Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor

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1 Correspondence Disciplines Biochemistry Biophysics Keywords Luciferase Bioluminescence Resonance Energy Transfer (BRET) Type of Observation Standalone Type of Link Orphan Data Submitted Jan 19, 2017 Published May 22, x Triple Blind Peer Review The handling editor, the reviewers, and the authors are all blinded during the review process. Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor Kabir H Biswas, Sandhya S Visweswariah Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Mechanobiology Institute, National University of Singapore, Singapore ; Department of Molecular Reproduction, Development and Genetics Indian Institute of Science Abstract Cyclic guanosine monophosphate (cgmp) hydrolyzing phosphodiesterase 5 (PDE5) is multidomain protein, in which cgmp binding to the regulatory GAFa domain allosterically increases the cgmp hydrolytic activity of the catalytic domain. In addition to cyclic nucleotides, GAF domains in phosphodiesterases have been proposed to be regulated by sodium (Na + ) ions. The current study was initiated to investigate the effect of Na + ions on the structure of the GAFa domain of PDE5. Bioluminescence Resonance Energy Transfer (BRET) experiments with various sensor constructs (the GFP 2 -GAFa- Rluc, GFP 2 -PDE5-Rluc containing the full-length PDE5, and a control, GFP 2 -Rluc, construct that does not contain any domains from PDE5) showed a general salt (NaCl and KCl)-induced reduction in the BRET efficiency. Independent determination of Rluc and GFP 2 emission of the GFP 2 -Rluc construct revealed that increased salt concentration enhances Rluc activity without concomitantly increasing GFP 2 fluorescence, thus providing a basis for the decrease in the BRET efficiency. The isolated GAFa domain sensor showed similar changes in BRET efficiency with varying concentrations of cgmp at both low (10 mm) and physiologically relevant (100 mm) NaCl concentrations. However, the full-length PDE5 sensor failed to respond to both cgmp and sildenafil at low (10 mm) NaCl concentration. These results suggest a role for NaCl in regulating the structural changes in PDE5, and thus NaCl should be included at 100 mm in BRET assays to detect ligand-induced conformational changes. Additionally, the increase in Rluc activity at higher salt concentrations reported here could be utilized to detect changes in ionic strengths in live cells. Objective To determine the effect of buffer NaCl concentration on ligand-induced changes in BRET efficiency of PDE5 constructs. Full Open Access Supported by the Velux Foundation, the University of Zurich, and the EPFL School of Life Sciences. 4.0 Creative Commons 4.0 This observation is distributed under the terms of the Creative Commons Attribution 4.0 International License. Introduction Phosphodiesterase 5 is a key regulator of cgmp signaling in a variety of cell types including vascular smooth muscle cells, and in which it functions to control their contractility [1]. It is a multidomain protein with two N-terminally located regulatory GAF domains (GAFa and GAFb) in tandem, and C-terminally located catalytic domain. While the GAFb domain is not known to bind any ligand, the GAFa domain binds cgmp with high specificity [2], and cgmp binding to the GAFa domain allosterically regulates the activity of the catalytic domain [3] [4]. The structural change induced upon cgmp binding to the GAFa domain has been utilized in developing highly specific, live cell cgmp sensors [2] [5] [6]. The catalytic domain of PDE5 specifically hydrolyzes cgmp [7] [8], and has been targeted with a number of pharmaceutical inhibitors, including sildenafil, for treatment of diseases such as penile erectile dysfunction and pulmonary hypertension [9] [10]. Structural studies have revealed that the binding of these inhibitors is associated with specific structural changes in the catalytic domain [11]. Ligand bindinginduced structural changes in both the GAFa and the catalytic domains in the full-length PDE5 have been extensively explored by developing a Bioluminescence Resonance Energy Transfer (BRET)-based intramolecular conformational sensor [12]. BRET is being increasingly utilized for monitoring activation of proteins and signaling DOI: /matters Matters (ISSN: ) 1

2 Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor pathways in live cells [13] [14] [15]. The underlying mechanism behind BRET is the non-radiative transfer of energy from a bioluminescent donor such as Renilla luciferase (Rluc) to a fluorescent acceptor such as GFP 2 (a variant of GFP optimized for excitation by Rluc emission) [16]. Key parameters that affect the resonance energy transfer efficiency include the distance between the donor and acceptor, their relative orientation, the quantum yield of the donor and the refractive index of the medium [17]. A typical BRET assay is performed by incubating live cells or cell lysates with the Rluc substrate, and measuring the GFP 2 fluorescence and Rluc emission. BRET efficiency is determined as a ratio of GFP 2 and Rluc emissions. a DOI: /matters Matters (ISSN: ) 2

3 Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor Figure Legend Effect of salt (NaCl or KCl) on the conformational change in BRET-based PDE5 sensors. (A, B & C) Graphs showing the BRET efficiencies (ratio of GFP 2 and Rluc emission at 37 ) determined from lysates prepared from cells expressing the GAFa (A), the fulllength PDE5 (B) and the GFP 2 -Rluc (C) sensor constructs at various salt concentrations. Note the concentration-dependent decrease in the BRET efficiency at higher salt concentrations. Insets in each panel show a plot of EC 50 values for individual constructs. (D) Graph showing the luciferase emission (counts per second; CPS) of lysates prepared from cells expressing the GFP 2 -Rluc sensor construct at various salt concentrations. Inset shows a plot of EC 50 values. (E) Graph showing the fluorescence of lysates prepared from cells expressing the GFP 2 -Rluc sensor construct at various salt concentrations. (F) Graph showing the luciferase activity of lysates prepared from cells expressing the Rluc protein at various salt concentrations. Inset shows a plot of EC 50 values. (G) Graph showing the percentage change in the BRET efficiency of lysates prepared from cells expressing the GAFa sensor construct with increasing concentrations of cgmp at low (10 mm) and relevant (100 mm) NaCl concentrations. Inset shows a plot of EC 50 values. (H & I) Graph showing the percentage change in the BRET efficiency of lysates prepared from cells expressing the PDE5 sensor construct with increasing concentrations of cgmp (H) and sildenafil (I) at low (10 mm) and relevant (100 mm) NaCl concentrations. Insets show a plot of EC 50 values (ND, could not be determined). Data shown are mean ± S.E.M of a minimum of two measurements from a representative experiment. Results & Discussion Cyclic nucleotide binding to the tandem GAF domains allosterically regulates the activity of the catalytic domain of CyaB1 and CyaB2, adenylyl cyclases from Anabaena [18] [19]. Additionally, Na + ions have been shown to inhibit the GAF domain-mediated activation of the catalytic domain in these proteins [20]. This inhibitory effect was found to be specific to Na + ions, and other monovalent cations did not induce such effects on the GAF domain [20]. 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 [20]. 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 [21] [22] leading to changes in the structural properties of the protein [23]. 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 [2]. Lysate prepared from HEK 293T cells transfected with the GFP 2 -GAFa-Rluc sensor construct was incubated with varying concentrations of NaCl, and intramolecular BRET efficiency (measured as a ratio of GFP 2 emission and Rluc emission) was monitored [2] [12]. 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 (EC 50 ) of 70 ± 43 mm (Figure A). Since BRET efficiency is dependent upon the structure of the protein [12] [24], 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 [1]. Similar to the isolated GAFa domain, the full-length PDE5 also showed a concentrationdependent reduction in the BRET efficiency with increasing NaCl concentrations with an EC 50 value of 218 ± 140 mm (Figure B). However, contrary to the specific inhibitory effect of Na + seen with the Anabaena proteins [20], increasing concentrations of KCl also reduced the BRET efficiency (EC 50 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 GFP 2 and Rluc domains, but no domains from PDE5, was used. As seen with the GAFa and full-length PDE5 sensors, the GFP 2 -Rluc construct also showed a dose-dependent reduction in the BRET efficiency (EC 50 values of 95 ± 55 mm and 93 ± 10 mm for NaCl and KCl, respectively) (Figure C). These results suggest DOI: /matters Matters (ISSN: ) 3

4 Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor that the higher salt concentrations in the buffer negatively impact the energy transfer between Rluc and GFP 2. 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 GFP 2 protein such that the fluorescence output of GFP 2 is reduced, even though a similar amount of non-radiative energy is transferred [17]. To delineate the mechanism behind this, the activity of the Rluc and the fluorescence of the GFP 2 proteins in the GFP 2 -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 EC 50 values of 183 ± 63 mm and 198 ± 62 mm for NaCl and KCl, respectively (Figure D), without significantly impacting the GFP 2 emission of the fusion protein (Figure E). Similar increase in the emission was also observed for a construct of the Rluc protein only (EC 50 of 154 ± 5 mm) (Figure F). 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 GFP 2 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 [25]. 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 [2] [12] to detect ligandinduced conformational changes. The salt concentration of 10 mm and 100 mm were chosen based on the EC 50 value of BRET efficiency reduction (Figure A). 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 (EC 50 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 (Figure H & I), which was seen at 100 mm NaCl concentration (EC 50 values of 104 ± 50 and 100 ± 52 nm for cgmp and sildenafil, respectively) (Figure H & 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. The present study is directed towards understanding the effect of NaCl concentration on the structure as well as ligand-induced conformational changes in PDE5-based constructs using the BRET technology. The enzymatic activity of Rluc protein used as a donor in the BRET technology is increased upon an increase in the buffer salt (NaCl as well as KCl) concentration resulting in a net decrease in the BRET efficiency of all GFP 2 and Rluc containing sensor constructs tested here. The GAFa domain-based cgmp sensor responds to cgmp at both low as well as physiologically relevant NaCl concentrations. However, the response of the full-length PDE5-based sensor is sensitive to NaCl concentrations, and fails to show changes in BRET efficiency at low NaCl DOI: /matters Matters (ISSN: ) 4

5 concentrations. Observations reported here will be useful in both designing in vitro conformational sensor assays [2] [12] [24] [26] as well as live cell assays involving changes in intracellular ionic strength [27] [28]. One of the limitations of the present study is the use of crude lysates for determining both the BRET efficiency as well as the luciferase activity. While lysates prepared from transfected cells is experimentally straightforward, the presence of other cellular components could potentially impact the measurements. To alleviate this issue, assays should be performed with purified proteins. Additionally, the effect of increased salt concentrations on the structures of the proteins should be assessed independently using assays such as circular dichroism [20] or hydrogen-deuterium exchange [26]. Intracellular ionic strength is tightly controlled in a living cell and an alteration in the intracellular ionic strength could activate specific signaling pathways in the cell such as activation of ion channels or transporters as a counter measure [27] [31]. Indeed, a specific class of proteins have been evolved for the purpose of detecting changes in the intracellular ionic strengths [28]. However, there is a lack of genetically encoded, synthetic reporter for intracellular ionic strength. The sensitive activation of Rluc upon increase in the salt concentration reported here points towards its utilization as a luminescence-based, live cell indicator of changes in the intracellular ionic strength. Additional Information Methods and Supplementary Material Please see This work was supported by the Department of Biotechnology, Government of India, and a fellowship to KHB from the Council for Scientific and Industrial Research, Government of India. We acknowledge members of the laboratory for useful discussions. KHB acknowledges the Senior Research Fellowship from the Mechanobiology Institute, National University of Singapore, Singapore. Ethics Statement Not applicable. Citations [1] Conti et al. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. In: Annu Rev Biochem 76 (2007), pp url: Citation&list_uids= [2] Biswas et al. The GAF domain of the cgmp-binding, cgmp-specific phosphodiesterase (PDE5) is a sensor and a sink for cgmp. In: Biochemistry (2008), pp url: http: // [3] Corbin et al. [3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cgmp. In: Mol Pharmacol 63.6 (2003), pp url: Citation&list_uids= [4] Rybalkina et al. Multiple affinity states of cgmp-specific phosphodiesterase for sildenafil inhibition defined by cgmp-dependent and cgmp-independent mechanisms. In: Mol Pharmacol 77.4 (2010), pp url: https: // [5] Nikolaev et al. Fluorescent sensors for rapid monitoring of intracellular cgmp. In: Nat Methods 3.1 (2006), pp url: Citation&list_uids= [6] Russwurm et al. Design of fluorescence resonance energy transfer (FRET)-based cgmp indicators: a systematic approach. In: Biochem J (2007), pp url: Citation&list_uids= [7] Turko et al. Studies of the molecular mechanism of discrimination between cgmp and camp in the allosteric sites of the cgmp-binding cgmp-specific phosphodiesterase (PDE5). In: J Biol Chem (1999), pp url: Citation&list_uids= [8] Zhang et al. A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases. In: Mol Cell 15.2 (2004), pp url:

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