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  • Meeting abstract
  • Open Access

Asymmetric properties of rod cGMP Phosphodiesterase 6 (PDE6): structural and functional analysis

  • 1Email author,
  • 1, 5,
  • 2, 6,
  • 1,
  • 1,
  • 1, 3,
  • 1, 7,
  • 2,
  • 1, 4,
  • 1 and
  • 1
Contributed equally
BMC Pharmacology and Toxicology201516 (Suppl 1) :A76

https://doi.org/10.1186/2050-6511-16-S1-A76

  • Published:

Keywords

  • Cryo Electron Microscopy
  • PDE6 Activation
  • cGMP Phosphodiesterase
  • Asymmetric Property
  • Full Enzyme Activity

Photoreceptor cGMP Phsophodiesterase 6 (PDE6) is the effector molecule of visual signal transduction and mediates fast response of light signals. The rod holo-PDE6 comprises catalytic (α, β; each ~ 90 kDa) and two identical inhibitiory (γ; ~ 10 kDa) subunits. The catalytic subunits comprise N-terminal tandem GAF domains followed by C-terminal catalytic domains and isoprenylations for membrane-association. Contrary to activation of other tandem GAF comprising PDEs, PDE6 activation does not occur via cGMP-induced concerted conformational changes. Rather two copies of the α-subunit of retinal G-Protein (Gα*), transducin, activate PDE6 by partially displacing the inhibitory subunits. The activation of PDE6 has therefore been described as a “de-inhibition”. The affinity of Gα* to PDE6 and the enzymatic activity of the intermediary 1:1 complex is highly disputed, therefore a conclusive activation model is lacking so far.

Our combined structural, enzymatic and computational investigations deal with the activation-mechanism of PDE6. Our cryo electron microscopy (EM) structure of PDEαβ catalytic core shows an elongated bell-shaped structure with symmetric side-by-side arrangement of the two subunits with flexible membrane-binding domains. A comparison with nearly full-length inactive PDE2A structure [1] suggests that less compaction of both subunits and higher degree of conformational freedom of the catalytic domains result in constitutive activation of PDE6αβ, which is kept inactive by the inhibitory γ subunits. Furthermore, the structure of PDE6 suggests Gα* binding-sites pointing to opposing faces. The enzymatic characterization using Gα* titration of the PDE6 however reveal striking asymmetry of the two catalytic subunits with a high and a low affinity binding site for Gα*. Occupancy of the PDE6 with one Gα* induces negligible activity, whereas occupancy with two copies of Gα* leads to full enzyme activity. Such an activation mechanism constitutes a “coincidence switch” that allows noise filtering (i.e., spontaneously produced Gα* do not activate PDE6). Our spatiotemporal simulation work indeed confirms that spontaneously generated Gα* lead to the formation of singly occupied PDE6 and only a high local concentration of Gα*, as produced by an active receptor (rhodopsin), leads to doubly Gα* occupied effector complex. Therefore the localized large concentration of Gα* combined with the asymmetric properties of PDE6 constitutes a “density switch” that allows suppression effector level noise and reliable reporting of single quantum events in rod photoreceptor cells.

Notes

Authors’ Affiliations

(1)
Biophysics and Medical Physics, Charité – Universitätsmedizin Berlin, 10117 Berlin, Germany
(2)
Department of Mathematics, Computer Science and Bioinformatics, Freie Universität Berlin, 14195 Berlin, Germany
(3)
Microscopy & Cryo Electron Microscopy Group, Max-Planck Institut für Molekulare Genetik, 14195 Berlin, Germany
(4)
Zentrum für Biophysik und Bioinformatik, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
(5)
Present address: Research Group Structural Dynamics of Proteins, Center of Advanced European Studies and Research (caesar), 53175 Bonn, Germany
(6)
Present address: Department of Molecular & Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA
(7)
Present address: FEI VSG (Visualization Science Group), Zuse Institut Berlin, 14195 Berlin, Germany

References

  1. Pandit J, Forman MD, Fennell KF, Dillman KS, Menniti FS: Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct. PNAS. 2009, 106: 18225-18230. 10.1073/pnas.0907635106.View ArticlePubMedPubMed CentralGoogle Scholar

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