Rosalie Baak1
(1) Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.
The nuclear receptor Peroxisome proliferator-activated receptor g (PPARg) is a ligand inducible transcription factor that is essential for adipocyte function, differentiation and maintenance. Compelling evidence for this comes from heterozygous loss-of-function PPARGmutations, resulting in lipodystrophy. Such natural mutants may help to elucidate the relevance of the various conformations that are evident from structural studies, but not supported by in vivo evidence yet.
Recently, we identified a novel PPARg lipodystrophy mutation K184N, located in the DNA-binding domain but not contacting the DNA directly. Initial analysis revealed that this mutant showed drastic reduced ability to activate transcription. To unravel the underlying molecular defect we systematically investigated 3 scenarios: 1) disturbance of heterodimerization with RXRa; 2) disturbance of interdomain interactions; 3) disturbance of DNA-binding. Firstly, protein complementation assays show that heterodimerization is still intact in the K184N mutant. Secondly, MD simulations showed disturbed interactions between K184 and LBD residues in the mutant situation, suggesting that this PPARg-DBD:PPARg-LBD interaction interface could be indirectly involved in DNA binding and subsequent activation. Lastly, DNA binding assays and reporter assays indicated that the K184N mutant interferes with DNA binding in a sequence-specific fashion: natural PPRE’s with imperfect repeats displayed reduced binding and subsequent activation, while perfect palindromic PPRE’s, either natural or synthetic, were unaffected. Taken together, these data support a model where the DBD can adopt multiple conformations depending on the PPRE sequence, possibly supported by PPARg-DBD:PPARg-LBD interactions.