Abstract
Purpose::
Five members of the PDE superfamily contain regulatory GAF domains. For PDE2 and PDE5, cGMP binding to the GAF domains induces an intramolecular allosteric transition that accelerates catalysis of the enzyme. The structural and biochemical similarities between PDE5 and PDE6 predict a similar allosteric mechanism for PDE6 catalytic subunits upon cGMP binding, but evidence for intramolecular allosteric communication between catalytic and GAF domains of PDE6 is lacking. The purpose of this work is to determine whether the cGMP binding sites on PDE6 play an allosteric role in the visual signaling pathway.
Methods::
Bovine rod PDE6 was purified from bovine retinas. The inhibitory Pγ subunit (Pγ) and various truncated Pγ peptides were chemically synthesized or purified following bacterial expression. A method to stably load cGMP onto the GAF domains of PDE6 catalytic dimer (Pαß) using Pγ1-45 and EDTA was developed. Radiotracer and phosphate release assays were used to measure catalytic activity, and membrane filtration employed for measurements of radiolabeled cGMP binding.
Results::
Using a combination of EDTA, Pγ1-45 and cGMP, we were able to bind 2 cGMP per PDE so that cGMP remained tightly bound following restoration of cGMP hydrolytic activity. cGMP-loaded Pαß showed no effects on Km or Vmax compared to Pαß with unoccupied GAF domains. However, cGMP-loaded Pαß showed a 2-fold increase of sildenafil inhibition potency. The affinity of Pγ63-87 for Pαß was also enhanced about 2-fold by cGMP binding to the GAF domains in presence of Pγ1-45. Addition of Pγ1-45 also greatly stimulated vardenafil binding to the catalytic domains of Pαß. This allosteric mechanism is reciprocal, in that vardenafil binding to the catalytic domains induced a high-affinity cGMP binding site within the GAF domains of Pαß.
Conclusions::
cGMP binding to the GAF domains not only regulates Pγ affinity (as shown in previous studies) but also directly communicates with the catalytic domains in a reciprocal manner. This intramolecular allosteric mechanism may play a role in the negative feedback mechanism of PDE6 during bright light adaptation during visual transduction.
Keywords: photoreceptors • enzymes/enzyme inhibitors • protein structure/function