockets of different cyclic nucleotide PDEs are made up of highly conserved residues, whereas the inhibition by Pγ is a unique attribute of PDE6. We speculated that to cover the catalytic pocket, the Pγ C terminus, besides Met758, interacts with additional nonconserved residues located at the perimeter of the entrance to the active site. The fact that the introduction of PDE6α′-(737–784) into PDE5/PDE6α′ chimera leads to a full inhibition of the PDE activity by Pγ suggests the PDE6α′-(737–784) segment contains most if not all residues interacting with the Pγ C terminus. In the PDE6α′ model, PDE6α′-(737–784) comprises about half of the catalytic cavity mouth. Residues at three positions within PDE6α′-(737–784) (Lys769, Phe777, and Phe781) are conserved among photoreceptor PDEs but have nonhomologous substitutions in PDE5. Supporting our hypothesis, replacement of two residues, Phe777 and Phe781, by Ala in Chi16 has resulted in mutant PDEs that in comparison with Chi16 were less potently and incompletely inhibited by Pγ. Phe777 and Phe781 are located next to each other, opposite to the Met758 side of the catalytic opening (Fig. 3, B and C). Thus, it appears that the Pγ C terminus makes a bridge over the catalytic pocket. Such a model provides an interesting explanation to the results of an earlier study that examined inhibition of PDE6 by C-terminally truncated Pγ mutants (21Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Truncations of one or two of the C-terminal Ile86-Ile87 residues led to substantial increases in the K i value, whereas further truncations, up to 8–11 C-terminal residues, reduced the maximal inhibition of PDE6 activity without significantly affecting theK i value (21Skiba N.P. Artemyev N.O. Hamm H.E. J. Biol. Chem. 1995; 270: 13210-13215Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A plausible interpretation is that PγIle86-Ile87 interact with residues on one side of the catalytic pocket and other residues, perhaps Pγ-(77–85), stretch over the catalytic cavity until Pγ reaches the opposite side. Accordingly, removal of PγIle86-Ile87decreases the affinity of Pγ for the PDE6 catalytic subunit, whereas progressive removal of Pγ-(77–85) residues gradually facilitates access of cGMP to the catalytic site. To determine the orientation of the Pγ C terminus against the catalytic site and identify point-to-point interactions with PDE6α′, we examined the inhibition of Chi16 and the M758A, F777A, and F781A mutants of Chi16 by two Pγ mutants, PγI86A and PγI87A. The simplest prediction is that if a C-terminal Ile of Pγ interacts with one of the three PDE6α′ residues, the corresponding mutant PDE would be inhibited comparably by Pγ and by the Pγ mutant. Complicating this prediction, side chains of Phe777 and Phe781 make a hydrophobic contact and thereby may support each other in the interaction with Pγ. The analysis of inhibition of Chi16 mutants by Pγ mutants indicates that Ile86 and Ile87 of Pγ interact with Phe777 and Phe781 of PDE6α′. Moderate increases in the K i values and reductions in the maximal inhibition of F777A and F781A caused by the PγI86A substitution suggest that Ile86 probably contacts one or both the PDE6α′ residues. The failure of PγI86A to inhibit M758A is consistent with the notion that Ile86 binds Phe777/781, but not Met758. The lack of inhibition is likely caused by the inability of M758A and PγI86A to establish at least two of the three critical contacts involving Met758, Phe777, and Phe781. The PγI87A mutant did not appreciably inhibit the activity of the M758A mutant PDE. PγI87A inhibited F781A stronger than F777A pointing to a probable contact between PγIle87 and Phe781of PDE6α′. The incomplete inhibition of mutant PDEs by Pγ or Pγ mutants most likely reflects equivalent partial inhibition of both active sites of the catalytic dimer, rather than the loss of inhibition at one site. The analysis of Pγ secondary structure predicts an α-helical structure for the C-terminal residues Pγ-(75–84) (30Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). The C terminus of Pγ, Pγ-(75–87), manually docked to the PDE6α′ catalytic site is shown in Fig. 3, B and C. The model assumes the helical structure of Pγ-(75–84) and the contacts between PγIle86-Ile87 and PDE6α′Phe777-Phe781. This orientation of Pγ is also consistent with Gln752 of PDE6α′ (6Granovsky A.E. Artemyev N.O. J. Biol. Chem. 2000; 275: 41258-41262Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) making a contact with a Pγ residue located N-terminally to Pγ-(75–87). The remarkable ability of photoreceptor PDEs to hydrolyze cGMP with a catalytic rate constant of ∼4000–5500 moles of cGMP per mole of PDE·s (12Mou H. Grazio III, H.J. Cook T.A. Beavo J.A. Cote R.H. J. Biol. Chem. 1999; 274: 18813-18820Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Leskov I.B. Klenchin V.A. Handy J.W. Whitlock G.G. Govardovskii V.I. Bownds M.D. Lamb T.D. Pugh E.N. Arshavsky V.Y. Neuron. 2000; 27: 525-537Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 14Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar, 15Dumke C.L. Arshavsky V.Y. Calvert P.D. Bownds M.D. Pugh Jr., E.N. J. Gen. Physiol. 1994; 103: 1071-1098Crossref PubMed Scopus (50) Google Scholar) is essential to the signal amplification in the visual cascade. All catalytic subunits of cyclic nucleotide PDEs contain two strictly conserved metal binding motifs, His-Asn-X-X-His (motif I) and His-Asp-X-X-His (motif II). In PDE6α′ these motifs are as follows:557His-Asn-Trp-Arg-His561 and597His-Asp-Ile-Asp-His601. The crucial role of the metal ions and the binding motifs for PDE catalytic activity has been recently supported by a crystallographic study of the PDE4 catalytic domain (20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar). Rather than forming separate metal binding sites, both motifs are involved in coordination of two bound metal ions, ME1 and ME2 (20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar). For example, ME1, most likely a tightly bound Zn2+, is coordinated by the His residue (His561of PDE6α′) from motif I, and the His and Asp residues from motif II (His597-Asp598). A model of cAMP docked in the PDE4 active site demonstrates that ME1 and ME2 bind the cyclic phosphate, position a potential water molecule for the nucleophilic attack, and would serve to stabilize the transition state (20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar). In view of the role of metal binding sites in hydrolysis of cyclic nucleotides, we have considered the motifs I and II as probable structural determinants of the catalytic properties of PDE6. Motifs I and II are practically identical in PDE5 and PDE6. Therefore, a spatial orientation of these sites might be a potential key factor for cGMP hydrolysis. Motif I comprises the N-terminal potion of the helix-α6, and motif II is in the loop connecting helices 7 and 8. A PDE5/PDE6α′ chimera, Chi20, was generated by replacing a PDE6α′ domain containing helices α6-α8 into PDE5. The analysis of Chi20 revealed a more than 10-fold increase in the maximal catalytic rate accompanied by a ∼5-fold increase in the K m value. Subsequent chimeric PDE, Chi21, containing only helix α6 of PDE6α′ displayed catalytic properties similar to those of Chi20. An alignment of sequences of photoreceptor PDEs and PDE5 corresponding to the helix-α6 shows a high degree of homology with the notable exception of residues at two positions corresponding to PDE6α′ Gly562 and Gly566. Gly562 of PDE6α′ is conserved only in the PDE6 family, but substituted by Ala in PDE5 (Fig. 3 A). Importantly, Gly562immediately follows His561 from motif I. His561, by analogy to PDE4, is involved in coordination of ME1, and in the positioning of His557 to accomplish the protonation of the O3′ leaving group (20Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (322) Google Scholar). To probe the role of the Gly residues, a doubly substituted PDE5 mutant, A608G/A612G, has been made. The k cat value of the A608G/A612G mutant was comparable with those of Chi20 and Chi21, and ∼10-fold higher then that of PDE5. These results suggest that the Gly residues are in part responsible for the catalytic characteristics of PDE6. Most likely, they allow for a positioning of motif I that is most favorable for cGMP hydrolysis. Other yet to be defined determinants contribute to the unique catalytic power of PDE6, because the achievedk cat value is still ∼40–50-fold lower thank cat described for native activated PDE6. Overall, our results suggest that a progressive incorporation of PDE6 domains or residues into PDE5 not only allows a structure-function analysis of PDE6, but also represents a realistic approach to generate a chimeric enzyme that would be functionally indistinguishable from PDE6. We thank Boyd Knosp for assistance with molecular modeling.

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