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

Academic Article

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.
  • Authors

  • Chu, Feixia
  • Hogan, Donna
  • Gupta, Richa
  • Gao, Xiong-Zhuo
  • Nguyen, Hieu T
  • Cote, Rick
  • Status

    Publication Date

  • September 6, 2019
  • Published In

    Keywords

  • allosteric regulation
  • chemical cross-linking
  • phosphodiesterase 6 (PDE6)
  • quantitative mass spectrometry
  • visual transduction
  • Digital Object Identifier (doi)

    Start Page

  • 3677
  • End Page

  • 3689
  • Volume

  • 431
  • Issue

  • 19