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

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=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-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, 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 (Kd=0.9-4 nm) and LCFAs (Kd=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 (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha 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-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.