Fig. 5: Mutation of distant, coevolving residues alters cGMP-induced allosteric regulation in the full-length PDE5.
From: Coevolving residues distant from the ligand binding site are involved in GAF domain function
A Schematic showing the mechanism of the BRET2-based, full-length PDE5A2 conformational biosensor in the absence and presence of cGMP. B Western blot analysis of the cell lysates prepared from HEK293T cells transfected with either the WT or mutant full-length PDE5A2 biosensor plasmid constructs and probed using an anti-GFP antibody showing the expression of biosensor constructs. Whole blot image used for generating the figure is shown in Supplementary Fig. 6. C Graphs showing bioluminescence spectra obtained from lysates prepared from cells expressing the WT and L267A and F295A mutant PDE5 biosensor constructs. Note the reduction in the GFP2 emission peaks in the L267A and F295A mutant PDE5 biosensors. Data shown are mean ± S.D. from a representative experiment, with experiments performed three times. D Bar graph showing the basal BRET values of the WT, L267A, and F295A mutant PDE5 biosensors. Note the significant reduction in the basal BRET values of the L267A and F295A mutants compared to the WT GAFa domain. E Graphs showing percentage decrease in BRET values of the WT, L267A, and F295A mutant PDE5 biosensors after 30 min incubation with the indicated cGMP concentrations. Note the decrease in the maximum % change in BRET as well as the rightward shift in the cGMP dose-response curves of the mutant biosensors. Inset, values indicating the EC50 of cGMP-induced conformational change for the respective GAFa domains in the full-length PDE5 biosensor. For (D, E), data shown are mean ± S.D. from three experiments, with each experiment performed in triplicates. F Graph showing log(EC50) (mean ± S.D.) values of cGMP-binding induced conformational change in the isolated GAFa domain and full-length PDE5 BRET-based biosensors against ΔG (mean ± S.D.) values obtained from the MD simulation runs of the isolated GAFa domain (WT and mutants). Data shown are mean ± S.D. from three experiments. All p-values shown in the figure were obtained from Student’s t-tests (two-tailed, unpaired, equal variance) performed for the L267A or F295A mutants against the WT GAFa domain. For all tests, a p-value of <0.05 was considered statistically significant.
