43 research outputs found
Investigation of ligand interactions with human RXRα by hydrogen/deuterium exchange and mass spectrometry
Transactivation of a DR-1 PPRE by a human constitutive androstane receptor variant expressed from internal protein translation start sites
Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (ΔNCARs). The ΔNCARs are expressed in liver and in vitro cell systems following translation from in-frame methionine AUG start codons at positions 76, 80, 125, 128, 168 and 265 within the full-length CAR mRNA. The resulting CAR proteins lack the N-terminal DNA-binding domain (DBD) of the receptor, yielding ΔNCAR variants with unique biological function. Although the ΔNCARs maintain full retinoid X receptor alpha (RXRα) heterodimerization capacity, the ΔNCARs are inactive on classical CAR-inducible direct repeat (DR)-4 elements, yet efficiently transactivate a DR-1 element derived from the endogenous PPAR-inducible acyl-CoA oxidase gene promoter. RXRα heterodimerization with CAR1, CAR76 and CAR80 isoforms is necessary for the DR-1 PPRE activation, a function that exhibits absolute dependence on both the respective RXRα DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRα, nor CAR's DBD. A new model of CAR DBD-independent transactivation is proposed, such that in the context of a DR-1 peroxisome proliferator-activated response element, only the RXRα portion of the CAR-RXRα heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex
Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR
The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions
Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase
Activation of RXR–PPAR heterodimers by organotin environmental endocrine disruptors
The nuclear receptor retinoid X receptor-α (RXR-α)–peroxisome proliferator-activated receptor-γ (PPAR-γ) heterodimer was recently reported to have a crucial function in mediating the deleterious effects of organotin compounds, which are ubiquitous environmental contaminants. However, because organotins are unrelated to known RXR-α and PPAR-γ ligands, the mechanism by which these compounds bind to and activate the RXR-α–PPAR-γ heterodimer at nanomolar concentrations has remained elusive. Here, we show that tributyltin (TBT) activates all three RXR–PPAR-α, -γ, -δ heterodimers, primarily through its interaction with RXR. In addition, the 1.9 Å resolution structure of the RXR-α ligand-binding domain in complex with TBT shows a covalent bond between the tin atom and residue Cys 432 of helix H11. This interaction largely accounts for the high binding affinity of TBT, which only partly occupies the RXR-α ligand-binding pocket. Our data allow an understanding of the binding and activation properties of the various organotins and suggest a mechanism by which these tin compounds could affect other nuclear receptor signalling pathways
Exploring Key Parameters to Detect Subtle Ligand-Induced Protein Conformational Changes Using Traveling Wave Ion Mobility Mass Spectrometry
Evidencing subtle conformational transitions in proteins
occurring
upon small modulator binding usually requires atomic resolution techniques
(X-ray crystallography or NMR). Recently, hyphenation of ion mobility
and mass spectrometry (IM-MS) has greatly enlarged the potentials
for biomolecular assembly structural characterization. Using the well
3D-characterized Bcl-xL/ABT-737 protein model, we explored in the
present report whether IM-MS can be used to differentiate close conformers
and monitor collision cross section (CCS) differences correlating
with ligand-induced conformational changes. Because comparing CCS
derived from IM-MS data with 3D-computed CCS is critical for thorough
data interpretation, discussing pitfalls related to protein construct
similarity and missing sequence sections in PDB files was of primary
importance to avoid misinterpretation. The methodic exploration of
instrument parameters showed enhanced IM separation of Bcl-xL conformers
by combining high wave heights and velocities with low helium and
nitrogen flow rates while keeping a high He/N<sub>2</sub> flow rate
ratio (>3). The robustness of CCS measurements was eventually improved
with a modified IM calibration method providing constant CCS values
regardless of instrument settings. Altogether, optimized IM-MS settings
allowed a 0.4 nm<sup>2</sup> increase (i.e., 2%) of Bcl-xL CCS to
be evidenced upon ABT-737 binding