Conformational Studies of Human Vitamin‐D Receptor by Antipeptide Antibodies, Partial Proteolytic Digestion and Ligand Binding

Abstract

We have studied conformational changes of human vitamin-D receptor by using antipeptide antibodies, partial proteolytic digestion and binding of the natural ligand calcitriol or its synthetic analogs. Before exposing either ['sS]methionine-labelled in vitro translated human vitamin-D receptor or a recombinant human vitamin-D receptor produced either in Escherichia coli or in 5 ' ' insect cells to limited proteolysis by trypsin or chymotrypsin, the proteins were treated with calcitriol or its synthetic analogs. The digestion products were analyzed by SDS/PAGE, immunoblotting with polyclonal antipeptide antibodies targeted against different domains of the receptor, and Edman N-terminal sequencing. After limited proteolysis with trypsin, two fragments of M , 21 000 and M , 34000 could be localized into N-terminus and C-terminus of the receptor, respectively, by antipeptide antibodies. We found that treatment with calcitriol or its synthetic analogs leads to differential resistance of the ligand-binding domain of the recombinant receptor to partial proteolysis in vitro. We suggest that this is due to distinct conformational changes in the domain induced by the different ligands. The short N-terminal region and the Zn-finger domain form, however, a protease-resistant structure which is independent on the presence or absence of the ligand. When the C-terminal fragment of M , 34000 was further analyzed by Edman N-terminal sequencing, the major cleavage site in the receptor between amino acids Arg173 and His174 was revealed. Recently, interest has arisen in numerous synthetized analogs of calcitriol, human VDR ligands, because of their potential uses for cancer and psoriasis therapies. Many of these analogs are several orders of magnitude more potent in their antiproliferative action than calcitriol, but their calcemic effects are not proportionally higher and their relative affinities for human VDR do not differ notable from that of calcitriol [17-221. It has been recently determined that the reason for the better antiproliferative action of the 20-epi analogs may be a ligand-induced conformational change of human VDR that differs from the conformational change induced by calcitriol. This change in conformation may enhance dimerization of human VDR with RXR and therefore render it more active in transcription [lo].We have studied the conformational changes of human VDR by using antipeptide antibodies, partial proteolytic digestion, and the natural ligand calcitriol or its synthetic analogs. We report here that human VDR has ligand-dependent resistance to proteolysis and that the whole ligand-binding domain, including the C-terminal end of the hinge region, participates in the obvious ligand-induced conformational change of human VDR. We suggest that the binding of the ligand leads to a conformational change similar to that found in RXR-a, RAR-y, and thyroidhormone receptor-a, and that treatment with specific ligands causes slightly different changes in the conformation of human VDR.

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

“…3A). Nevertheless, the work of Vaisanen et al [21] was substantiated by the R173Q-1,25D protease sensitivity assay (PSA) result, where the presence of hVDRwt-like ∼34 kDa (c1), ∼32 kDa (c2), or ∼30 kDa (c3) fragments were not observed. Alternatively, three new higher molecular weight bands were observed (a1-a3, Fig.…”

“…The trypsin site exposed when H12 is in the closed position (hVDR-c1, Fig. 1C) has been successfully mapped to R173 [21] (insertion loop, Fig. 1B), while the trypsin sites exposed when sterols stabilize hVDRwt-c2 and hVDRwt-c3 remain unidentified.…”

“…3B) confirms that all three hVDRwt conformers share the same N-terminus [21]. To identify the C-terminus of the hVDRwt-c2 and hVDRwt-c3 fragments, the cationic R-group of prospective trypsin sites in H11/H12 and/or the hinge domain (Figs 1B and 1C) were identified using in silico techniques (expasy.org) and mutated.…”

“…3A) in a 14% SDS-PAGE gel. R173 is known to be the hVDRwt-c1 trypsin site [21]. Based on the hVDR primary sequence, trypsin cleavage at R173 produces a ∼29 kDa C-terminal fragment (VDR-c1, expasy.org) and a ∼21 kDa N-terminal fragment; therefore, the hVDRwt-c1 fragment does not migrate at its projected molecular weight (Fig.…”

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

“…The different proteolysis pattern of VDR in the presence of the natural ligand and the 20-epi compounds has been interpreted as reflecting large conformational changes of the receptor on binding of the latter class of molecules. It has been suggested that the 20-epi compounds induce an alternate conformation of the VDR-LBD, rendering the receptor more resistant to protease digestion (8,11,12,(14)(15)(16). To investigate the binding mode of the 20-epi analogs to the VDR-LBD, we determined the high resolution crystal structures of the VDR in complex with MC1288 and KH1060 ( Fig.…”