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...

We have detected two paralogs of the tRNA endonuclease gene of Methanocaldococcus jannaschii in the genome of the crenarchaeote Sulfolobus solfataricus. This finding has led to the discovery of a previously unrecognized oligomeric form of the enzyme. The two genes code for two different subunits, both of which are required for cleavage of the pre-tRNA substrate. Thus, there are now three forms of tRNA endonuclease in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaea and the Nanoarchaea. The last-named enzyme, arising most likely by gene duplication and subsequent ''subfunctionalization,'' requires the products of both genes to be active. molecular evolution ͉ RNA-protein interactions ͉ tRNA splicing G ene duplication is the primary source of new genes. The ''subfunctionalization'' hypothesis argues that duplicate genes experience degenerate mutations that divide the activity encoded by a single ancestral gene among its descendants (1). Here, we report a striking example of subfunctionalization.In Archaea, the tRNA endonuclease plays a key role in assuring the correct removal of the intron from pre-tRNAs and pre-rRNA (2-6), which constitute the core of the translation machinery. Crystal structures of the tRNA endonucleases from Methanocaldococcus jannaschii (METJA) and Archaeoglobus fulgidus (ARCFU), both belonging to the phylum Euryarchaeota, are available (7,8). These structures differ in a remarkable way. The structure of the homotetrameric endonuclease from METJA reveals two different functional roles for the monomeric units. The METJA endonuclease is organized as a dimer of dimers, with one subunit from each dimer participating in the catalytic cleavage reaction (the catalytic subunit) and the other (structural) subunit acting to place the two catalytic subunits correctly in space.The crystal structure of the ARCFU endonuclease, by contrast, shows it to be a homodimer. Each subunit contains two similar repeating domains that are homologous to the subunit structure of the homotetrameric enzyme from METJA; the C-terminal repeat (CR) is the active domain, and the N-terminal repeat (NR) acts to stabilize the dimer.The overall shape and size of the homodimeric ARCFU endonuclease resembles that of the homotetrameric METJA enzyme.Both METJA and ARCFU belong to the Euryarchaeota. Nothing is known about the tRNA endonuclease of Crenarchaeota, the other main family of Archaea. To determine the properties of a crenarchaeal endonuclease, we searched the genome sequence of Sulfolobus solfataricus (SULSO) for homologs of the METJA endonuclease and found two candidate sequences.Characterization of the two gene products reveals that both are required for tRNA endonuclease activity, each presumably functioning like one-half of the ARCFU enzyme. Detailed analysis of the amino acid sequences of the two proteins supports the idea that they evolved by the process called subfunctionalization (1, 9, 10). Materials and MethodsIdentification of the Homologs. Th...

The plethora of actions of 1alpha,25(OH)(2)D(3) in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to reduce the calcemic side effects while maintaining the transactivation potency of these ligands. Here, we report the crystal structures of VDR ligand binding domain bound to two vitamin D agonists of therapeutical interest, calcipotriol and seocalcitol, which are characterized by their side chain modifications. These structures show the conservation of the VDR structure and the adaptation of the side chain anchored by hydroxyl moieties. The structure of VDR-calcipotriol helps us to understand the structural basis for for the switching of calcipotriol to a receptor antagonist by further side chain modification. The VDR-seocalcitol structure, in comparison with the structure of VDR-KH1060, a superagonist ligand closely related to seocalcitol, shows adaptation of the D ring and position of C-21 in order to adapt its more rigid side chain.