Fig. 4 | Nature Communications

Fig. 4

From: Mechanistic insights into the role of prenyl-binding protein PrBP/δ in membrane dissociation of phosphodiesterase 6

Fig. 4

Interaction of PrBP/δ with the PDE6. a Size-exclusion chromatography of PDE6–PrBP/δ complex. Analytical size-exclusion chromatography confirms the stoichiometry of the complex as PDE6–PrBP/δ2. Runs for PDE6 only (black; molecular weight (MW) of ~217 kDa) and PrBP/δ only (red; MW of ~17 kDa). Gel filtration reveals complex formation and peak shift for PDE6–PrBP/δ at ratios of 1:2 (green) and 1:3 (blue). Excess PrBP/δ at the ratio of 1:3 (blue) elutes as a separate peak at MW of ~17 kDa (Supplementary Fig. 4). b Electron microscopy of the PDE6–PrBP/δ complex. Averages of all classes for PDE6 and PDE6–PrBP/δ complexes obtained by electron microscopy show a bell-shaped PDE6 molecule, where the densities at the top represent the more massive catalytic domains. Representative class-averages of PDE6 sample show one or two additional protruding densities for around 5% particles, which likely represents endogenous PrBP/δ (~17 kDa) of the native sample. Representative class-averages of PDE6–PrBP/δ sample show one or two additional protruding densities for around 78% particles, which represents PDE-bound PrBP/δ. Additional protruding densities are highlighted by red arrows in the lower right box of each panel of classes. c Activation assay of PDE6 as a monitor of PrBP/δ induced solubilization of PDE6. Gα*-induced activity of PDE6 (50 nM; circles) was measured as a function of PrBP/δ concentration in the presence of isolated disc membranes (10 µM) and Gα* (1 µM). Increasing concentration of PrBP/δ progressively decreases PDE6 activity till it was equal to the PDE6 activity measured in solution (dotted red line). Data points were fitted to a model with two different PrBP/δ binding sites on PDE6 (solid black line; K1 = 0.04 ± 0.01 µM and K2 = 0.7 ± 0.9 µM; R2 = 0.996) or with two identical binding sites (dashed gray line; K = 0.07 ± 0.01 µM; R2 = 0.994). Inset: semi-logarithmic replot of the data. All data points are replicates of three measurements depicted with standard error

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