Molecular Determinants for Cyclic Nucleotide Binding to the Regulatory Domains of Phosphodiesterase 2A

Wu, Albert Y.; Tang, Xiao; Martinez, Sergio E.; Ikeda, Kaori; Beavo, Joseph A.

doi:10.1074/jbc.m404287200

Cited by 77 publications

(101 citation statements)

References 54 publications

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“…S1). It is catalytically active, with a V max of 32 Ϯ 11 mol/min/mg for cGMP hydrolysis, which is comparable to values reported previously for the native enzyme purified from bovine heart (22) or for purified recombinant mouse PDE2A holoenzyme (23). Activation by cGMP is also similar: approximately 4-fold at a half-maximal cGMP concentration of 1.4 M (Fig.…”

Section: Resultssupporting

confidence: 71%

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Pandit

Forman

Fennell

et al. 2009

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

151

159

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We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long ␣-helices. The subunits cross at the GAF-B/ catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.cGMP activation ͉ GAF domains ͉ PDE-2A T he cyclic nucleotides cAMP and cGMP are ubiquitous intracellular signaling molecules that mediate a vast array of biological processes throughout the body. The means by which these two molecules participate in diverse functions, in different cell types and within single cells, involves tight regulation of the spatial and temporal residence of their concentrations. The phosphodiesterases (PDEs) are a superfamily of enzymes that metabolically inactivate cAMP and cGMP (1) to play key roles in both these aspects of regulation. The PDEs are modular enzymes characterized by a relatively conserved C-terminal catalytic domain and a more variable N-terminal domain involved in regulation of activity, subcellular localization, and interactions with other proteins. There are 11 PDE gene families, with different families encoded by one to four genes, and further diversity derived from alternative splicing. The PDE families differ broadly in specificity and affinity for cAMP and cGMP. Much has been learned about the molecular bases for these differences from studies of X-ray crystal structures of the catalytic domains of

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Section: Resultssupporting

confidence: 71%

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Pandit

Forman

Fennell

et al. 2009

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

151

159

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“…The Asp to Ala mutation in a GST fusion of PDE5 GAF-A modestly decreased binding (21). In PDE2A GAF-B, all five NKFDE residues were mutated to Ala (24). Each one abolished binding of cGMP and cAMP.…”

Section: Discussionmentioning

confidence: 99%

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.

Self Cite

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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“…[20][21][22] Strains expressing these enzymes regain mating competence, which is deficient in strains lacking PDE activity, 23 indicating that these PDEs are functional when expressed in S. pombe (data not shown).…”

Section: Construction Of Fission Yeast Strains Expressing Mammalian Pdesmentioning

confidence: 99%

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

et al. 2008

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Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3′,5′-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens. (Journal of Biomolecular Screening 2008:62-71)

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Molecular Determinants for Cyclic Nucleotide Binding to the Regulatory Domains of Phosphodiesterase 2A

Cited by 77 publications

References 54 publications

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

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

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

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