Allosteric Regulation of Rod Photoreceptor Phosphodiesterase 6 (PDE6) Elucidated by Chemical Cross-Linking and Quantitative Mass Spectrometry. Issue 19 (6th September 2019)

• Record Type: Journal Article
• Authors: Chu, Feixia; Hogan, Donna; Gupta, Richa; Gao, Xiong-Zhuo; Nguyen, Hieu T.; Cote, Rick H.
• Is Part Of: Journal of molecular biology. Volume 431:Issue 19(2019)
• Page Start: 3677
• Page End: 3689
• Publication Date: 2019-09-06
• Subjects: phosphodiesterase 6 (PDE6) -- chemical cross-linking -- quantitative mass spectrometry -- visual transduction -- allosteric regulation; PDE6 phosphodiesterase-6 -- EM electron microscopy -- qCX-MS chemical cross-linking and quantitative mass spectrometric analysis -- DSS disuccinimidyl suberate -- DE-XL dead-end cross-link; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2019.07.035
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ2 ) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ2, and Pαβγ2 –cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases. Graphical abstract: Unlabelled Image Highlights: PDE6 is regulated by the binding of inhibitory Pγ and noncatalytic cGMP. cGMP binding in GAFa domains changes the conformation of catalytic domains. Pγ and cGMP display asymmetric binding to the GAFa domains of rod PDE6. The Pαβ catalytic dimer adapts an open conformation in different allosteric states.