Two of the most important regulatory second messengers for cardiac function are the cyclic nucleotides, cAMP and cGMP. These molecules work together to regulate calcium and thereby the rate and force of contraction. In the cardiocyte, a major control point for cAMP and cGMP is at the level of cyclic nucleotide phosphodiesterase 2A (PDE2A). In cardiac stromal cells the major PDE is PDE1A, a calcium/calmodulin regulated enzyme. These enzymes serve as an intracellular receptor and regulators for cGMP and a regulator for both cAMP and cGMP levels in the cell. Two regulatory allosteric binding sites for cGMP are found on PDE2 in what are now recognized as N-terminal GAF domains. Binding of cGMP to these domains """"""""activate"""""""" the catalytic domain of the enzyme. However, very little is known about the 3 dimensional structure of these regulatory domains nor the molecular mechanisms by which they regulate the catalytic domain except by analogy to other enzymes. In PDE1A, the N-terminal domain also regulates catalytic activity but in this case the regulator is Ca++/CaM. Again, nothing is known about the 3 dimensional structure or mechanistically how binding of CaM relieves inhibition of the enzyme. In the first part of this application we propose to determine by X-ray crystallographic and nuclear magnetic resonance techniques the primary structure of the regulatory GAF domains of PDE2A. We also propose to use this information along with that obtained from direct binding and mutagenesis to explore the molecular mechanism by which ligand occupancy allows the GAF domains to regulate catalytic activity of the enzyme. In pilot experiments we have been able for the first time to establish large-scale bacterial expression and purification protocols for the cGMP binding GAF domains of PDE2. We have also worked out smaller but adequate expression protocols for the PDE1A and 2A holoenzymes from Sf9 cells. More importantly, very recently, we have been able to determine conditions for reproducibly producing crystals for the GAF domain that diffract to approximately 3.0 angstroms. We are proposing here to refine the structure of this GAF domain to higher resolution in both the presence and absence of regulatory ligand. As we have so far only been able to obtain crystals in the prese3nce of the GAF domain ligand, cGMP, we further propose to determine the three dimensional structure of the non-liganded GAF domain by NMR techniques After completing the initial structures of the PDE2A GAF domains, we intend to use similar methods to compare them to the structure of the cGMP binding GAF domains of the other GAF domain containing PDEs, PDE5A, PDE10A and PDE11A. In the second part, we propose to determine the crystal structure of the catalytic domain of the cardiac Ca2+/CaM dependent PDE1A and the solution structure of the isolated CaM-binding/inhibitory domain of PDRE1A. This enzyme, which is expressed predominantly in stromal cells of the hear5t, is thought to regulate pools of cAMP and cGMP that effect matrix deposition. This enzyme serves as a pivotal point of cross talk between the Ca2+ and cyclic nucleotide pathways. We have recently worked out an isolation and purification procedure for the catalytic subunit of PDE1A and have obtained small crystals that diffract to approximately 8 angstroms. We propose to produce better crystals and refine the structure of this PDE. Finally, we propose to determine the sites and mechanisms by which the inhibitory domain of PDE1A interacts with the catalytic subunit by a series of NMR structural determinations and by complementary binding/function experiments.
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