Structural Determinants of the Agonist-independent Association of Human Peroxisome Proliferator-activated Receptors with Coactivators

Ferdinand Molnár,Merja Matilainen,Carsten Carlberg

doi:10.1074/jbc.m502463200

Ferdinand Molnár, Merja Matilainen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m502463200

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Abstract

Lipid homeostasis is controlled by various nuclear receptors (NRs), including the peroxisome proliferator-activated receptors (PPARalpha, delta, and gamma), which sense lipid levels and regulate their metabolism. Here we demonstrate that human PPARs have a high basal activity and show ligand-independent coactivator (CoA) association comparable with the NR constitutive androstane receptor. Using PPARgamma as an example, we found that four different amino acid groups contribute to the ligand-independent stabilization of helix 12 of the PPAR ligand-binding domain. These are: (i) Lys329 and Glu499, mediating a charge clamp-type stabilization of helix 12 via a CoA bridge; (ii) Glu352, Arg425, and Tyr505, directly stabilizing the helix via salt bridges and hydrogen bonds; (iii) Lys347 and Asp503, interacting with each other as well as contacting the CoA; and (iv) His351, Tyr(355), His477, and Tyr501, forming a hydrogen bond network. These amino acids are highly conserved within the PPAR subfamily, suggesting that the same mechanism may apply for all three PPARs. Phylogenetic trees of helix 12 amino acid and nucleotide sequences of all crystallized NRs and all human NRs, respectively, indicated a close relationship of PPARs with constitutive androstane receptor and other constitutive active members of the NR superfamily. Taking together, the ligand-independent tight control of the position of the PPAR helix 12 provides an effective alternative for establishing an interaction with CoA proteins. This leads to high basal activity of PPARs and provides an additional view on PPAR signaling.

Highlights

The ligand-binding domain (LBD) of most nuclear receptors (NRs) is a characteristic three-layer antiparallel ␣-helical sandwich formed by 11–13 ␣-helices
This distance reflects an optimal position of the two charge residues for effective CoA interaction of the LBD, as found in ligand-activated NRs, such as vitamin D3 receptor (VDR) [41]
In this study we demonstrate that in the absence of high affinity ligands peroxisome proliferator-activated receptor (PPAR) interact with CoA proteins, stabilize the agonistic LBD conformation c1, so that the addition of agonist provides only a rather weak increase in complex formation and conformation stabilization, and show high basal activity in reporter gene assays

Summary

Introduction

The LBDs of most NRs is a characteristic three-layer antiparallel ␣-helical sandwich formed by 11–13 ␣-helices. We show that the ligand-independent interaction of the PPARs with CoA proteins is caused by intramolecular stabilization of the helix 12 in the active LBD conformation even in the absence of agonist. Using PPAR␥ as an example, we determined that four different amino acid groups contribute to the stabilization of helix 12 These interactions are conserved within the PPAR subfamily, and this suggests that the same mechanism applies for all three PPARs. phylogenetic trees of helix 12 amino acid and nucleotide sequences of all crystallized NRs and all human NRs, respectively, indicated a close relation of PPARs with CAR and other constitutive active members of the NR superfamily

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