title: Critical role of residues defining the ligand binding pocket in hepatocyte nuclear factor-4alpha.
abstract: Hepatocyte nuclear factor-4alpha (HNF-4alpha), a member of the nuclear receptor superfamily, is a crucial regulator of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism. Unlike other members of the superfamily, HNF-4alpha activates transcription in the absence of exogenously added ligand. Recently published crystallographic data show that fatty acids are endogenous ligands for HNF-4. Transcriptional analysis of point mutations of the residues that are located in helices H3, H5, H10, and H11, which have been shown to come in contact with the ligand, resulted in a dramatic decrease in activity, without affecting DNA binding and dimerization. Our results show the importance of residues Ser-181, Met-182 in H3, Leu-219, Leu-220 and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 that line the ligand-binding domain pocket in HNF-4alpha and impair its transactivation potential. Structural modeling reveals that the mutations do not cause any large scale structural alterations, and the observed loss in transactivation can be attributed to local changes, demonstrating that these residues play a significant role in maintaining the structural integrity of the HNF-4alpha ligand binding pocket.
authors.affiliation: Aristotle University of Thessaloniki, Faculty of Sciences, School of Biology, Department of Genetics, Development and Molecular Biology, GR-54124, Thessaloniki, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Aggelidou
authors.forename: Panagiota
authors.initials: P
authors.lastname: Iordanidou
authors.forename: Panayota
authors.initials: P
authors.lastname: Tsantili
authors.forename: Georgios
authors.initials: G
authors.lastname: Papadopoulos
authors.forename: Margarita
authors.initials: M
authors.lastname: Hadzopoulou-Cladaras
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; D010750:Phosphoproteins; D014157:Transcription Factors; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase