tailieunhanh - Báo cáo khoa học: Functional characterization of hepatocyte nuclear factor-4a dimerization interface mutants

Hepatocyte nuclear factor-4 (HNF-4a), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization con-trols important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. | iFEBS Journal Functional characterization of hepatocyte nuclear factor-4a dimerization interface mutants Eleni Aggelidou Panagiota lordanidou Constantinos Demetriades Olga Piltsi and Margarita Hadzopoulou-Cladaras Department of Genetics Development and Molecular Biology Laboratory of DevelopmentalBiology Schoolof Biology Aristotle University of Thessaloniki Greece Keywords coactivator dimerization HNF-4 nuclear receptor transcription Correspondence M. Hadzopoulou-Cladaras Department of Genetics Development and Molecular Biology Laboratory of Developmental Biology Schoolof Biology Aristotle University of Thessaloniki Thessaloniki 54124 Greece Fax 30 2310 998298 Tel 30 2310 998303 E-mail cladaras@ These authors contributed equally to this study. Received 31 August 2005 revised 21 February 2006 accepted 2 March 2006 doi Hepatocyte nuclear factor-4 HNF-4a 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-4a 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-4a 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-4a activity and are responsive to .