Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies

Juntunen, Kaisa; Rochel, N.; Moras, Dino; Vihko, Pirkko

doi:10.1042/bj3440297

Cited by 17 publications

(10 citation statements)

References 33 publications

(28 reference statements)

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“…We have previously reported the dissociation constants K d of vitamin D for the VDRwt ( K d = 0.55 n m ) and VDRmt ( K d = 0.37 n m ) [13] and have shown that there is no significant difference between values for the two proteins. These values agreed well with those previously obtained for recombinant VDR from E. coli , yeast and insect cells [25–31]. To compare the relative binding affinities for VDRmt of 1,25(OH) 2 D 3 and several 1,25(OH) 2 D 3 agonists analogs, MC903, EB1089 and the 20‐epi KH1060 (Fig.…”

Section: Resultssupporting

confidence: 87%

“…In previous reports on the expression and purification of E. coli VDR, the protein was solubilized by renaturation of inclusion bodies [25,26] or by expression of the VDR LBD fused to the glutathione S ‐transferase [27] or the maltose binding protein [28]. Lower amounts of the protein has also been purified from yeast [29] or insect cells using baculovirus [30,31]. The insertion region is not well ordered according to secondary structure prediction and presents only few short β strands.…”

Section: Resultsmentioning

confidence: 99%

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Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor

Rochel¹,

Tocchini-Valentini²,

Egea³

et al. 2001

European Journal of Biochemistry

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Vitamin D nuclear receptor mediates the genomic actions of the active form of vitamin D, 1,25(OH) 2 D 3 . This hormone is involved in calcium and phosphate metabolism and cell differentiation. Compared to other nuclear receptors, VDR presents a large insertion region at the N-terminal part of the ligand binding domain between helices H1 and H3, encoded by an additional exon. This region is poorly conserved in VDR in different species and is not well ordered as observed by secondary structure prediction. We engineered a VDR ligand binding domain mutant by removing this insertion region. Here we report its biochemical and biophysical characterization. The mutant protein exhibits the same ligand binding, dimerization with retinoid X receptor and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Solution studies by small angle X-ray scattering shows that the conformation in solution of the VDR mutant is similar to that observed in the crystal and that the insertion region in the VDR wildtype is not well ordered.Keywords: nuclear receptor; VDR LBD; ligand binding; conformation; transactivation.Steroids, retinoids, thyroid hormone and vitamin D 3 mediate their pleitoric effects through ligand-dependent transcription factors, the nuclear receptors [1,2]. These receptors control cell growth and differentiation, homeostasis, development and other physiological processes by modulating target genes. Members of the nuclear receptors superfamily exhibit the same modular structure with a variable N-terminal domain (A /B region), a conserved DNA binding domain (DBD, C region), a flexible hinge region (D region) and a moderately conserved ligand binding domain (LBD, E/F region). The A /B region is the most divergent among the nuclear receptors and contains a ligand independent activation function AF-1. The LBD contains a dimerization interface and a ligand-dependent transcriptional activation domain AF-2. The crystallographic structures of ligand binding domains of several liganded and unliganded nuclear receptors (reviewed in [3,4]) have shown that they share a common fold with 11±13 a helices sandwiched' in three layers. Ligand binding induces a conformational change in the orientation of the AF-2 core motif that allows the interaction with coactivators that facilitate the interaction between the nuclear receptor and the basal transcription machinery [3]. Several coactivators [5±7] have been identified as the SRC coactivators family, the DRIP/ TRAP coactivators or cointegrators (CBP/P300). They all present a LXXLL motif, a requirement for the interaction with the AF-2 domain of nuclear receptors. Efficient transcriptional regulation is achieved through the interaction of nuclear receptors with coregulator and the recruitment to the promoter of diverse functional domains responsible of acetylation, deacetylation, phophorylation, ligation or proteolysis [6].The vitamin D nuclear receptor (VDR) mediates the genomic actions upon binding to the active form ...

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor

Rochel¹,

Tocchini-Valentini²,

Egea³

et al. 2001

European Journal of Biochemistry

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“…Other full-length nuclear receptors, for example the GR, have been puri®ed to homogeneity via column chromatography [Warren et al, 1996]. Another group [Juntunen et al, 1999] recently puri®ed baculovirus expressed hVDR to homogeneity in preparation for crystallization, and hVDR also has been isolated using amylose resin af®nity puri®ca-tion of an E. coli-overexpressed hVDR-maltose binding protein fusion construct [Mottershead et al, 1996]. To probe speci®c hVDR±DNA interactions in the current study, puri®ed BEVS overexpressed receptor was utilized in electrophoretic mobility shift assays to evaluate the binding of hVDR, both alone and in conjunction with nuclear extracts, to the rat osteocalcin VDRE, an imperfect DR3 element with the sequence GGGTGAatgAGGACA.…”

Section: Discussionmentioning

confidence: 99%

Isolation of baculovirus‐expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor

Jurutka

MacDonald

Nakajima

et al. 2002

J of Cellular Biochemistry

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Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the possible significance of VDR homodimers and the functional role of receptor phosphorylation. To address these issues, milligram quantities of baculovirus-expressed hVDR were purified to 97% homogeneity, and then tested for binding to the rat osteocalcin vitamin D responsive element (VDRE) via electrophoretic mobility shift and half-site competition assays in the presence or absence of a CV-1 nuclear extract containing retinoid X receptor (RXR). Methylation interference analysis revealed that both the hVDR homodimer and the VDR-RXR heterodimer display similar patterns of VDRE G-base protection. However, in competition studies, the relative dissociation of the homodimeric hVDR complex from the VDRE was extremely rapid (t1/2 < 30 s) compared to the dissociation of the heteromeric complex (t1/2 > 5 min), thus illustrating the relative instability and low affinity of homodimeric VDR binding to DNA. These results indicate that VDR-RXR heterodimers are the preferred VDRE binding species. Further, two dimensional gel electrophoresis of hVDR demonstrated several isoelectric forms of the receptor, suggesting that it is subject to multiple phosphorylation events. In vitro kinase assays confirmed that purified hVDR is an efficient substrate for protein kinases A and Cbeta, as well as casein kinase II. In vivo studies of the expressed receptor in intact cells, namely baculovirus vector infected Sf9 insect cells and transfected mammalian COS-7 cells, demonstrated that hVDR was phosphorylated in a hormone-enhanced fashion. Functional consequences of hVDR phosphorylation were suggested by the observations that: (i) potato acid phosphatase (PAP)-treated hVDR no longer interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, and (ii) treatment of transfected COS-7 cells with a phosphatase inhibitor (okadaic acid) along with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) resulted in a synergistic enhancement of both hVDR phosphorylation and transactivation of a VDRE-linked reporter gene, compared to the effect of treatment with either agent alone. These studies point to a significant role for phosphorylation of VDR in regulating high-affinity VDR-RXR interactions with VDREs, and also in modulating 1,25(OH)2D3-elicited transcriptional activation in target cells.

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“…Full length WT His-hVDR and WT Flag-hRXRα were expressed in Baculovirus system and purified by Ni-NTA/SEC or Flag/SEC, respectively, as described previously. [41]…”

Section: Methodsmentioning

confidence: 99%

Methods for the analysis of high precision differential hydrogen–deuterium exchange data

Chalmers

Pascal

Willis

et al. 2011

International Journal of Mass Spectrometry

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Hydrogen/deuterium exchange (HDX) mass spectrometry has been widely applied to the characterization of protein dynamics. More recently, differential HDX has been shown to be effective for the characterization of ligand binding. Previously we have described a fully automated HDX system for use as a ligand screening platform. Here we describe and validate the required data analysis workflow to facilitate the use of HDX as a robust approach for ligand screening. Following acquisition of HDX data at a single on-exchange time point (n ≥ 3), one way analysis of variance in conjunction with the Tukey multiple comparison procedure is used to establish the significance of any measured difference. Analysis results are graphed with respect to a single peptide, ligand or group of ligands, or displayed as an overview within a heat map. For the heat map display, only Δ%D values with a Tukey-adjusted P value less than 0.05 are colored. Hierarchical clustering is used to bin compounds with highly similar HDX signatures. The workflow is evaluated with a small data set showing the ligand binding domain (LDB) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) screened against 10 functionally selective ligands. More significantly, data for the vitamin D receptor (VDR) in complex with 87 ligands are presented. To highlight the robustness and precision of our automated HDX platform we analyzed the data from 4191 replicate HDX measurements acquired over an eight month timeframe. Ninety six percent of these measurements were within 10 percent of the mean value. Work has begun to integrate these analysis and graphing components within our HDX software suite.

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Large-scale expression and purification of the human vitamin D receptor and its ligand-binding domain for structural studies

Cited by 17 publications

References 33 publications

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor

Functional and structural characterization of the insertion region in the ligand binding domain of the vitamin D nuclear receptor

Isolation of baculovirus‐expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor

Methods for the analysis of high precision differential hydrogen–deuterium exchange data

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