Distinct Conformational Changes Induced by 20-epi Analogues of 1α,25-Dihydroxyvitamin D3 Are Associated with Enhanced Activation of the Vitamin D Receptor

Journal of Biological Chemistry

Mahinthichaichan

et al. 2009

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The steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1,25D) 2 (see Fig. 1) and the nuclear vitamin D receptor (VDR) forms a complex that modulates the transcription of genes containing a VDR element. This process is termed a genomic response and involves 1,25D binding to the VDR, formation of a heterodimer with retinoid X receptor, and recruitment of nuclear co-activators (NCoAs) and the basal transcription machinery (1).The VDR is a member of the nuclear hormone superfamily of transcription factors, all of which share similar domain partitioning, ligand binding domain (LBD) tertiary fold and localization of their ligand binding pocket (LBP). The current inducedfit model describing nuclear receptor (NR) activation (i.e. the mouse-trap model) is founded on the comparison of apo-and holo-NR x-ray crystal structures. The mouse trap model posits that closure of the NR activation helix (i.e. helix-12) is induced by ligand binding to an opened-like helix-12 apo-NR conformer. The closed helix-12 conformation completes the activation function II domain (AF2), which serves as the high affinity binding site for recruitment of various NCoAs (1-4) (Fig. 2). Thus, the steric blockage of NR helix-12 closure is the basis for the design of traditional NR genomic antagonists (5-9).

Section: Discussionmentioning

confidence: 51%

“…The ϳ34-kDa hVDRwt fragment stabilized by 1,25D represents the active, closed conformation of helix-12 (hVDR-c1, Fig. 5A) (20,30). The MK-H305F and MK-H305F/H397F PSA results demonstrated that MK did stabilize a strong hVDR-c1 band (Fig.…”

Section: Mk Stabilizes the Active Closed Hvdr Helix-12 Conformation mentioning

confidence: 87%

On the Mechanism Underlying (23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone Antagonism of hVDRwt Gene Activation and Its Switch to a Superagonist

Journal of Biological Chemistry

Mahinthichaichan

et al. 2009

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“…The reaction tubes were incubated for 2 h at 30°C, then placed on ice. The 1,25D or analog (10 Ϫ5 M) was added, the tubes were incubated at room temperature for 20 min (28), and then 15 g͞ml trypsin was added to the tubes and incubated for 20 min at room temperature, followed by a 5-min incubation at 80°C with SDS. After SDS͞PAGE, bands were visualized by autoradiography.…”

Section: Methodsmentioning

confidence: 99%

Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1α,25(OH) ₂ -vitamin D ₃ signaling

Keidel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2004

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154

200

T he steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1,25D) (Fig. 1A), other steroid hormones, retinoids, and thyroid hormones form the family of ligands for the nuclear receptor (NR) superfamily (1), the members of which produce genomic responses through selective interaction of the liganded receptor with promoters of appropriate genes and basal transcription machinery. Many of these hormones also activate rapid, nongenomic (NG), cellular signaling cascades (2, 3) (except retinoids) that range from activation of ion channels (4, 5) to promoting kinase and other cytosolic signaling cascades (6-9). Defining the structure-function requirements for 1,25D and 17␤-estradiol (E 2 ) rapid actions has been aided by the synthesis of analogs that are NG agonists like 1␣,25(OH) 2 -lumisterol (JN) (ref. 10 and Fig. 1 A) and 4-estren-3␣,17␤-diol (EST) (11) or antagonists like 1␤,25(OH) 2 -vitamin D 3 (HL) (ref. 12 and Fig. 1 A), but that are only weak genomic transactivators (13). Thus, important structural attributes of the sterol dictate its agonistic properties and subsequent genomic vs. NG signaling profile (14, 15).When 1,25D rapid signaling cascades were first discovered, it was hypothesized that the observed activities were propagated by a novel membrane protein(s) (16), because analogs JN and HL did not compete well with [ 3 H]1,25D for binding to the nuclear vitamin D receptor (VDR) (17). Recently, in studies using a VDR knockout (KO) mouse (18) and a naturally occurring human VDR mutation (19), 1,25D-mediated rapid responses were shown to require a functional VDR. Both the VDR and estrogen receptor (ER) have been found localized to the plasma membrane in caveolae (7, 20); therefore, it has been proposed that the VDR and ER propagate some NG signaling (6,9,18,21,22). However, given the poor affinity of JN and HL for the VDR, it is difficult to understand how these sterols can facilitate their activities through the VDR.Results obtained from the modeling (INSIGHT 2000.1) of JN, 1,25D, and HL in the VDR ligand-binding domain (LBD) showed that the VDR could possibly accept and form favorable nonbonding interactions with vitamin D sterols in a distinct ligand-binding pocket [an alternative ligand-binding pocket (A pocket)] from the genomic pocket (G pocket) that was previously defined by x-ray crystallography (23, 24). Our proposed A-pocket accepts ligands that differ in shape from those in the classical G pocket (3,23,24).The data from these models has led to the proposal that the VDR can function as a rapid response receptor through a conformational ensemble mechanism (3, 25) whereby the flexible 1,25D steroid hormone samples an ensemble of energetically similar protein conformations (26). In addition, the ensemble model and existence of an A pocket may provide an explanation for the observed sex-nonspecific, nongenotropic signaling through the ER␣ receptor by EST (3, 9, 11). The physiological relevance of the ensemble model and functional importance of an A pocket within the VDR is further substantiated by applying the m...

“…Currently the most actively used method for assaying VDR H12 conformational heterogeneity in solution, is the partial trypsin digest experiment (protease sensitivity assay, PSA) [17]. Unlike x-ray crystallography, PSA provides insight into solution state H12 dynamics and ultimately yields insight into the population distribution of apo/holo-VDR (H12) isomers under steady state conditions.…”

Section: Introductionmentioning

confidence: 99%

New insights into Vitamin D sterol-VDR proteolysis, allostery, structure–function from the perspective of a conformational ensemble model

The Journal of Steroid Biochemistry and Molecular Biology

Bishop

et al. 2007

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Recently, we have developed a vitamin D sterol (VDS)-VDR conformational ensemble model. This model can be broken down into three individual, yet interlinked parts: a) the conformationally flexible VDS, b) the apo/holo-VDR helix-12 (H12) conformational ensemble, and c) the presence of two VDR ligand binding pockets (LBPs); one thermodynamically favored (the genomic pocket, Gpocket) and the other kinetically favored by VDSs (the alternative pocket, A-pocket). One focus of this study is to use directed VDR mutagenesis to 1) demonstrate H12 is stabilized in the transcriptionally active closed conformation (hVDR-c1) by three salt-bridges that span the length of H12 (cationic residues R154, K264 and R402), 2) to elucidate the VDR trypsin sites [R173 (hVDRc1), K413 (hVDR-c2) and R402 (hVDR-c3)] and 3) demonstrate the apo-VDR H12 equilibrium can be shifted. The other focus of this study is to apply the model to generate a mechanistic understanding to discrepancies observed in structure-function data obtained with a variety of 1α,25(OH) 2 -vitamin D 3 (1,25D) A-ring and side-chain analogs, and side-chain metabolites. We will demonstrate that these structure-function conundrums can be rationalized, for the most part by focusing on alterations in the VDS conformational flexibility and the elementary interaction between the VDS and the VDR A-and G-pockets, relative to the control, 1,25D.