27 research outputs found
Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society
Regulatory function of conserved sequences upstream of the long-wave sensitive opsin genes in teleost fishes
Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant
Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix–loop–helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met–Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met–Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors
Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI‑A by Ferrocene Functionalization
A series of novel ferrocene (Fc)
functionalized RuÂ(III) complexes was synthesized and characterized.
These compounds are derivatives of the anti-metastatic RuÂ(III) complex
imidazolium [<i>trans</i>-RuCl<sub>4</sub>(1<i>H</i>-imidazole) (DMSO-<i>S</i>)] (<b>NAMI-A</b>) and
are derived from its pyridine analogue (<b>NAMI-Pyr</b>), with
direct coupling of Fc to pyridine at the 4 or 3 positions, or at the
4 position via a two-carbon linker, which is either unsaturated (vinyl)
or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV–vis
spectroscopic studies of the ligand exchange processes of the compounds
in phosphate buffered saline (PBS) report similar solution behavior
to <b>NAMI-Pyr</b>. However, the complex with Fc substitution
at the 3 position of the coordinated pyridine shows greater solution
stability, through resistance to the formation of oligomeric species.
Further EPR studies of the complexes with human serum albumin (hsA)
indicate that the Fc groups enhance noncoordinate interactions with
the protein and help to inhibit the formation of protein-coordinated
species, suggesting the potential for enhanced bioavailability. Cyclic
voltammetry measurements demonstrate that the Fc groups modestly reduce
the reduction potential of the RuÂ(III) center as compared to <b>NAMI-Pyr</b>, while the reduction potentials of the Fc moieties
of the four compounds vary by 217 mV, with the longer linkers giving
significantly lower values of <i>E</i><sub>1/2</sub>. EPR
spectra of the compounds with 2-carbon linkers show the formation
of a high-spin FeÂ(III) species (<i>S</i> = 5/2) in PBS with
a distinctive signal at <i>g</i> = 4.3, demonstrating oxidation
of the FeÂ(II) ferrocene center and likely reflecting degradation products.
Density functional theory calculations and paramagnetic <sup>1</sup>H NMR describe delocalization of spin density onto the ligands and
indicate that the vinyl linker could be a potential pathway for electron
transfer between the Ru and Fe centers. In the case of the ethyl linker,
electron transfer is suggested to occur via an indirect mechanism
enabled by the greater flexibility of the ligand. In vitro assays
with the SW480 cell line reveal cytotoxicity induced by the ruthenium
ferrocenylpyridine complexes that is at least an order of magnitude
higher than the unfunctionalized complex, <b>NAMI-Pyr</b>. Furthermore,
migration studies with LNCaP cells reveal that Fc functionalization
does not reduce the ability of the compounds to inhibit cell motility.
Overall, these studies demonstrate that <b>NAMI-A</b>-type compounds
can be functionalized with redox-active ligands to produce both cytotoxic
and anti-metastatic activity