Regulation 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 the rod PDE6 catalytic dimer is composed 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 the α4-helix of GAFa (close to the cGMP binding site) and the β1/β2 loop of GAFb as key motifs that have been previously proposed to communicate with the catalytic domains of PDE6. 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 certain retinal degenerative diseases.
Molecular, Cellular and Biomedical Sciences
Digital Object Identifier (DOI)
Gupta, Richa; Liu, Yong; Wang, Huanchen; Nordyke, Christopher T.; Puterbaugh, Ryan Z.; Varga, Krisztina; Chu, Feixia; Ke, Hengming; Vashisth, Harish; and Cote, Rick H., "Structural analysis of allosteric regulation of the regulatory GAF domains of cone photoreceptor phosphodiesterase-6 (PDE6) by cGMP and the inhibitory γ-subunit" (2019). Faculty Publications. 705.