The Journal of Steroid Biochemistry and Molecular Biology
A perspective on how the Vitamin D sterol/Vitamin D receptor (VDR) conformational ensemble model can potentially be used to understand the structure–function results of A-ring modified Vitamin D sterols
Introduction
1α,25-Dihydroxyvitamin D3 (1,25D) is an essential steroid hormone required for maintenance of calcium and phosphorous homeostasis as well as proper development and maintenance of hair follicles and bone [1], [2]. Clinical manifestations of 1,25D deficiency, including rickets, osteomalacia, muscle weakness, and the development of secondary hyperparathyroidism in patients with chronic kidney disease (CKD) are well recognized. Recent evidence suggests a dearth of Vitamin D may also be associated with additional physiological abnormalities including some forms of cancer, high blood pressure, depression, and immune-system disorders such as multiple sclerosis, rheumatoid arthritis, and diabetes [3], [4], [5], [6], [7], [8], [9], [10], [11].
Vitamin D3, the inactive precursor to 1,25D, can be obtained exogenously from the diet (e.g. fish oils and milk, etc.), and endogenously via photolytic ring opening of 7-dehydrocholestrol upon exposure of the skin to sunlight [12], [13]. Vitamin D from these sources is not biologically active until it is activated by the body. Activation is achieved by two sequential hydroxylations catalyzed by the cytochrome-P450 enzymes CYP27A (25-hydroxylase), in the liver, producing 25(OH)-Vitamin D3 (25D) [14], [15], and CYP27B (1α-hydroxylase), in the kidney [16], [17], producing the most active hormonal form of Vitamin D3, 1α,25(OH)2-Vitamin D3 (1,25D) (Fig. 1A) [18].
However, 1,25D is not the only active metabolite of Vitamin D3 [19], [20], [21], [22]. One of the most recently discovered 1,25D metabolites is 3-epi-1,25D (HJ, Fig. 1A) [23]. 3-epi-1,25D (HJ) has been found to be produced, by a non-P450-related enzyme [24], in a variety of primary and transformed cell lines [21], [23], [25], [26]. In addition, the 3-epi homologs of 1,25D, 24,25(OH)2-D3, and 25(OH)D3 have been found in rats administered pharmacological doses of the parent Vitamin D metabolite [27], [28], [29]. Also different drug forms of 1,25D have been shown to undergo 3-epimerization [30], [31], [32]. Physiologically 3-epi-1,25D is unique because it is nearly as potent as 1,25D in suppressing PTH secretion [21], yet it interestingly lacks 1,25D's induction of hypercalcemia [33]. Alternatively, epimerization of both A-ring stereocenters (β,α; HH, Fig. 1) produces a Vitamin D sterol that significantly enhances PTH secretion [21] and lacks a hypercalcemic response [33]. It has been previously proposed by scientists in the field that HJ's intriguing potential pharmacological properties may be a direct result of its increased affinity for the serum Vitamin D binding protein (DBP) [33], [34].
The synthetic 1β,25(OH)2-Vitamin D3 (HL, Fig. 1A) is an A-ring stereoisomer of 1,25D that has no significant effect on PTH secretion [21], but is the only A-ring diastereomer of 1,25D that is well recognized to be a potent antagonist of 1,25D's non-genomic responses [35], [36]. Chemically and structurally 1,25D, 3-epi-1,25D (HJ), 1-epi-1,25D (HL), and 1β,3α-1,25D (HH) have the same molecular volume and intrinsic conformational flexibility in their side-chain and seco-B-ring regions [13], [34], [35]. However, it has been known for a long time that epimerization of either carbons 1 or 3 alters the A-ring chair equilibrium to favor a diaxial orientation in hydrophobic NMR solvents (e.g. CDCl3, see Fig. 1A and B) and a diequatorial orientation in hydrophilic NMR solvents (e.g. acetone-d6) [13], [37], [38]. All three of 1,25D's A-ring diastereomers show poor affinity for the VDR as measured in a steroid competition assay [35], [36] (Fig. 1A).
Recently our laboratory has demonstrated using in silico techniques [39], [40] that the VDR may contain an alternative ligand binding pocket (A-pocket) that 1,25D can fit, and make favorable non-bond interactions with, in three of four A-ring (α or β)/seco-B-ring (cis or trans) combinations. The presence of an A-pocket that physically overlaps with the A-ring domain of the genomic pocket (G-pocket, Fig. 2B) and the concept that helix-12 (H12) of the VDR is mobile in the absence of ligand, has led to the proposal that a conformational ensemble model [39], [40], [41] rather than a classical induced-fit model [42] may better fit the known Vitamin D sterol/VDR structure–function results.
In this study, 1,25D (1α,3β) and its three A-ring diastereomers [HJ (1α,3α), HH (1β,3α), HL (1β,3β)], as well as its 3-deoxy analogs (CF, Fig. 1A) and its 1-deoxy (25D, Fig. 1A) precursor metabolites were docked in the VDR A- and G-pockets (see Section 2). Analysis and comparison of the putative low energy molecular models and MD simulations indicates that non-bond contacts made by the sterols and their potential stabilities are different in the G-pocket, but HJ, HH, and 1,25D all bind the putative A-pocket similarly. A mechanism that centers on the ability of the C1 and C3 hydroxyls to interact with Y143, S237, R274, S275, and S278 (Fig. 2A) in an exchangeable manner is proposed, that provides a unique molecular rationale for why these sterols functional potencies do not always correlate with their VDR affinity, as measured in a steroid competition assay.
Section snippets
Reagents
1α,25(OH)2-Vitamin D3 (1,25D), 25(OH)-Vitamin D3 (25D), and 1β,25(OH)2-Vitamin D3 (HL) were gifts from Dr. Milan Uskokovic (Hoffmann La Roche, Nutley, NJ). See Norman et al. [33] for other 1,25D A-ring diastereomers. 3-Deoxy-1,25D (CF) was a gift from Professor William H. Okamura (University of California, Riverside).
RCI assay
See Wecksler and Norman [43].
Discovery of the putative VDR alternative ligand binding site (A-pocket)
See Mizwicki et al. [39].
Basic minimization protocol
See Mizwicki et al. [39] or contact [email protected].
Docking the A-ring analogs to the VDR G- and A-pockets
The positions of the VDR backbone and other heavy atoms in the
Relative competitive index (RCI) in vitro assay
The VDR binding affinities, as measured in a steroid competition assay (RCI), for 1,25D and the other three A-ring diastereomers have the following rank order: 1,25D (α,β) [100%] > HJ (α,α) [24%] > HL (β,β) [1.0%] > HH (α,β) [0.2%] (Fig. 1A) [33]. This rank order is consistent with results from other laboratories [21], [45]. 3-Deoxy-1,25D (CF, Fig. 1A) has a VDR RCI value of ∼6.0%, while removal of the 1α-OH of 1,25D severely abrogates the sterol's VDR RCI value (25D RCI = 0.15%).
Partial trypsin digest or protease sensitivity in vitro assay (PSA)
The VDR conformations
Discussion
Elucidating the mechanism(s) underlying the lack of correlation between VDR RCI (affinity) and Vitamin D sterol/VDR structure–function experimental results is currently a challenging scientific endeavor. The existence of other known Vitamin D sterol binding proteins (e.g. the serum Vitamin D binding protein (DBP) and Vitamin D sterol metabolic enzymes) may provide a rational explanation for this lack of correlation; however, it is also theoretically possible that the detailed way the A-ring
Conclusions
It was shown here that the Vitamin D sterol/VDR conformational ensemble model can be used to provide a plausible mechanistic understanding for the in vitro and intracellular activities of 1,25D, its three A-ring diastereomers, and 1- and 3-deoxy-1,25D. It is noted that further structural and kinetic validation of the proposed model is required, because the model is currently based on extrapolations from energy minimized and crystallographic ligand–receptor complexes that show total energy
Acknowledgment
This work was supported by NIH grant DK-03012-38 (AWN).
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