REGULATION OF PHOTORECEPTOR PHOSPHODIESTERASE AND ITS ACTIVATION BY TRANSDUCIN ELUCIDATED BY STRUCTURAL AND BIOCHEMICAL APPROACHES

Xiongzhuo Gao, University of New Hampshire, Durham

Abstract

The photoreceptor phosphodiesterase (PDE6) is the central enzyme in the visual signaling pathway. Rod PDE6 is a heterotetramer which consists of a catalytic dimer (Pαβ) and two inhibitory γ subunits (Pγ). PDE6 differs from other phosphodiesterases (PDEs) in its unique inhibitory Pγ subunit and its interactions with a G protein, transducin (Tα) during PDE6 activation. The activity of PDE6 has to be strictly regulated for light detection by rod and cone photoreceptors. The hypotheses of my research are: (1) the overall structure of PDE6 is similar to other GAF- containing PDEs but it has some unique structural features which allow Pγ and Tα to bind; (2) multiple regions of Pγ and Tα are involved in regulation of PDE6 activity during visual transduction.

The first aim of my research is to define the important regions of Pγ stabilizing its binding to Pαβ and stimulating cGMP binding to GAFa domain of Pαβ. In order to reach this goal, we designed a series of N-terminal truncated Pγ mutants. We expressed those Pγ mutants in E. coli cells and purified them to over 90% purity. Through inhibition assays of Pαβ using these Pγ truncation mutants, we identified two regions of Pγ (a.a. 27-30 and 35-38) that primarily contribute to stabilizing Pγ binding to Pαβ; in contrast, the poly-proline region of Pγ (a.a. 18 to 26) contributes little to binding. Using these Pγ truncation mutants, we also showed that residues 27-30 are required for maximally stimulating cGMP binding to the GAFa domain of PDE6, and that neighboring amino acids (a.a. 21- 26) enhance the interactions between Pγ and GAFa domain of Pαβ that result in stimulation of cGMP binding. Experiments relying on short, synthetic Pγ peptides supported the above observations.

The second aim is to elucidate the structure of PDE6 holoenzyme using chemical cross-linking and mass spectrometry (MS) analysis in conjunction with homology modeling. The refined PDE6 homology model revealed an “open” conformation in its catalytic domain, which is different from the structure of another GAF-containing PDE (PDE2). The cross-linking map of Pγ with Pαβ showed that Pγ binds to Pαβ in an extended linear pattern with the N-terminus of Pγ asymmetrically interacting with the GAFa domain of Pαβ. Reconstituted PDE6 using Pαβ and photoactivatable Pγ at specific sites demonstrated that Pγ position 23 specifically cross-linked to Pα GAFa domain and Pγ position 30 preferentially binds to GAFb domain. Other positions of Pγ did not show binding preference.

The final aim of my thesis is to characterize the interaction of PDE6 with Tα. We prepared lipid vesicles to improve the interaction of these two proteins. Under the optimized conditions, we observed a cross-linked PDE6-Tα complex which migrates at 150 kDa on SDS-PAGE. By analyzing those bands, we identified multiple interaction sites between Tα and PDE6 The cross-linking results indicate that two Tα molecules bind to PDE6 to fully activate the enzyme. We also found that association of Tα with PDE6 causes an interaction shift of N-terminus of Pγ from GAFa domain to GAFb domain. Cysteine scanning mutagenesis identified that Pγ G59 and several residues in the Pγ C-terminal α-helical regions are important for PDE6-Tα interaction. This study revealed that PDE6 has a complicated regulatory mechanism by changing Pγ interaction with Pαβ and Tα during light activation.