Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. We have solved the x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations.     

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).     

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.     

Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4alpha1: modulation by the F domain

For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4alpha1 (HNF4alpha1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4alpha1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4alpha-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4alpha1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4alpha1 in vitro and repress HNF4alpha1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4alpha1 and GRIP1, CBP, or p300, SMRT can titer out HNF4alpha1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4alpha can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.     

Thyroxine-thyroid hormone receptor interactions

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.