Abstract
Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.
Highlights
Cyclic nucleotide phosphodiesterases (PDEs)1 regulate cAMP and cGMP signaling pathways by controlling the intracellular levels of cyclic nucleotides
The current study reports high affinity cAMP binding to the TbPDE2B GAF-A domain, characterizes the binding, and provides data that suggest this cAMP binding is important for regulation of the trypanosome PDE activity
TbPDE2B Binds cAMP through Its GAF-A Domain— HEK293T cells were transiently transfected with a full-length native TbPDE2B expression plasmid or a TbPDE2B catalytic domain fragment plasmid
Summary
Materials—[5,8-3H]cAMP and [8-3H]cGMP were purchased from PerkinElmer Life Sciences. cAMP and cGMP (sodium salts), phenylmethylsulfonyl fluoride, dithiothreitol, and isopropyl 1-thio--D-galactopyranoside were obtained from Sigma. The cells were resuspended and lysed in lysis buffer (phosphate-buffered saline (Invitrogen) plus 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, pH 7.5) by microfluidization (10,000 p.s.i.) (Microfluidics) and centrifuged at 16,000 ϫ g for 30 min. Immunoprecipitation—Transfected and control (untransfected) cell supernatants were first cleared by incubation with 15 l of protein G-agarose beads at 4 °C for 45 min (with gentle mixing). Cleared supernatants were incubated with the anti-V5 antibody (Invitrogen) and 30 l of protein G-agarose beads for 2–3 h in a volume of 1 ml (with gentle mixing). Determination of Binding Equilibrium—To determine the time necessary for cNMP binding to reach equilibrium (for both the purified holoenzyme as well as the N terminus ϩ GAF-A construct), binding assays were done as described earlier with 10 nM [3H]cAMP and 6 nM purified protein. Protein levels in Western blots (with an anti-V5 antibody) were normalized using ImageJ (NIH) to measure the relative densities of the detected bands
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