Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.
Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.
Eeckhoute
Jérôme
J
INSERM U459, Faculté de Médecine Henri Warembourg, 1 Place de Verdun, F-59045 Lille Cedex, France.
Oxombre
Bénédicte
B
Formstecher
Pierre
P
Lefebvre
Philippe
P
Laine
Bernard
B
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D018698:Glutamic Acid / Q000235:genetics; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; C403128:MLX protein, human; D010750:Phosphoproteins; D012996:Solutions; D014157:Transcription Factors; D018698:Glutamic Acid; D004247:DNA; D001120:Arginine