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

Abstract

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1␣,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A-to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17␤-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain. A different class of proteins has been characterized that stimulate the transcriptional activity of liganded NRs in an activation function 2 (AF2)-dependent way. These coactivators are thought to bridge the NRs to the transcriptional apparatus. In particular, VDR is regulated by coactivators belonging to the steroid receptor activator (SRC)͞p160 family of proteins, which contain several LXXLL motifs (6). This motif has been shown to form an amphipatic ␣-helical structure that can interact with the AF2 region of NRs (7). Another class of coactivators [vitamin D receptor-interacting protein (DRIP), thyroid hormone receptor-associated protein (TRAP), activator-recruited cofactor (ARC); refs. 8-11] has been isolated as multiprotein complexes and strongly potentiated transcription mediated by VDR͞RXR in a ligand-dependent manner on DNA templates assembled into chromatin (8). One of their components, DRIP205, interacts directly with the ligand-binding domain (LBD) in the presence of ligand and anchors the rest of the subunits to the receptor.Among the several synthetic analogs of vitamin D, the 20-epi compounds, which exhibit an inverted stereochemistry at position 20 in the flexible aliphatic chain, have attracted much attention. They are potent growth inhibitors and inducers of cell differentiation, while showing an affinity similar to vitamin D for VDR (11). KH1060 ( Fig. 1), a member of this 20-epi family, exhibits similar properties, with decreased calcemic side effects. These compounds induce VDR-dependent transcription at concentrations at least 100-fold lower than the natural ligand and present antiproliferative activity several orders of magnitude higher than the natural ligand (11-13). The differences in biological activity of 1␣,25(OH) 2 D 3 and the 20-epi molecules in general, and KH1060 in particular, are known to be VDR-LBD dependent, but are not yet understood. Th...

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

“…Like the x-ray crystal structures of VDR superagonists [14] and 1,25D [11], the recently solved KH-VDR (VDR from zebrafish) complex [12] clearly demonstrates that the ligand binding pocket adapts to the KH sterol so it stabilizes the closed VDR H12 conformation (hVDR-c1). The KH-VDR x-ray result does correlate well with its ability to transactivate with similar or greater efficacy relative to 1,25D; however, the structure does not correlate with KH's solution state PSA results or reduced VDR affinity [18].…”

“…1C, yellow circles). Residues N424, E425, I426, and S427 are unresolved in the 1,25D-VDR x-ray structures [11,12,13,14,15], but when added in an α-helical orientation to the x-ray construct (pdb code: 1DB1), prior to assembly minimization, it was observed that R402 (H11) formed Coulombic interactions with either E425 and S398 or S427 and S398 (see methods; Fig. 1C, pink ribbon in yellow circle).…”

“…1B), have shown that agonists occupy the same relative steric space in the VDR LBD, termed the genomic pocket (G-pocket), and stabilize only the closed helix-12 conformation (VDR-c1, Fig. 1C) [11,12,13,14,15]. Analysis of these VDR structures and molecular models with complete AF2-domains, reveal that unlike any other hormonally regulated NR, H12 is held in the closed conformation (c1) by three salt-bridges: 1) K264---E420 2) R154---K413/ R154---L414/ R154---D232 and 3) R402---S427/ S398/ E425 (Fig.…”

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

“…Crucial to 1,25D-hVDR Transactivation-Based on the hVDR x-ray crystallographic results (5,(25)(26)(27) His-305 and His-397 form hydrogen bonds (H-bonds) with the 25-OH group of 1,25D (28,29) (Fig. 2).…”

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

“…Our proposed A-pocket accepts ligands that differ in shape from those in the classical G pocket (3,23,24).…”