Combined Simulation and Mutagenesis Analyses Reveal the Involvement of Key Residues for Peroxisome Proliferator-activated Receptorα Helix 12 Dynamic Behavior

Abstract

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor ␣ (PPAR␣) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPAR␣ helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPAR␣ helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPAR␣ transcription activation potential. Finally, we disclose important functions of residues in PPAR␣ AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPAR␣ constitutive activity, without changing PPAR␣ ligand-dependent activation potential.The three peroxisome proliferator-activated receptor isotypes (PPARs) 5 ␣, ␤/␦, and ␥ (NR1C1, NR1C2, and NR1C3, respectively (1)) form a distinct subfamily of nuclear hormone receptors (2, 3). They are key regulators of lipid and glucose homeostasis, inflammation, cancer, and tissue repair (4 -7). The effect of PPARs on the expression of their target genes results from three events: recognition and binding of the receptor to response sequences in the promoter of the target genes, ligand binding, and co-repressor/co-activator exchange. Two regions exhibit a high degree of similarity in all members of the superfamily, namely the DNA binding domain, which is located toward the N terminus, and the ligand and cofactor binding domain (LBD). Ligand binding by the receptor is generally thought to be the trigger for transcriptional activation, but alternative mechanisms such as modulation of receptor activity by phosphorylation have also been reported (8, 9). Particularly, PPAR␣ shows a high constitutive activity, but the molecular basis and role in vivo of this activity are unclear (10, 11). A wide variety of natural or synthetic compounds, including fatty acids and eicosanoids, was identified as PPAR ligands (12-15). Among the synthetic ligands, the lipid-lowering drugs fibrates and the insulin sensitizers thiazolidinediones are PPAR␣ and PPAR␥ agonists, respectively; these underscore the important role of PPARs as therapeutic targets. Besides its role in ligand binding and dimerization, the LBD is also the site of the major and ligand-dependent transcriptional activation function of the nuclear receptors, called the activation function 2 (AF-2) domain (16). The PPAR LBD consists of 12 ␣ helices forming the characteristic three-l...

Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture

“…The ligand and cofactor binding domain (LBD) is another conserved region across the nuclear hormone receptor superfamily members. This domain includes the ligand-dependent activation function, AF-2, which is located at the C-terminus of the receptors (Nolte et al, 1998;Michalik et al, 2007).…”

Molecular recognition of long chain fatty acids by peroxisome proliferator-activated receptor α

“…The agonists are shown as stick models and colored according to the atom types: blue-nitrogen, white-carbon, red-oxygen, green-sulphur receptors (Markt et al, 2007;Michalik et al, 2007) and ligands that might interfere with impaired lipid and glucose homeostasis have been designed and synthesized (Kasuga et al, 2006a(Kasuga et al, , 2006bMiyachi, 2007). Derivatives of natural substances such as LCFAs are believed to be safer drugs for controlling chronic diseases than existing synthetic drugs.…”

The molecular basis of IGF-II/IGF2R recognition: a combined molecular dynamics simulation, free-energy calculation and computational alanine scanning study

“…Using the GB model, it was then possible to compute the binding free-energy contribution of each residue at the interface between two interacting proteins [16,19,20,22,32,41,42]. The contribution of a given residue to the binding free energy can be obtained by summing the contribution of each atom of this residue.…”

“…As experimental methods are very laborious and time-consuming, efforts have been made to produce fast and accurate computational methodologies. In recent years, molecular dynamics (MD) simulation has been widely used to study protein-protein interactions [15][16][17][18][19][20][21][22]. This simulation approach can provide not only plentiful dynamic structural information on protein complex structures in solution, but also a wealth of energetic information.…”