NMR assignments of tryptophan residue in apo and holo LBD-rVDR (original) (raw)
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Reviews in Endocrine & Metabolic Disorders - REV ENDOCR METAB DISORD, 2001
The integrated operation of the vitamin D-endocrine system which produces the steroid hormone, 1a,25dihydroxyvitamin D 3 [1a,25(OH) 2 D 3 ], is dependent on the correct functioning of three different proteins, namely the plasma vitamin D-binding protein (DBP), the nuclear receptor (VDR nuc) for 1a,25(OH) 2 D 3 and a putative membrane receptor (VDR mem) responsible for rapid (nongenomic) biological responses. Although these three proteins have inherent in their secondary and tertiary structure a ligand-binding domain (LBD) that allows the stereospeci®c binding of 1a,25(OH) 2 D 3 or related analogs as agonist or antagonist ligands, these LBDs have no amino acid sequence homology or secondary or tertiary structure similarities with one another. Accordingly, a challenging structure-function problem has been to de®ne the precise shape of the unusually conformationally¯exible 1a,25(OH) 2 D 3 as it functions as a ligand for DBP, VDR nuc , and VDR mem. This article reviews 20 new analogs of 1a,25(OH) 2 D 3 with respect to how they provide insight into de®ning the LBD requirements of the DBP, the VDR nuc , and the VDR mem to facilitate the identi®cation of potential new drug forms of 1a,25(OH) 2 D 3 .
Ligand-Specific Structural Changes in the Vitamin D Receptor in Solution
Biochemistry, 2011
Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25dihydroxyvitamin D 3 [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D 3 [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.
Journal of Medicinal Chemistry, 2012
Actual use of the active form of vitamin D (calcitriol or 1α,25-dihydroxyvitamin D 3) to treat hyperproliferative disorders is hampered by calcemic effects, hence the continuous development of chemically modified analogues with dissociated profiles. Structurally distinct nonsecosteroidal analogues have been developed to mimic calcitriol activity profiles with low calcium serum levels. Here, we report the crystallographic study of vitamin D nuclear receptor (VDR) ligand binding domain in complexes with six nonsecosteroidal analogues harboring two or three phenyl rings. These compounds induce a stimulated transcription in the nanomolar range, similar to calcitriol. Examination of the protein−ligand interactions reveals the mode of binding of these nonsecosteroidal compounds and highlights the role of the various chemical modifications of the ligands to VDR binding and activity, notably (de)solvation effects. The structures with the tris-aromatic ligands exhibit a rearrangement of a novel region of the VDR ligand binding pocket, helix H6.
Journal of Bone and Mineral Research, 2003
R ECENTLY TWO BREAKTHROUGHS have been achieved with respect to understanding the three-dimensional protein structures of both the vitamin D binding protein (DBP) and the nuclear receptor (VDR) for the steroid hormone 1␣,25(OH) 2 -vitamin D 3 [1␣,25(OH) 2 D 3 ] and the detailed shape of their respective bound ligands. The determination of the crystal structure of the ligand binding domain (LBD) of the VDR bound to its natural ligand, 1␣,25(OH) 2 D 3, and to several superagonist analogs of 1␣,25(OH) 2 D 3
The Journal of Steroid Biochemistry and Molecular Biology, 2010
Ab initio fragment molecular orbital calculation van der Waals dispersion interaction energy Alanine-scanning mutational analysis Interfragment interaction energy analysis a b s t r a c t To provide physicochemical insight into the role of each residue in the ligand-binding pocket (LBP) of the vitamin D receptor (VDR), we evaluated the energies of the interactions between the LBP residues and 1␣,25(OH) 2 D 3 by using an ab initio fragment molecular orbital (FMO) method at the Møller-Plesset second-order perturbation (MP2) level. This FMO-MP2 method can be used to correctly evaluate both electrostatic and van der Waals dispersion interactions, and it affords these interaction energies separately. We deduced the nature of each interaction and determined the importance of all the LBP residues involved in ligand recognition by the VDR. We previously reported the results of alanine-scanning mutational analysis (ASMA) of all 34 non-alanine residues lining the LBP of the human VDR. The theoretical results in combination with the ASMA results enabled us to assign the role of each LBP residue. We concluded that electrostatic interactions are the major determinant of the ligand-binding activity and ligand recognition specificity and that van der Waals interactions are important for protein folding and, in turn, for cofactor binding.
Vitamin D Receptor Agonists Specifically Modulate the Volume of the Ligand-binding Pocket
Journal of Biological Chemistry, 2006
Existing crystal structure data has indicated that 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ) and its analogues bind the ligandbinding pocket (LBP) of the human vitamin D receptor in a very similar fashion. Because docking of a ligand into the LBP is a more flexible process than crystallography can monitor, we analyzed 1␣,25(OH) 2 D 3 , its 20-epi derivative MC1288, the two side-chain analogues Gemini and Ro43-83582 (a hexafluoro-derivative) by molecular dynamics simulations in a complex with the vitamin D receptor ligand-binding domain and a co-activator peptide. Superimposition of the structures showed that the side chain of MC1288, the first side chain of the conformation II of Gemini, the second side chain of Ro43-83582 in conformation I and the first side chain of Ro43-83582 in conformation II take the same agonistic position as the side chain of 1␣,25(OH) 2 D 3 . Compared with the LBP of the natural hormone MC1288 reduced the volume by 17%, and Gemini expanded it by 19%. The shrinking of the LBP of MC1288 and its expansion to accommodate the second side chain of Gemini or Ro43-83582 is the combined result of minor movements of more than 30 residues and major movements of a few critical amino acids. The agonist-selective recognition of anchoring OH groups by the conformational flexible residues Ala-303, Leu-309, and His-397 was confirmed by in vitro assays. In summary, variations in the volume of agonists lead to adaptations in the volume of the LBP and alternative contacts of anchoring OH-groups.
The Journal of Steroid Biochemistry and Molecular Biology, 2007
Most of the biological effects of 1,25-dihydroxyvitamin D 3 (hormone D) are mediated through the nuclear vitamin D receptor (VDR). Hormone binding induces conformational changes in VDR that enable the receptor to activate gene transcription. It is known that residues S237 and R274 form hydrogen bonds with the 1-hydroxyl group of hormone D, while residues Y143 and S278, and residues H305 and H397 form hydrogen bonds with the 3-hydroxyl and the 25-hydroxyl groups of the hormone. A series of VDR mutations were constructed (S237A, R274A, R274Q, Y143F, Y143A, S278A, H305A, and H397F; double mutants: S237A/R274A, Y143F/S278A, Y143A/S278A, and H305A/H397F). The relative binding affinities of the wild-type and variant VDRs were assessed. All of the mutants except H397F resulted in lower binding affinity compared to wild-type VDR. Binding to hormone was barely detectable in Y143F, H305A, and H305A/H397F mutants, and undetectable in mutants R274A, R274Q, Y143A, S237A/R274A, and Y143A/S278A, indicating the importance of these residues. Ability to activate gene transcription was also assessed. All of the VDR mutants, except the single mutant S278A, required higher doses of hormone D for half-maximal response. Defining the role of hormone D-VDR binding will lead to a better understanding of the vitamin D signal transduction pathway.
The Journal of Steroid Biochemistry and Molecular Biology, 2004
The steroid hormone 1␣,25(OH) 2-Vitamin D 3 [1␣,25(OH) 2 D 3 ] exerts a wide variety of biological actions through one or more receptors/binding proteins. The nuclear Vitamin D receptor (VDR) when bound to its natural ligand, 1␣,25(OH) 2 D 3 , can stimulate transcription of a wide variety of genes. The synthesis of 1␣,25(OH) 2 D 3 analogs allows the study of structure-function relationships between ligand and the VDR. 1␣,25(OH) 2 D 3 is a conformationally flexible molecule; specifically the side-chain of the hormone can display a large variety of shapes for its receptor. Here, we describe and analyze the properties of 10 1␣,25(OH) 2 D 3 analogs modified at the side-chain of which five lack carbon-19 (19-nor) but have a novel 20-cyclopropyl functionality. Analog NG [20,21-methylene-23-yne-26,27-F 6-19-nor-1␣,25(OH) 2 D 3 ] possesses a respectable binding affinity for the VDR and exhibits a high transcriptional activity (EC 50 ∼10 pM), while retaining low induction of hypercalcemia in vivo in the mouse, making it a primary candidate for further analyses of its anti-proliferative and/or cell differentiating properties.
The Journal of Steroid Biochemistry and Molecular Biology, 2010
Molecular modeling results indicate that the VDR contains two overlapping ligand binding pockets (LBP). Differential ligand stability and fractional occupancy of the two LBP has been physiochemically linked to the regulation of VDR-dependent genomic and non-genomic cellular responses. The purpose of this report is to develop an unbiased molecular modeling protocol that serves as a good starting point in simulating the dynamic interaction between 1␣,25(OH) 2-vitamin D 3 (1,25D3) and the VDR LBP. To accomplish this goal, the flexible docking protocol developed allowed for flexibility in the VDR ligand and the VDR atoms that form the surfaces of the VDR LBP. This approach blindly replicated the 1,25D3 conformation and side-chain dynamics observed in the VDR X-ray structure. The results are also consistent with the previously published tenants of the vitamin D sterol (VDS)-VDR conformational ensemble model. Furthermore, we used flexible docking in combination with whole-cell patch-clamp electrophysiology and steroid competition assays to demonstrate that (a) new non-vitamin D VDR ligands show a different pocket selectivity when compared to 1,25D3 that is qualitatively consistent with their ability to stimulate chloride channels and (b) a new route of ligand binding provides a novel hypothesis describing the structural nuances that underlie hypercalceamia.
The role of residue C410 on activation of the human vitamin D receptor by various ligands
The Journal of Steroid Biochemistry and Molecular Biology, 2012
Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in biologically important processes. The human vitamin D receptor (hVDR) is a member of the NR superfamily and is responsible for maintaining calcium and phosphate homeostasis. This receptor is activated by its natural ligand, 1␣, 25-dihydroxyvitamin D 3 (1␣, 25(OH) 2 D 3), as well as bile acids such as lithocholic acid (LCA). Disruption of molecular interactions between the hVDR and its natural ligand result in adverse diseases, such as rickets, making this receptor a good target for drug discovery. Previous mutational analyses of the hVDR have mainly focused on residues lining the receptor's ligand binding pocket (LBP) and techniques such as alanine scanning mutagenesis and site-directed mutagenesis. In this work, a rationally designed hVDR library using randomized codons at selected positions provides insight into the role of residue C410, particularly on activation of the receptor by various ligands. A variant, C410Y, was engineered to bind LCA with increased sensitivity (EC 50 value of 3 M and a 34-fold activation) in mammalian cell culture assays. Furthermore, this variant displayed activation with a novel small molecule, cholecalciferol (chole) which does not activate the wild-type receptor, with an EC 50 value of 4 M and a 25-fold activation. The presence of a bulky residue at this position, such as a tyrosine or phenylalanine, may contribute towards molecular interactions that allow for the enhanced activation with LCA and novel activation with chole. Additional bulk at the same end of the pocket, such as in the case of the variant H305F; C410Y enhances the receptor's sensitivity for these ligands further, perhaps due to the filling of a cavity. The effects of residue C410 on specificity and activation with the different ligands studied were unforeseen, as this residue does not line the hVDR's LBP. Further investigating of the structure-function relationships between the hVDR and its ligands, including the mutational tolerance of residues within as well as outside the LBP, is needed for a comprehensive understanding of the functionality and interactions of the receptor with these ligands and for development of new small molecules as potential therapeutic drugs.