Role of regulatory F-domain in hepatocyte nuclear factor-4α ligand specificity

The F-domain of rat HNF-4 alpha 1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4 alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4 alpha ( amino acids 1 - 455) has high affinity (K-d = 0.06 - 12 nM) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (K-d = 58 - 296 nM) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer ( FRET) from full-length HNF-4 alpha tryptophan ( FRET donor) to bound cis-parinaroyl-CoA ( FRET acceptor), which yielded an intermolecular distance of 33 angstrom, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (K-d = 0.9 - 4 nM) and LCFAs (K-d = 93 - 581 nM). ( iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity ( i. e. high affinity for LCFAs (K-d = 1.5 - 32 nM) and low affinity for LCFA-CoAs (K-d = 54 - 302 nM)). No FRET from HNF-4 alpha-E ( amino acids 132-370) tryptophan ( FRET donor) to bound cis-parinaroyl-CoA ( FRET acceptor) was detected, whereas an intermolecular distance of 28 angstrom was calculated from FRET between HNF-4 alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4 alpha only when the F-domain was present. ( v) cis-Parinaric acid bound to HNF-4 alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4 alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4 alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4 alpha.