Analysis of protein dimerization and ligand binding of orphan receptor HNF4α

Hepatocyte nuclear factor 4 alpha (HNF4 alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXR alpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4 alpha are combined with a structural model of the HNF4 alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4 alpha 1 were converted to the analogous residues in RXR alpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4 alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4a construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4 alpha LBD are also important in dimerization. The structural model of the HNF4 alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4 alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4 alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4 alpha ligands, are not good candidates for a traditional ligand for HNF4 alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4 alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1). (C) 2000 Academic Press.