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 cGMP- and sildenafil-induced conformational changes. The sildenafil-induced 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.
Highlights
Inherent dynamics, proteins sample multiple conformations in solution [4]
The PDE5 sensor expressed in HEK293T cells (Fig. 1B) was a dimer (Fig. 1C) and catalytically active (Km for cGMP 6.6 Ϯ 0.8 M; Fig. 1D), similar to that reported for native PDE5 [12]
Addition of cGMP to lysates prepared from cells in the presence of EDTA led to a decrease (ϳ40%) in bioluminescence resonance energy transfer (BRET) of the wildtype sensor, whereas mutation of the Phe-163 residue (which has a stacking interaction with cGMP in the GAFa domain [27, 35]) to alanine abrogated the BRET decrease (Fig. 2A)
Summary
Inherent dynamics, proteins sample multiple conformations in solution [4]. A “shift” in the ensemble of conformations, involving conformational selection and induced-fit mechanisms [5], is generally thought to result in allosteric regulation [6]. The mammalian cyclic nucleotide phosphodiesterases (PDEs) serve as examples of multidomain proteins where different N-terminal regulatory domains sense a variety of ligands, and allosterically modulate the hydrolysis of cAMP or cGMP [11]. Cyclic GMP binding to the GAFa domain allosterically activates the catalytic domain by increasing both the Vmax and affinity for cGMP, resulting in increased cGMP hydrolysis [17,18,19]. The sensor reveals structural changes and associated allostery induced by cGMP and sildenafil binding to the full-length PDE5. We show that metal ion binding to the catalytic site of PDE5 negatively modulates cGMP-induced and positively modulates sildenafil-induced conformational changes. The conformational changes induced by cGMP and sildenafil are structurally and thermodynamically distinct This novel sensor monitors dynamic changes in PDE5 conformation in intact cells, and provides new insights into allostery in a multidomain protein
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