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

This paper is dedicated by Professor Anthony W. Norman to his major Professor, Hector F. Deluca, on the occasion of his 75th birthday in April 2005. I had the privilege of being one of Hector's first graduate students in 1959 and benefited from a wonderful education in his laboratory as well as receiving an introduction to my favorite lifetime research topic, Vitamin D.
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Abstract

The steroid hormone 1α,25(OH)2-Vitamin D3 (1,25D) activates both genomic and non-genomic intracellular signaling cascades. It is also well recognized that co-incubation of 1,25D with its C-1 epimer, 1β,25D (HL), suppresses the efficiency of the non-genomic signal activated by 1,25D alone and that its C-3 epimer, 3α-1,25D (HJ) is nearly as potent as 1,25D in suppressing PTH secretion, believed to be propagated by 1,25D's genomic signaling. Both these sterols lack the hypercalcemic effect induced by pharmacological doses of 1,25D and have reduced VDR affinity compared to 1,25D, as measured in a steroid competition assay. Recent functional studies suggest that the VDR is required for both non-genomic and genomic signaling. Along these lines we have recently proposed a Vitamin D sterol/VDR conformational ensemble model that posits the VDR contains two distinct, yet overlapping ligand binding sites, and that the potential differential stabilities of 1,25D and HL in these two pockets can be used to explain their different non-genomic signaling properties. The overlapping region is predominantly occupied by the sterol's A-ring when it is bound to either the genomic ligand binding pocket (G-pocket), defined by X-ray crystallography, or the alternative ligand binding pocket (A-pocket), discovered using in silico techniques (directed docking). Therefore, to gain further insight into the potential application of this model we docked the other A-ring diastereomer [(1β,3α) = HH] of 1,25D and its 1- and 3-deoxy forms (25D and CF, respectively) to the A- and G-pockets to assess their potential stabilities in the pockets, relative to 1,25D. The models were then used to provide putative mechanistic arguments for their known structure–function experimental results. This model may provide new insights into how Vitamin D sterols that uncouple the unwanted hypercalcemic effect from attractive growth inhibitory/differentiation properties can do so by differentially stabilizing different subpopulations of VDR conformational ensemble members.

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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