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

Granovsky, Alexey E.; Natochin, Michael; McEntaffer, Randall L.; Haik, Tamara L.; Francis, Sharron H.; Corbin, Jackie D.; Artemyev, Nikolai O.

doi:10.1074/jbc.273.38.24485

Cited by 58 publications

(78 citation statements)

References 42 publications

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“…A role of this motif in binding cyclic nucleotide is supported by several publications describing the effects of several mutations to the motif, nearly all to Ala. In a chimera containing PDE6 GAF-A͞B and PDE5 catalytic domain, an Asn-to-Ala mutation in either GAF domain abolished noncatalytic cGMP binding (22). The Asp to Ala mutation in PDE5 in GAF-A or -B reduced affinity for cGMP (23).…”

Section: Discussionmentioning

confidence: 77%

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

Martinez

Bruder

Schultz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

103

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In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A͞B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A͞B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-Å crystal structure of the tandem GAF-A͞B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A͞B. Unexpectedly, the cyaB2 GAF-A͞B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the ␤2-␤3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed.cAMP ͉ phosphodiesterase

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Section: Discussionmentioning

confidence: 77%

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

Martinez

Bruder

Schultz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

103

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“…The original PDE6/PDE5 chimera retained the catalytic properties of PDE5 and the cGMP binding properties of PDE6. 23 Site-directed mutagenesis using the PDE6/PDE5 chimera has identified two sites on PDE5 (Ala(608) and Ala(612)) that accelerate catalysis 10-fold when substituted for glycine residues (found at positions 562 and 566 of cone PDE6). 24 These residues are near the metal binding motif 15 that represents the catalytic site of the enzyme.…”

Section: Similarities and Differences Between Pde5 And Pde6mentioning

confidence: 99%

“…20,21 (While PDE5 has been reported to copurify with the PDE6 g subunit, 22 the physiological significance is uncertain because the g subunit lacks binding or inhibitory activity toward PDE5 in vitro. 23 )…”

Section: Similarities and Differences Between Pde5 And Pde6mentioning

confidence: 99%

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

2004

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Phosphodiesterase 6 (PDE6) is highly concentrated in the retina. It is most abundant in the internal membranes of retinal photoreceptors, where it reduces cytoplasmic levels of cyclic guanosine monophosphate (cGMP) in rod and cone outer segments in response to light. The rod PDE6 holoenzyme comprises a and b catalytic subunits and two identical inhibitory c subunits. Each catalytic subunit contains three distinct globular domains corresponding to the catalytic domain and two GAF domains (responsible for allosteric cGMP binding). The PDE6 catalytic subunits resemble PDE5 in amino-acid sequence as well as in three-dimensional structure of the catalytic dimer; preference for cGMP over cyclic adenosine monophosphate (cAMP) as a substrate; and the ability to bind cGMP at the regulatory GAF domains. Most PDE5 inhibitors inhibit PDE6 with similar potency, and electroretinogram studies show modest effects of PDE5 inhibitors on visual function-an observation potentially important in designing PDE5-specific therapeutic agents.

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“…The tandem GAF domains in mammalian PDEs 2, 5, and 6, which bind cGMP, have been analyzed intensively (2,(7)(8)(9)(10)(11)(12). The studies have been hampered by the fact that cGMP concurrently serves as a substrate and as an allosteric regulator creating an unsolvable kinetic conundrum.…”

mentioning

confidence: 99%

Characterization of the Tandem GAF Domain of Human Phosphodiesterase 5 Using a Cyanobacterial Adenylyl Cyclase as a Reporter Enzyme

Bruder

Schultz

2006

Journal of Biological Chemistry

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We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K D for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.In essentially all eukaryotic cells, cAMP and cGMP act as second messengers. Therefore the concentration of these nucleotides is meticulously regulated by the rates of biosynthesis and breakdown (1, 2). Although for years most studies have focused on mammalian ACs, 2 more recently PDEs have come into focus. Based on biochemical properties and sequence alignments, mammalian PDEs have been grouped into 11 families (PDE1-PDE11). All catalytic domains are similar (20 -45% identity (3)), and peculiar regulatory features are mediated by differing N-terminal domains. PDEs 2, 5, 6, 10, and 11 contain N-terminal tandem GAF domains (the acronym derives from proteins of initial identification: mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli transcription factor FhlA (4, 5)). To date, GAF domains have been identified in more than 3000 proteins. They bind a variety of small ligands and promote protein dimerization (2, 6 -8).The tandem GAF domains in mammalian PDEs 2, 5, and 6, which bind cGMP, have been analyzed intensively (2, 7-12). The studies have been hampered by the fact that cGMP concurrently serves as a substrate and as an allosteric regulator creating an unsolvable kinetic conundrum. Because the tandem GAF domains of mammalian PDEs are closely related to those of cyanobacterial ACs (6, 13, 14), we have replaced the cyanobacterial tandem GAF domain in the cyaB1 AC, which imparts cAMP regulation, with that of rPDE2a. The chimera is regulated by cGMP acting via the GAF B domain and uses ATP as a substrate (6).Here, we successfully replaced the tandem GAF domain of the cyaB1 AC with that of the hPDE5, again and surprisingly generating a cGMP-regulat...

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

Cited by 58 publications

References 42 publications

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

Characterization of the Tandem GAF Domain of Human Phosphodiesterase 5 Using a Cyanobacterial Adenylyl Cyclase as a Reporter Enzyme

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