The crystal structures of the ligand binding domain of human vitamin D receptor (VDR) complexed with its natural ligand or the superagonists MC1288 or KH1060 have recently been reported. The crystallized ligand binding domain (LBD) of VDR, however, differs from the full-length VDR with respect to deletion of 50 amino acids between its helices 2 and 3. In this study, we investigated structurally and functionally important amino acid interactions within the ligand binding pocket of the fulllength VDR in the presence of several synthetic vitamin D 3 analogs. We used site-directed mutagenesis scanning combined with limited proteolytic digestion, electrophoretic mobility shift assay, and reporter gene assay and correlated the findings with the crystal structures of truncated VDR LBD. Our results suggest that structurally different agonists have distinct ligandreceptor interactions and that the amino acid residues H229, D232, E269, F279, and Y295 are critical for the agonistic conformation of the VDR. Our biological data, which were obtained with the full-length VDR, fit well with the crystal structure of the truncated VDR LBD and suggest that removal of the insertion domain between helices 2 and 3 of the receptor does not markedly influence the functionality of the VDR.The biologically active form of vitamin D 3 , 1␣,25-dihydroxyvitamin D 3 (calcitriol), regulates several important physiological and biochemical functions in mammals, such as calcium and phosphorus homeostasis as well as cellular growth, differentiation, and apoptosis (DeLuca et al., 1990;Walters, 1992). Binding of calcitriol to its receptor (VDR) induces a conformational change in the receptor and promotes formation of a heterodimer with retinoid X receptor (RXR) as well as binding of other transcriptional cofactors to the receptor. The VDR-RXR heterodimer attaches to a specific segment of DNA, the vitamin D response element (VDRE), which is located in the promoter regions of calcitriol target genes (Carlberg, 1995(Carlberg, , 1996Toell et al., 2000).The VDR belongs to the nuclear receptor superfamily. Many members of this superfamily have conserved amino acid sequences in the C terminus of the receptor (Wurtz et al., 1996;Haussler et al., 1998). VDR, however, makes an exception in that it has a long, unordered loop structure between helices 2 and 3. The LBDs of several nuclear receptors have already been successfully crystallized (Bourguet et al., 1995;Renaud et al., 1995;Wagner et al., 1995;Brzozowski et al., 1997;Williams and Sigler, 1998;Pike et al., 1999;Gampe et al., 2000;Egea et al., 2000;Clayton et al., 2001;Watkins et al., 2001), and a clear homology can be observed in their three-dimensional structures because they share a common helical sandwich structure with three layers of ␣-helices surrounding the hydrophobic ligand binding pocket. When the crystal structure of truncated VDR LBD was solved by Rochel et al. (2000), it could be observed that VDR does not make an exception to the structural principles of related receptors. However,...

1,25-Dihydroxyvitamin D3 (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D3 analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo+ ++-1alpha,25(OH)2D3), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 microM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRbeta proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRbeta-complex with additional nuclear proteins.

A growing number of calcitriol (1a,25-dihydroxyvitamin D 3 ) analogs have become available in recent years. Many of these analogs exhibit lower calcemic effects than calcitriol and inhibit cell proliferation and enhance cell differentation more efficiently than calcitriol. We have compared structure-function relationships of a series of new C-20 epimer (20-epi) vitamin D 3 analogs with their natural C-20 counterparts. In human MG-63 osteosarcoma cells, quantification of cellular osteocalcin mRNA levels by Northern blot analysis and osteocalcin biosynthesis by radioimmunoassay indicated that most studied analogs at a concentration of 10 nm induced osteocalcin gene expression more efficiently than the parent compound, calcitriol. Interestingly, when the biological responses were compared with the binding affinities of the analogs to in-vitro translated human vitamin D receptor and with their ability to protect the receptor against partial proteolytic digestion, significant correlations were not observed. Further, molecular modelling of the compounds by energy minimization did not reveal marked differences in the three-dimensional structures of the analogs. These results suggest that higher than normal ligand binding affinity or`natural' conformation of the ligand-receptor complex are not necessarily required for the`superagonist' transactivation activity. The mechanism of action of the efficient analogs may involve stabilization and/or differential binding of transcriptional coactivators or transcription intermediary factors to the hVDR during transactivation.

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The most important allergenic protein in cow dander and urine is Bos d 2. It is proposed to belong to the family of lipocalins, which are proteins capable of binding small hydrophobic molecules. The allergenic properties of Bos d 2 indicate an interaction between the accessible regions of the native protein and IgE. In this work, a three-dimensional model was created for Bos d 2 by comparative modeling, and features characteristic of outlier lipocalins were observed. The protruding regions of the surface were characterized and used in predicting the possible B-cell epitopes. There is a pocket inside the core and its size is appropriate for small molecules. The model shows a hydrophilic amino acid side chain of glutamic acid 115 on the inner surface of the hole and a phenylalanine as the "gatekeeper" instead of tyrosine, which is common in experimentally modeled lipocalins.