Cyclic nucleotide binding to the tandem GAF domains allosterically regulates the activity of the catalytic domain of CyaB1 and CyaB2, adenylyl cyclases from Anabaena. Additionally, Na+ ions have been shown to inhibit the GAF domain-mediated activation of the catalytic domain in these proteins. This inhibitory effect was found to be specific to Na+ ions, and other monovalent cations did not induce such effects on the GAF domain. Importantly, the inhibitory effect of Na+ ion was observed even when the GAF domains from the Anabaena adenylyl cyclase was exchanged for the GAF domains of rat PDE2 indicating the conservation of this mode of regulation across GAF domains. Specific interaction aside, altering the salt concentration will result in an alteration in the ionic strength of the buffer, unless controlled for. An alteration in the ionic strength could affect interactions between amino acids residues in a protein by electrostatic screening leading to changes in the structural properties of the protein. Therefore, an attempt was made to determine the role of Na+ ions on the structure of the cGMP-binding GAFa domain of PDE5. A BRET-based GAFa conformational sensor construct was utilized for this. Lysate prepared from HEK 293T cells transfected with the GFP2-GAFa-Rluc sensor construct was incubated with varying concentrations of NaCl, and intramolecular BRET efficiency (measured as a ratio of GFP2 emission and Rluc emission) was monitored. Increasing NaCl concentration in the buffer resulted in a concentration-dependent reduction in the BRET of the GAFa construct with an effective concentration value for 50% reduction in BRET efficiency (EC50) of 70 ± 43 mM (Fig. 1A). Since BRET efficiency is dependent upon the structure of the protein, this result suggests the possibility of a structural regulation of the GAFa domain by Na+ ions. This was then followed up with experiments with the full-length PDE5 (A2 isoform) domain that contains two GAF domains in tandem. Similar to the isolated GAFa domain, the full-length PDE5 also showed a concentration-dependent reduction in the BRET efficiency with increasing NaCl concentrations with an EC50 value of 218 ± 140 mM (Fig. 1B). However, contrary to the specific inhibitory effect of Na+ seen with the Anabaena proteins, increasing concentrations of KCl also reduced the BRET efficiency (EC50 value of 268 ± 80 mM) suggesting that it could be a general effect of the increase in the salt concentration in the buffer. To confirm this, a construct containing only the GFP2 and Rluc domains, but no domains from PDE5, was used. As seen with the GAFa and full-length PDE5 sensors, the GFP2-Rluc construct also showed a dose-dependent reduction in the BRET efficiency (EC50 values of 95 ± 55 mM and 93 ± 10 mM for NaCl and KCl, respectively) (Fig. 1C). These results suggest that the higher salt concentrations in the buffer negatively impact the energy transfer between Rluc and GFP2.
The reduction in the BRET efficiency at higher salt concentrations could occur due to either a change in the structure of the proteins resulting in a reduction in the energy transfer or a decrease in the quantum yield of GFP2 protein such that the fluorescence output of GFP2 is reduced, even though a similar amount of non-radiative energy is transferred. To delineate the mechanism behind this, the activity of the Rluc and the fluorescence of the GFP2 proteins in the GFP2-Rluc construct were independently monitored. Increasing the salt concentration of the buffer resulted in a dose-dependent increase in the emission of the Rluc protein with EC50 values of 183 ± 63 mM and 198 ± 62 mM for NaCl and KCl, respectively (Fig. 1D), without significantly impacting the GFP2 emission of the fusion protein (Fig. 1E). Similar increase in the emission was also observed for a construct of the Rluc protein only (EC50 of 154 ± 5 mM) (Fig. 1F). These results suggests that the decrease in the BRET efficiency of the PDE5 constructs is due to an increase in Rluc emission without a concomitant increase in the fluorescence of the GFP2 protein. The increase in the Rluc emission at higher salt concentrations is contrary to the sensitivity of the Firefly luciferase, which is an ATP-consuming enzyme, to higher ionic strengths.
While the general increase in the Rluc activity observed in the presence of higher salt concentrations is interesting in itself, it is not obvious as to how alterations in the salt concentration would impact the ligand-binding induced conformational change in a sensor protein. Therefore, experiments were performed to understand the impact of NaCl in the ability of BRET-based, PDE5 conformational sensors to detect ligand-induced conformational changes. The salt concentration of 10 mM and 100 mM were chosen based on the EC50 value of BRET efficiency reduction (Fig. 1A). Incubation of lysates prepared from cells expressing the GAFa sensor construct with varying concentrations of cGMP in the presence of low (10 mM) or physiologically relevant (100 mM) NaCl revealed similar increases in the BRET efficiency (EC50 values of 38 ± 3 vs. 22 ± 5 nM at 10 and 100 mM NaCl, respectively). On the other hand, incubation of lysates prepared from cells expressing the full-length PDE5 sensor construct with varying concentrations of cGMP or sildenafil at low (10 mM) NaCl concentration revealed a lack of change in the BRET efficiency of the sensor (Fig. 1H & I), which was seen at 100 mM NaCl concentration (EC50 values of 104 ± 50 and 100 ± 52 nM for cGMP and sildenafil, respectively) (Fig. 1H & I).
The lack of change in the BRET efficiency of the full-length PDE5 sensor with cGMP in the presence of low NaCl concentration could be a result of enzymatic hydrolysis of cGMP by the catalytic domain of PDE5. The use of non-hydrolyzable cGMP analogues may thus confirm the effect of salt on PDE5 conformational change. However, experiments described here were performed in the presence of ethylenediaminetetraacetic acid (EDTA), which would chelate metal ions in the buffer, and thus render the catalytic domain of PDE5 inactive. Therefore, the absence of change in BRET efficiency is not a consequence of hydrolysis of cGMP added during the assay. It is also important to note that no change in BRET was observed in the presence of sildenafil citrate at low NaCl concentration. Therefore, the requirement for 100 mM NaCl for the detection of ligand-induced conformational changes in PDE5 suggests a role for Na+ ions by either specific binding to PDE5, or buffer ionic strength in regulating the structure of PDE5. Since the GAFa sensor responded equally well to cGMP in the two NaCl concentrations, it is possible that the GAFb domain in PDE5 is responsible for the sensitivity to NaCl concentration.