Trypanosome cyclic nucleotide phosphodiesterase 2B binds cAMP through its GAF-A domain.
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.
Laxman
Sunil
S
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
Rascón
Ana
A
Beavo
Joseph A
JA
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D011994:Recombinant Proteins; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517275:PDE2A protein, human; D006152:Cyclic GMP; D000645:Ammonium Sulfate