Functional characterization of hepatocyte nuclear factor-4 alpha dimerization interface mutants.
Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.
Aggelidou
Eleni
E
Department of Genetics, Development and Molecular Biology, Laboratory of Developmental Biology, School of Biology, Aristotle University of Thessaloniki, Greece.
Iordanidou
Panagiota
P
Demetriades
Constantinos
C
Piltsi
Olga
O
Hadzopoulou-Cladaras
Margarita
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D051557:Hepatocyte Nuclear Factor 4 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D051381:Rats
D051557:Hepatocyte Nuclear Factor 4; D004247:DNA