Structural Basis for the Accommodation of Bis- and Tris-Aromatic Derivatives in Vitamin D Nuclear Receptor

Actual use of the active form of vitamin D (calcitriol or 1α,25-dihydroxyvitamin D₃) to treat hyperproliferative disorders is hampered by calcemic effects, hence the continuous development of chemically modified analogues with dissociated profiles. Structurally distinct nonsecosteroidal analogues have been developed to mimic calcitriol activity profiles with low calcium serum levels. Here, we report the crystallographic study of vitamin D nuclear receptor (VDR) ligand binding domain in complexes with six nonsecosteroidal analogues harboring two or three phenyl rings. These compounds induce a stimulated transcription in the nanomolar range, similar to calcitriol. Examination of the protein–ligand interactions reveals the mode of binding of these nonsecosteroidal compounds and highlights the role of the various chemical modifications of the ligands to VDR binding and activity, notably (de)­solvation effects. The structures with the tris-aromatic ligands exhibit a rearrangement of a novel region of the VDR ligand binding pocket, helix H6

Reconstruction of Quaternary Structure from X‑ray Scattering by Equilibrium Mixtures of Biological Macromolecules

A recent renaissance in small-angle X-ray scattering (SAXS) made this technique a major tool for the low-resolution structural characterization of biological macromolecules in solution. The major limitation of existing methods for reconstructing 3D models from SAXS is imposed by the requirement of solute monodispersity. We present a novel approach that couples low-resolution 3D SAXS reconstruction with composition analysis of mixtures. The approach is applicable to polydisperse and difficult to purify systems, including weakly associated oligomers and transient complexes. Ab initio shape analysis is possible for symmetric homo-oligomers, whereas rigid body modeling is applied also to dissociating complexes when atomic structures of the individual subunits are available. In both approaches, the sample is considered as an equilibrium mixture of intact complexes/oligomers with their dissociation products or free subunits. The algorithms provide the 3D low-resolution model (for ab initio modeling, also the shape of the monomer) and the volume fractions of the bound and free state(s). The simultaneous fitting of multiple scattering data sets collected under different conditions allows one to restrain the modeling further. The possibilities of the approach are illustrated in simulated and experimental SAXS data from protein oligomers and multisubunit complexes including nucleoproteins. Using this approach, new structural insights are provided in the association behavior and conformations of estrogen-related receptors ERRα and ERRγ. The possibility of 3D modeling from the scattering by mixtures significantly widens the range of applicability of SAXS and opens novel avenues in the analysis of oligomeric mixtures and assembly/dissociation processes

PGC-1α RID1 binding to ERRα and ERRγ measured by MST.

<p>Unlabeled PGC-1α RID1 protein was titrated into a fixed concentration of (<b>A</b>) labeled ERRα LBD and (<b>B</b>) labeled ERRγ LBD. Top panels: raw thermophoresis data recorded at 20°C using the LED at 50% and IR-laser at 80%. Bottom panels: isotherms averaged over three consecutive measurements and fitted according to the law of mass action to yield the apparent K_D,1. For the determination of K_D,1, the concentration of unlabeled PGC-1α RID1 was varied between 30 µM and 3 nM, while the concentration of ERR LBD was kept fixed (50 nM). Insets: isotherms for titration series comprising higher unlabeled PGC-1α RID1 concentrations (300 µM to 10 nM) with a fixed ERR LBD concentration (20 nM), showing the two binding events of binding affinities K_D,1 and K_D,2.</p

PGC-1α RID interacts through an identical region with ERRα and ERRγ LBD.

<p>NMR spectra of PGC-1α RID fragments in interaction with (<b>A–B</b>) ERRα LBD and (<b>C</b>) ERRγ LBD. ¹H-¹⁵N HSQC spectra of PGC-1α RID1 (<b>A and C</b>) and RID2 (<b>B</b>) alone (black) and following addition of unlabeled ERR LBD (red). The sets of attenuated cross-peaks are broadly similar. The additional peaks in RID2 are not affected by binding to either ERR LBD. Cross-peaks in regions expected for Gly and Ser/Thr residues are boxed and the cross-peak that probably arises from the C-ter residue is indicated (CT). The cross-peak corresponding to W189 is boxed, showing the broadening or shift upon complex formation.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-8

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-6

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-3

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-5

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-4

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis-10

<p><b>Copyright information:</b></p><p>Taken from "Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis"</p><p>Environmental Health Perspectives 2004;113(3):278-284.</p><p>Published online 9 Dec 2004</p><p>PMCID:PMC1253752.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p