Structural, biophysical, and biochemical characterization of the GAF domains from phosphodiesterases 5 and 6

Phosphodiesterases 5 and 6 control intracellular levels of cGMP through hydrolysis and regulate numerous signal transduction pathways in physiological processes such as smooth muscle cell contraction (PDE5) and vision (PDE6). The catalytic domains of both proteins are regulated by allosteric binding of cGMP to an N-terminal GAF domain (GAF A) of a tandem GAF domain pair. In my thesis, I present atomically detailed structures of both cGMP-bound GAF A domains as determined by NMR (PDE5A) and X-ray crystallography (PDE6C). Each domain adopts a conserved overall fold with well-defined cGMP binding pockets. However, the nucleotide coordination is distinct with a series of differing binding contacts. Nucleotide binding specificity is provided in each by the orientation of an Asp/Asn residue that is within hydrogen bond distance of the guanine ring. In PDE5A, a D196A mutation disrupts cGMP binding and increases cAMP affinity causing an altered cAMP-bound structural conformation in constructs containing only GAF A. NMR studies reveal that both GAF domains undergo significant cGMP-dependent conformational changes. In PDE5A, GAF B stabilizes the highly dynamic multi-state apo-GAF A domain, presumably via direct interaction. In contrast, cGMP-free GAF A from PDE6C is more defined and in a single "open" state with flexible elements. Biophysical characterization of the GAF domains by circular dichroism and analytical ultracentrifugation further underlines the differences of the conformational changes and dimerization properties between the two PDEs. Structural features of the GAF domains from PDE5 and PDE6 revealed here provide a basis for future investigations of the regulatory mechanism of both PDEs and the design of GAF-specific small molecule inhibitors of PDE function. Preliminary hits from small molecule library screens by saturation transfer difference NMR highlight the potential of GAF domains as drug targets that could be further pursued for the identification of drug lead compounds for the treatment of various PDE-related disease conditions. The two structures of the GAF domains extend our understanding of how GAF domains bind small molecules and how they function as regulatory units.