Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

Abstract

The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMPand sildenafil-induced conformational changes. The sildenafilinduced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.

Sustained α -catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

“…Cell-based assays have utilized either binding of the conformation-specific a18 antibody to a-catenin (11) or a FRET-based a-catenin force sensor (13). Although they generally report a force-induced structural transition, each of these tools reports a distinct conformation or conformational transition in a-catenin (53)(54)(55)(56). This is clear from results obtained with the FRET-based a-catenin sensor and a vinculin construct without the autoinhibitory tail (13).…”

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

“…We have earlier shown that not only is BRET a useful tool to monitor cyclic nucleotide-dependent conformational changes in an isolated GAF domain (41), but it can also report on allostery in the full-length cGMPbinding, cGMP-specific phosphodiesterase, PDE5 (31). These results formed the basis for our attempt to monitor conformational changes using BRET in full-length KATms, where biochemical evidence indicated that cAMP binding regulated the associated catalytic domain that possessed acetyltransferase activity (29,30).…”

“…In Vitro BRET Assays-HEK 293T cells were transfected with pGFP 2 -CNBLIN-Rluc or pGFP 2 -FL-Rluc plasmids and, 72 h following transfection, lysed in 50 mM HEPES (pH 7.5), containing 2 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 10 mM sodium pyrophosphate, 80 M glycerophosphate, 1 mM benzamidine, 1 g/ml aprotinin, 1 g/ml leupeptin, 5 g/ml soybean trypsin inhibitor, 100 M sodium orthovanadate, and 10% glycerol (31). Following brief sonication, lysates were centrifuged at 13,000 ϫ g, and the cytosol was removed.…”

“…have recently utilized BRET as a means of monitoring conformational changes in proteins that interact with cyclic nucleotides (31,41). BRET is based on energy transfer between luminescence donor and fluorescent acceptor proteins (e.g.…”

“…In this study, we monitor the dynamic conformational changes that occur upon cAMP binding to MSMEG_5458 (henceforth referred to as KATms) using bioluminescence resonance energy transfer (BRET) (31) as well as HDXMS. cAMP binding to full-length KATms induced a conformational change that could be detected by an increase in BRET.…”