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B. M. Barren, L. Gakhar, H. Muradov, S. Ramaswamy, N. O. Artemyev; Structural Studies of Phosphodiesterase 6 Inhibition by the -subunit. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2996.
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© ARVO (1962-2015); The Authors (2016-present)
Phosphodiesterase 6 (PDE6) is the key effector enzyme in the vertebrate phototransduction cascade. The atomic structures of chimeric PDE5/6 catalytic domain complexed with catalytic-site drugs and the inhibitory domain of the PDE6 γ-subunit (Pγ) were investigated in order to gain structural insights into PDE6 function.
The construct for chimeric PDE5/6 catalytic domain (PDE5/6cd) containing the M-loop/helix15 region of human cone PDE6 was generated and the protein was expressed in E. coli. Purified PDE5/6cd was analyzed for the enzymatic activity and the ability to interact with the inhibitory Pγ peptide Pγ70-87. Protein crystals of PDE5/6cd complexed with sildenafil (Viagra) or IBMX and Pγ70-87 were grown using the hanging drop vapor diffusion technique and analyzed by X-ray crystallography. The structures of PDE5/6cd-sildenafil and PDE5/6cd-IBMX-Pγ70-87 were solved by molecular replacement using a known structure of PDE5 catalytic domain as a template.
Purified recombinant PDE5/6cd hydrolyzed cGMP with the KM value of 4.2 µM and was inhibited by sildenafil with a Ki of 2.5 nM. Pγ-70-87 inhibited PDE5/6cd and trypsin-activated bovine rod PDE6 with comparable potencies indicating that PDE5/6cd retains nearly all of the interactions between PDE6 and the Pγ C-terminus. The crystal structures show the relationships between the Pγ inhibitory interaction site within the M-loop of PDE6 and proximate drug-binding sites in the catalytic pocket.
The PDE5/6cd-Pγ70-87 structure yielded important molecular details on the critical inhibitory interaction of the Pγ C-terminus with the PDE6 catalytic subunits. The interplay of the Pγ- and the sildenafil-binding sites at the catalytic pocket of PDE6 will help to understand side effects of the drug on vision.
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