Molecular determinants of cGMP-binding to chicken cone photoreceptor phosphodiesterase

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Molecular determinants of cGMP-binding to chicken cone photoreceptor phosphodiesterase

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Title: Molecular determinants of cGMP-binding to chicken cone photoreceptor phosphodiesterase
Author: Huang, Daming, 1971-
Abstract: The photoreceptor phosphodiesterase (PDE6) is a key regulator in the signaling of photo-transduction. It possesses two GAF domains, GAF-A and GAF-B. Sequence homology alignment between the GAF domains of PDE2, 5 and 6 suggests that GAF-A is the non-catalytic cGMP binding site of PDE6. Based on 3D-modeling of the GAF-A domain of chicken cone PDE6 with the crystal structure of mouse PDE2A GAF-B domain, residues making side-chain contact with cGMP were identified. Mutagenesis of these residues indicated that the overall architecture of the GAF cGMP-binding pocket is largely conserved between PDE2, 5 and 6. Residues F123, D169, T172, T176 are critical for the binding of cGMP and their corresponding alanine mutants lost the ability to bind cGMP completely. Three of the 4 crucial residues map to the H-4 helical structure of GAF domain. It is speculated that this region is a necessary structure unit for cGMP binding. The GAF-A domain was demonstrated to be the non-catalytic cGMP binding site of PDE6. In addition, we have characterized the photoreceptor PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion-exchange chromatograph. The first and second peaks were identified as chicken cone and rod photoreceptor PDEs respectively by mass spectrometry. Chicken photoreceptor PDEs have an ion-exchange profile, cGMP-binding and enzyme kinetics similar to the bovine photoreceptor PDEs. Our characterization of chicken photoreceptor PDEs suggests that the cGMP-binding and enzymatic characteristics are well conserved between birds and mammals. Furthermore, the cGMP-binding of chicken cone PDE6 holo-enzyme was shown to have very similar cGMP-binding kinetics to bacteria expressed individual GAF-A or GAF-A/B domains.
Description: Thesis (Ph. D.)--University of Washington, 2004
URI: http://hdl.handle.net/1773/5095

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