Fig. 4: Mutation of the distant, coevolving residues alters cGMP-induced allosteric conformational change in the isolated GAFa domain of PDE5.
From: Coevolving residues distant from the ligand binding site are involved in GAF domain function
A Cartoon representation of apo (PDB: 3MF016) and holo (PBD: 2K3122) PDE5 GAFa domain structures aligned using all Cα-atoms (left panel) and using Cα-atoms of α2 helix (right panel) highlighting structural changes (apo: gray, holo: black) induced upon cGMP binding. B Schematic showing the BRET2-based GAFa domain conformational biosensor that shows increased energy transfer upon cGMP binding likely due to the structural juxtaposition of helix α5 against helix α2, and thus, bringing the BRET donor and acceptor closer. C Cartoon representation of the PDE5 GAFa domain highlighting the distant, coevolving residues L267 and F295 (spheres in blue) that were mutated to A. The ligand, cGMP, is shown in the stick representation. D Western blot analysis of the cell lysates prepared from HEK293T cells transfected with either the WT or mutant GAFa domain biosensor plasmids 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. 4. E Graphs showing bioluminescence spectra obtained from lysates prepared from cells expressing either RLuc alone (control) or the indicated GAFa biosensor constructs (containing the WT or the mutant PDE5 GAFa domains sandwiched between GFP2 and RLuc proteins). Note the appearance of a second peak in the WT GAFa domain biosensor, indicating energy transfer from RLuc (donor) to GFP2 (acceptor), while this peak is reduced in both L267A and F295A mutant GAFa domain biosensors. Data shown are representative of multiple experiments and were fitted using either a single (RLuc alone) or two Gaussian (GAFa domain biosensors) distributions. F Graph showing BRET values (ratio of GFP2 and RLuc emissions) of the WT, L267A, and F295A mutant GAFa domain biosensors in the absence and presence of cGMP (1 μM). Note the significant reduction in the basal BRET values of the L267A and F295A mutant biosensors compared to the WT biosensor. Data shown are the mean ± standard deviation (S.D.; error bars) from five measurements of a representative experiment, with experiments performed four times. G Graphs showing percentage increase in BRET values for the WT, L267A, and F295A mutant GAFa domain biosensors with the indicated cGMP concentrations. Note the shift in the cGMP dose-response curves of the L267A and F295A mutant GAFa domain biosensors in comparison to the WT GAFa domain. Data shown are the mean ± standard deviation (SEM.; error bars) from a representative experiment, with experiments performed four times. H Graph showing log(EC50) values for cGMP-induced conformational change in the WT, L267A, and F295A mutant GAFa domain biosensor. Data shown are mean ± S.D. obtained from four independent experiments. Inset, values on top indicate the cGMP EC50 values (mean ± S.D.) of the respective GAFa domain biosensors. (I, J) Graph showing melting temperatures (Tm) and slope of the melting temperature curves of the WT and L267A and F295A mutant GAFa domain biosensors. Data shown are mean ± S.D. obtained from three independent melting temperature curves (curves are provided in Supplementary Fig. 9). 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.
