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Structural analysis of the regulatory GAF domains of cGMP phosphodiesterase elucidates the allosteric communication pathway" (2019). Faculty Publications. 705. AbstractRegulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that the physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6.Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network that communicates with the PDE6 catalytic domains. Molecular dynamics (MD) simulations of cone GAFab revealed asymmetry in the two GAFab subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases. domain is preceded by N-terminal tandem GAF domains (individually referred to as GAFa and GAFb). The tandem GAFab domains are believed to serve several functions for PDEs, including enhancing dimerization of PDE catalytic subunits and allosterically communicating with the catalytic domain to regulate catalytic activity 3 . Whereas structural determinations have revealed the domain organization and dimerization interface of PDE2 and PDE5 GAFab dimers 6,7 , nearly full-length PDE2 8 , and the rod photoreceptor PDE6 holoenzyme 9-11 , much remains to be learned about the allosteric mechanisms underlying GAF domain regulation of PDE catalytic activity. This gap in knowledge arises from challenges in comparing the conformational dynamics of PDEs in various liganded states, thereby hindering the design of allosteric modulators targeting the regulatory GAF domains of PDEs for therapeutic applications 12 . All five GAF-containing PDEs are believed to bind only a single cyclic nucleotide molecule per catalytic subunit. In the case of PDE5, PDE6, and PDE11, cGMP (or cGMP analogs) selectively bind to the first (GAFa) domain, whereas PDE2 and PDE10 selectively bind cGMP and cAMP, respectively, to the se...