Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 432, Issue 21, Pages 5765-5783Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2020.08.026
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Funding
- National Eye Institute [R01 EY05798]
- National Institute of General Medical Sciences [P20 GM113131, GM059791, P20GM103449]
- National Science Foundation [CLF 1307367]
- National Institute of Child Health and Human Development [R01 HD093783]
- National Science Foundation Major Research Instrumentation Award [DBI-1828319]
- Intramural Research Program of the NIH, National Institute of Environmental Health Sciences [ACI-1548562]
- NSF [ACI-1548562]
- Extreme Science and Engineering Discovery Environment (XSEDE) Comet resource at San Diego Supercomputer Center [TGMCB160183]
- National Science Foundation EPSCoR grant [OIA-1757371]
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Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 angstrom resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory gamma-subunit (P gamma). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network. Molecular dynamics simulations of cone GAFab revealed differences in conformational dynamics of the two subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of P gamma to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled P gamma identified the central polycationic region of P gamma interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases. (C) 2020 Elsevier Ltd. All rights reserved.
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