Probing domain functions of chimeric PDE6alpha'/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.

A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.

Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases.

Three methods have been used to assess the conformational effects associated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and relative compactness (Stokes radius) of the protein molecule. The combined data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein.

Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding.

cGMP-binding phosphodiesterases contain two kinetically distinct cGMP-binding sites (a and b), and each site contains a conserved N(K/R)XnFX3DE sequence. N276A, K277A, K277R, D289A, and E290A mutants in the N276KX7FX3DE290 sequence of site a (higher affinity site) of bovine cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) were expressed in High Five cells and purified. The cGMP-binding affinities of three mutants [K277A (Kd approximately 12 microM), D289A (Kd approximately 24 microM), and N276A (Kd approximately 60 microM)] were decreased in comparison with wild-type enzyme (Kd = 1.3 microM), which suggested an important role for Asn276, Lys277, and Asp289 in cGMP binding. These residues could be presented as a putative NKXnD motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, Km for cGMP, and IC50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.

An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase.

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.