Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.
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 alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha 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 PPARalpha 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 PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.
Michalik
Liliane
L
Center for Integrative Genomics and National Research Center "Frontiers in Genetics," University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland.
Zoete
Vincent
V
Swiss Institute for Bioinformatics, University of Lausanne, CH-1015 Lausanne, Switzerland.
Krey
Grigorios
G
National Agricultural Research Foundation, Fisheries Research Institute, Nea Peramos, GR-64007 Kavala, Greece.
Grosdidier
Aurélien
A
Swiss Institute for Bioinformatics, University of Lausanne, CH-1015 Lausanne, Switzerland; Ludwig Institute for Cancer Research and National Research Center "Molecular Oncology," CH-1066 Epalinges, Switzerland.
Gelman
Laurent
L
Center for Integrative Genomics and National Research Center "Frontiers in Genetics," University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland.
Chodanowski
Pierre
P
Swiss Institute for Bioinformatics, University of Lausanne, CH-1015 Lausanne, Switzerland.
Feige
Jérôme N
JN
Center for Integrative Genomics and National Research Center "Frontiers in Genetics," University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland.
Desvergne
Béatrice
B
Center for Integrative Genomics and National Research Center "Frontiers in Genetics," University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland.
Wahli
Walter
W
Center for Integrative Genomics and National Research Center "Frontiers in Genetics," University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland. Electronic address: walter.wahli@unil.ch.
Michielin
Olivier
O
Swiss Institute for Bioinformatics, University of Lausanne, CH-1015 Lausanne, Switzerland; Ludwig Institute for Cancer Research and National Research Center "Molecular Oncology," CH-1066 Epalinges, Switzerland; Multidisciplinary Oncology Center (CePO), Lausanne University Hospital, CH-1011 Lausanne, Switzerland. Electronic address: olivier.michielin@unil.ch.
D000595:Amino Acid Sequence; D000818:Animals; D019295:Computational Biology; D003198:Computer Simulation; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D047493:PPAR alpha / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014158:Transcription, Genetic; D014982:Xenopus laevis
D008024:Ligands; D009687:Nuclear Proteins; D047493:PPAR alpha