On the Mechanism Underlying (23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone Antagonism of hVDRwt Gene Activation and Its Switch to a Superagonist

(23S)-25-Dehydro-1α(OH)-vitamin D3-26,23-lactone (MK) is an antagonist of the 1α,25(OH)2-vitamin D3 (1,25D)/human nuclear vitamin D receptor (hVDR) transcription initiation complex, where the activation helix (i.e. helix-12) is closed. To study the mode of antagonism of MK an hVDR mutant library was designed to alter the free molecular volume in the region of the hVDR ligand binding pocket occupied by the ligand side-chain atoms (i.e. proximal to helix-12). The 1,25D-hVDR structure-function studies demonstrate that 1) van der Waals contacts between helix-12 residues Leu-414 and Val-418 and 1,25D enhance the stability of the closed helix-12 conformer and 2) removal of the side-chain H-bonds to His-305(F) and/or His-397(F) have no effect on 1,25D transactivation, even though they reduce the binding affinity of 1,25D. The MK structure-function results demonstrate that the His-305, Leu-404, Leu-414, and Val-418 mutations, which increase the free volume of the hVDR ligand binding pocket, significantly enhance MK antagonist potency. Surprisingly, the H305F and H305F/H397F mutations turn MK into a VDR superagonist (EC50 ∼ 0.05 nm) but do not concomitantly alter MK binding affinity. Molecular modeling studies demonstrate that MK antagonism stems from its side chain energetically preferring a pose in the VDR ligand binding pocket where its terminal C26-methylene atom is far removed from helix-12. MK superagonism results from an energetically favored increase in interaction between Leu-404/Val-418 and C26, resulting in an increase in the stability and population of the closed, helix-12 conformer. Finally, the results/model generated, coupled with application of a VDR ensemble allosterics model, provide an understanding for the species specificity of MK.

The steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1,25D) 2 (see Fig. 1) and the nuclear vitamin D receptor (VDR) forms a complex that modulates the transcription of genes containing a VDR element. This process is termed a genomic response and involves 1,25D binding to the VDR, formation of a heterodimer with retinoid X receptor, and recruitment of nuclear co-activators (NCoAs) and the basal transcription machinery (1).
The VDR is a member of the nuclear hormone superfamily of transcription factors, all of which share similar domain partitioning, ligand binding domain (LBD) tertiary fold and localization of their ligand binding pocket (LBP). The current inducedfit model describing nuclear receptor (NR) activation (i.e. the mouse-trap model) is founded on the comparison of apo-and holo-NR x-ray crystal structures. The mouse trap model posits that closure of the NR activation helix (i.e. helix-12) is induced by ligand binding to an opened-like helix-12 apo-NR conformer. The closed helix-12 conformation completes the activation function II domain (AF2), which serves as the high affinity binding site for recruitment of various NCoAs (1-4) (Fig. 2). Thus, the steric blockage of NR helix-12 closure is the basis for the design of traditional NR genomic antagonists (5)(6)(7)(8)(9).
1,25D-VDR-induced genomic responses can be blocked by co-incubation with a 10-fold excess of analog MK or ZK159222 (ZK, Fig. 1); however, the mode of antagonism is different for these two molecules (7,10). The ZK side-chain molecular volume is significantly enhanced relative to 1,25D; therefore, ZK acts as a traditional NR antagonist by blocking VDR helix-12 closure (5). Two mechanisms have been proposed to explain MK antagonism; both are based on the inability of MK to function as an antagonist in the rodent VDR (rVDR). One current hypothesis suggests that the ␣/␤-unsaturated exo-cyclic methylene of MK (Fig. 1) undergoes a 1,4-Michael addition (i.e. formation of a covalent MK-VDR adduct) with Cys-403 and/or Cys-410 of the human VDR (hVDR), neither of which are present in the rVDR primary sequence (10). Yet another hypothesis proposes that the energetic stability of the rVDR closed helix-12 conformer is enhanced relative to hVDR (7). Thus the detailed allosterics underlying MK antagonism of 1,25D-hVDR-mediated transactivation remains an important, unresolved question in light of the proposed use of MK or its analogs as a potential treatment for Paget disease (12).
To date no x-ray complex of the MK⅐hVDR complex is available. Therefore, to gain a molecular understanding to MK antagonism of 1,25D-hVDR gene activation and species specificity (7,10,(13)(14)(15)(16)(17) we applied traditional structure-function assays, molecular modeling, and a vitamin D sterol-VDR conformational ensemble model (18,19). Our comprehensive structure-function studies demonstrate that MK antagonism hinges on its reduced side-chain molecular volume allowing it to bind favorably to two distinct regions within the hVDRwt LBP. Furthermore, we show for the first time in the NR field that a known antagonist (MK) can be converted into a superagonist ligand by a conservative single point mutation and provide further evidence in support of the VDR ensemble model (18 -21).

EXPERIMENTAL PROCEDURES
Molecular Volume Calculations-The molecular volume of 1,25D, MK, and ZK was determined using Discovery Studio 2.0 (Molecular Attributes, Accelrys Inc.) and atom typing with the cff forcefield. For 1,25D and ZK the conformation used for the calculation was taken from the pose observed in their reported VDR LBD x-ray .pdb files. For MK the lowest energy complex determined using the flexible docking protocol (see below) was used to calculate the molecular volume. The molecular volumes of the hVDRwt ligand binding pocket (LBP) and the His-305, His-397, Leu-404, Leu-414, and Val-418 mutants were calculated following mutant generation and side-chain refinement in DS2.0. Following refinement the Receptor-Ligand Interaction/ Binding Site/Find Sites from Receptor Cavities tool in DS2.0 was used to determine the molecular volume of the LBP.
EC 50 Determination in CV1 Cells-Cells were seeded at 0.4 ϫ 10 5 cells per well on 24-well plates (BD Biosciences) in minimal Eagle medium with Earle's buffered salts and non-essential amino acids, with 10% fetal bovine serum (Mediatech Inc., Herndon, VA). After 24-h incubation, phosphate-buffered saline-washed cells, at ϳ60% confluence, were transfected using a 10-min pre-treatment with 0.2 mg/ml DEAE-dextran (Sigma) in phosphate-buffered saline. Pre-treated cells were then washed in phosphate-buffered saline and incubated for 30 min with phosphate-buffered saline containing 0.1 g/well activator pcDNA3.1(Ϫ)Nhe1(Ϫ)VDR or mutant plasmid and 0.5 g/well reporter OC-pSEAP2 (secreted alkaline phosphatase) (22). Transfected cells were incubated in 80 M chloroquine in growth media with 5% charcoal-stripped fetal bovine serum for 4 h followed by the same culture medium without chloroquine for 24 h. At 28 h post transfection, the cell medium was replaced with the same medium containing various concentrations of 1,25D or its analogs, with a final ethanol concentration of 0.1%. In all antagonism studies first the 1,25D EC 50 in the given VDR construct was experimentally derived (n Ն 3), then a 10-fold excess of MK was co-incubated with the EC 50 concentration of 1,25D (see supplemental materials). At 22 h after analog treatment, an aliquot of the cell medium was harvested to measure secreted alkaline phosphatase using the Phospha-Light TM Kit (Tropix, Bedford, MA). The concentration of steroid producing 50% (EC 50 ) of the maximal activity was then determined using Prism v5.0.
Protease Sensitivity Assay-[ 35 S]-VDR was generated from pcDNA3.1(Ϫ)Nhe1(Ϫ)VDR or mutant VDR plasmid constructs using the Promega TNT reticulocyte lysate kit. The reaction tubes were incubated for 2 h at 30°C then placed on ice. 1,25D or analog was added, and the tubes were incubated at room temperature for 20 min. 15 g/ml trypsin was added to the tubes and incubated for 20 min at room temperature followed by 5.0-min incubation at 80°C with SDS. Reaction mix-tures were then loaded in a 12.5% or 15% SDS-PAGE gel and subjected to electrophoresis. Bands were visualized by autoradiography. The molecular weight of each band was calculated using Stratagene EagleSight software (v3.0.001).
To determine the affinity of ligands for the hVDR, 0.05 pmol of [ 3 H]1,25D in EtOH was added to borosilicate glass tubes (Fisher). Increasing concentrations of cold ligands were added to the tubes in duplicate, and total EtOH was adjusted to 20 l. 230 l (25 g of total protein, determined by Bradford) of the diluted, clarified VDR-LBD was added to each tube, briefly vortexed, and placed at 4°C for 4 -24 h (buffer same as lysis buffer minus protease inhibitor cocktail and pH 8.0). Following incubation, 250 l of 50% (v/v) hydroxyapatite solution in TED (10 mM Tris-base, 1.5 mM EDTA, 1 mM dithiothreitol) was added and washed three times with 1 ml of TED plus 0.5% Triton X-100. Bound [ 3 H]1,25D was eluted with 1 ml of ethanol and counted in 7 ml of Liquiscint (National Diagnostics) scintillation mixture using a Beckman LS6500. Data from duplicate samples were averaged and plotted as % max bound [ 3 H]1,25D versus the log of the concentration of the competitor used. Non-linear regression using Prism (GraphPad Software, San Diego, CA) was performed to determine the IC 50 , correlation coefficient, and R 2 values. Data were also plotted to generate a relative competitive index value (21).
PC_Model v8.0 Calculations-Whole molecule GMMX (Global-MMX) experiments were carried out on 1,25D and MK allowing for flexibility in the A-ring, seco-B-ring, and sidechain single bonds of both molecules (Fig. 1). The following GMMX variables were used: search on bonds only, -correction turned on, energy window of 11.0 kcal/mol, and 1000 total conformers generated. After the manual check of the sp 2 -hybridized atoms, the 1,25D and MK conformer sets were typed with the cff forcefield (DS2.0) and used for the flexible docking simulation.
Flexible Docking Protocol-The complex formation between the apo-hVDR molecules and the PC_Model ligand conformers was simulated by applying a flexible docking protocol (DS2.0). To simulate the 1,25D and MK complex formation with the hVDRwt LBP and its mutants, 50 hVDR LBP conformations were first generated (this includes the hVDRwt LBD). Tyr-401, Leu-404, Leu-414, Thr-415, Val-418, and Phe-422 (i.e. the flexible docking residues, these residues form the VDR LBP). In all of the calculations the VDR conformational isomers were generated and the protocol stopped, so that the same set of VDR LBP conformers was sourced in all the simulations. The flexible docking protocol variables changed from the default parameters were as follows: a 10.0-Å docking sphere was defined by searching the apo-VDR molecule for putative binding sites; the number of flexible docking residues allowed for structural refinement was set to 12; the top 5 hits were saved for each protein-ligand pair, and a simulated annealing step was performed. The Generalized Born with Molecular Volume method (23,24) was used to determine the ⌬G binding value (⌬G binding ϭ ⌬G holo-VDR Ϫ ⌬G apo-VDR Ϫ ⌬G ligand ), where the only default values changed were the cutoff distances, which were changed from 14, 12, and 10 Å, to 10, 8, and 6 Å, respectively. For a more detailed description of the computational techniques/protocols employed herein see the supplemental materials.
To study the impact that expanding or shrinking the free molecular volume of the VDR LBP proximal to the helix-3/ helix-12 interface (Fig. 2) has on 1,25D-hVDR transactivation, we generated seven conservative hVDR point mutants. These were used to determine the 1,25D effective concentration (EC 50 ) required to induce the expression of an osteocalcin-driven SEAP reporter, in CV1 cells ( Table 1). The transactivation results demonstrated that all of the hVDR mutant constructs that increase the free molecular volume proximal to the helix-3/helix-12 interface suppressed 1,25D transactivation. In contrast, mutations made that reduced the free molecular volume, had little or no effect on the 1,25D EC 50 ( Table 1).
Effect of Helix-3/Helix-12 Interface Mutations on MK Antagonist Potency-Co-incubation of 10 nM MK with 1 nM 1,25D significantly hindered the ability of 1,25D to act as a potent genomic agonist in CV1 cells transiently transfected with  The hVDR (LBD, aa 120 -427, ⌬165-215) is rendered to depict its ribbon structure and the relative hydrophobicity of its amino acid R-groups (blue, hydrophobic; red, polar, charged; pink-white, polar, uncharged). 1,25D is surface-rendered to illustrate its van der Waals (vdW) surface (green) and the location of the hVDR LBP. The position of the 1,25D C25 hydroxyl group and the C26/C27 side-chain methyl groups are labeled. The methyl groups lie proximal to Leu-227 helix-3 (H3), Leu-404 (H11), Leu-414 (H12), and Val-418 (H12), which form the hVDR LBP helix-3/ helix-12 interface. The 25-OH group of 1,25D forms H-bonds with His-305 and His-397. The His-305 and Leu-404 residues form the bottom of the hVDR LBP. The upper surface of the nuclear co-activator (NCoA) binding site is highlighted by rendering the surface of amino acids 233 (H3) through 264 (H5) visible. The bottom of the NCoA surface is formed by helix-12 (H12). Helices 10 (H10) and 11 (H11) are labeled for reference. hVDRwt (Fig. 3). When a 10-fold excess of MK was combined with an EC 50 dose of 1,25D in the helix-3/helix-12 interface mutants (Table 1), MK antagonist potency was significantly enhanced by all of the hVDR mutants that increased the free molecular volume of the hVDR LBP (Figs. 2 and 3). These results were somewhat expected given MK had a reduced sidechain molecular volume (344 Å 3 ) when compared with 1,25D (360 Å 3 (Fig. 1)).
hVDR-His-305 and/or His-397 Mutations Alter 1,25D Agonist Potential and hVDR Binding Affinity-Located opposite the helix-3/helix-12 interface in the hVDR LBP are the basic His-305 and His-397 residues (Fig. 2). To study the effect that expanding or shrinking the hVDR LBP molecular volume at the 305 and 397 loci has on 1,25D agonist potential, H305F, H305A, H397F, H305A/H397F, and H305F/H397F mutants were assayed. Of these mutants, the H305A, H397F, and H305A/H397F modifications increased the free volume of the LBP and had a weakened ability to respond to 1,25D (Table 2). However, only the H305A and H305A/H397F mutants were nearly as effective in attenuating 1,25D-induced transactivation when compared with the helix-3/helix-12 interface mutants (compare Tables 1 and 2).
In contrast, the H305F and H305F/H397F mutations reduced the free volume of the hVDR LBP and showed an hVDRwt-like response to 1,25D ( Table 2). This functional result correlated well with the measured affinity of 1,25D in H305F, but not H305F/H397F, where the ability of 1,25D to bind the H305F/H397F was weakened by ϳ15-fold (Table 2).
MK⅐hVDR Antagonist/Agonist Activities Are Significantly Altered by the His-305 and/or His-397-hVDR Mutations-When the H305A and H305A/H397F mutants were co-incubated with an EC 50 dose of 1,25D and a 10-fold excess of MK, MK functioned as a complete antagonist (Յ5% of the 1,25Dreporter activity (Fig. 4, A-C). MK also functioned as a complete antagonist in the R402E hVDR construct, which has been previously shown to intrinsically reduce the stability of the closed hVDR helix-12 conformation (20) (Fig. 4D). The only mutant that increased the free molecular volume of the hVDR LBP that did not adhere to the enhanced antagonistic trend of MK was H397F (Fig. 4E).
Surprisingly, MK antagonism was lost in the H305F and H305F/H397F hVDR mutant constructs (Fig. 4, F and G, respectively). In fact, MK was a 29-and 33-fold more efficient agonist than 1,25D and therefore showed a superagonist response in these mutants (Table 2, 4th column). MK's efficacy (i.e. EC 50 ) with H305F and H305F/H397F were the same; however, the agonist potency of 10 nM MK was approximately five orders of magnitude better in the H305F/H397F construct (Fig.  4G). These results indicate that reducing the free molecular volume of the hVDR LBP provides a physicochemical environment capable of overcoming the reduced molecular volume of the MK side chain (Fig. 1).
Given that the MK side chain has more -bond character when compared with 1,25D, we tested if the switch of MK into a superagonist was due to the H305F modification increasing MK's VDR affinity. The measured MK-VDR affinity (i.e. IC 50 ) in H305F and H305F/H397F indicated that it was only slightly better in the H305F construct and worse in the H305F/H397F construct when compared with the MK⅐hVDRwt affinity ( Table  2). Thus the measured MK-H305F/H397F EC 50 and IC 50 values do not correlate, an observation that was also true for 1,25D (Table 2). Because a correlation between affinity and function did not exist, we next determined if MK is capable of stabilizing the transcriptionally active, closed helix-12 conformation in H305F and H305F/H397F by performing partial trypsin digest (i.e. PSA) experiments.  Each hVDRwt mutation was made, and the molecular volume was calculated using the Discover Studio software package (see "Experimental Procedures" for protocol). CV cells were transiently co-transfected with an osteocalcin-driven SEAP reporter and a hVDR construct and the 1,25D EC 50 was measured (n values presented in table). All EC 50 values are statistically (p Յ 0.05) greater than hVDRwt with the exception of V418L. Representative dose-response curves are provided in supplemental Fig. 1.   -c1) in the H305F and H305F/H397F Constructs-Three major hVDR fragments were generated by PSA. The ϳ34-kDa hVDRwt fragment stabilized by 1,25D represents the active, closed conformation of helix-12 (hVDR-c1, Fig. 5A) (20,30). The MK-H305F and MK-H305F/H397F PSA results demonstrated that MK did stabilize a strong hVDR-c1 band (Fig. 5,  A and B). Furthermore, the hVDR-c1 band was observed at very low doses of MK only in the H305F/H397F construct (Fig. 5,  compare A and B). The hVDR-c1 band was also observed at low doses of 1,25D (Fig. 5C) indicating that the enhanced stability of the hVDR-c1 conformer stems from a favorable intramolecular effect introduced by the H305F/H397F double mutation. This was confirmed by a significant enhancement in the protection of the apo-H305F/H397F construct against proteolytic degradation when compared with apo-hVDRwt (Fig. 5D).

Construct
Formation of MK⅐hVDRwt Covalent Adducts-Consistent with the proposal that the MK molecule can form a covalent adduct with the hVDR LBP (10), the MK⅐hVDRwt PSA dose experiment demonstrated that the c1, c2, and c3 can be covalently modified by MK in a dose-dependent manner (see white arrows, Fig. 6). The covalent adducts were not observed in any of the His-305 or His-397 point mutations, as determined by PSA (Fig. 5 and supplemental Fig. 4), suggesting that either His residue is capable of forming an MK covalent adduct. To test the validity of this hypothesis and to gain a more precise structural/molecular understanding for the switch of MK into a superagonist/complete antagonist and its known species specificity (7, 10), we developed a flexible docking molecular modeling protocol.
Simulating the Dynamics and Energetic Stability of the 1,25D⅐hVDR Complex-To simulate the binding and stability of flexible vitamin D sterols to a flexible hVDR LBP we combined two techniques. Whole molecule PC_Model GMMX conformational search calculations were employed to generate conformational isomers of 1,25D and MK. For 1,25D this computation produced 502 conformational isomers and for MK 133, consistent with the reduction in the rotational degrees of freedom of the MK side chain (Fig. 1). The conformational isomers were then docked to different hVDR LBP conformers using the flexible docking protocol of Discovery Studio 2.0 (DS2.0, see "Experimental Procedures").
Using this approach, the 1,25D conformational isomer observed in the x-ray structure was selected as the most energetically favored ligand pose in the flexible hVDR LBP (Fig. 7A). The ⌬G binding value calculated for the energy optimized 1,25D-hVDR x-ray complex was Ϫ47.1 kcal/mole, whereas the complex predicted by the flexible docking protocol was Ϫ50.7 kcal/ mole (Table 3), indicating a minor refinement to the x-ray complex. Furthermore, the 1,25D ⌬G binding values calculated for H305F, H305F/H397F, H397F, and H305A qualitatively correlated with the measured 1,25D EC 50 values in these mutants ( Table 2).
In the Region 2 MK⅐hVDRwt complex the C26 atom lies closer to Leu-227 (3.27 Å)/Leu-404 (helix 11, 3.93 Å) and away from the helix-12 residues Leu-414 (5.05 Å)/Val-418 (6.98 Å, helix-12 residues, Fig. 7D). This indicates that, when the MK side chain is bound to the LBP in the Region 2 orientation, MK functions as an antagonist. Importantly, the MK side-chain heterogeneity within the LBP was not observed when the same set of MK conformational isomers were flexibly docked to the rodent VDR (rVDRwt) LBP using an identical flexible docking protocol (Fig. 7E).
The average calculated ⌬G binding values for the top five Region 1 and 2 MK⅐hVDR LBP complexes indicate that MK affinity for hVDR is significantly stronger when the side chain occupies Region 2 (Ϫ25.4 versus Ϫ39.3 kcal/mole, respectively, Table 3). This computational result correlated well with the fact that MK functioned as an antagonist ligand in the hVDRwt construct (Figs. 3 and 4A). Furthermore, when the MK side chain was bound to Region 2 of the LBP, an approach to the

,25D/MK EC 50 values, and VDR affinity in the His-305 and/or His-397 point mutants
The LBP molecular volumes were calculated using DS 2.0 (see "Experimental Procedures"). 1,25D and MK EC 50 values were measured in CV cells co-transfected with a SEAP-reporter plasmid and listed VDR construct. All 1,25D EC 50 values are statistically (p Յ 0.05) greater than hVDRwt with the exception of H305F and H305F/H397F. The MK EC 50 values in H305F and H305F/H397F are statistically (p Յ 0.01) less than the 1,25D/MK EC 50 in hVDRwt and 1,25Ds EC 50 in the given mutant. ND indicates that this value was not determined. See supplemental Figs. 1 and 2 for sample 1,25D and MK dose-response curves obtained in these mutants. The relative competitive index values indicate how well MK competes off 0.05 pmol of ͓ 3 H͔1,25D in a recombinant hVDR LBD (aa 118 -427, ⌬165-215) clarified extract. All studies incorporate a HAP workup protocol (31) to separate bound ligand from free. 1.25D and MK inhibitory concentrations (IC 50 , half of ͓ 3 H͔1,25D competed off) were determined in the recombinant hVDR LBD. These values indicate what concentration of MK or 1,25D is required to compete off 50% of the ͓ 3 H͔1,25D. The HAP protocol was used to separate bound from free in these experiments. The IC 50 curves are provided in supplemental Fig. 3.   DECEMBER 25, 2009 • VOLUME 284 • NUMBER 52

Physicochemical Control of 1,25D/MK-VDR Structure-function
formation of an MK-C26/His-305 covalent adduct (3.32 Å) could be observed (Fig. 7D). Structural/Molecular Basis for MK Complete Antagonism-The H305A mutation substantially increased the free volume of the LBP opposite the helix-3/helix-12 interface. Therefore, the average distance between the MK C26 atom and Leu-414 (5.34 Å) and Val-418 (7.08 Å) in the H305A LBP was significantly increased when compared with those same measurements in hVDRwt (4.58 and 5.89 Å, supplemental Table 1). This was due to the side-chain lactone exclusively occupying Region 2 of the H305A LBP ( Fig. 7F and Table 3). These same trends were observed in the H305A/ H397F flexible docking results (supplemental Table 1).
Structural/Molecular Basis for MK Superagonism-The MK-H305F flexible docking results indicate a shift of the MK C26 atom to exclusively occupy Region 1 of the H305F LBP (Table 3 and Fig. 7G). A concomitant increase in MK affinity was also observed (Ϫ47.2 kcal/mole, Table 3). The increased stability of this complex was imparted by aoverlap between Phe-305 and the MK side-chain lactone (supplemental Fig. 7). These same trends were observed in the H305F/H397F LBP flexible docking results (Ϫ47.4 kcal/mol, Table 3).

DISCUSSION
This communication shows that MK antagonism of the 1,25D-hVDRwt genomic response is unique when compared with the traditional mode of NR antagonism. This is due to the reduced side-chain molecular volume of MK when compared with 1,25D and its unique side-chain chemistry (Fig. 1). We demonstrated using traditional structure-function analyses and molecular modeling that the reduced molecular volume allows for heterogeneity in the MK side-chain conformations accepted by the hVDRwt LBP, when compared with 1,25D (Regions 1 and 2, see Fig. 7, A and B). To our knowledge the flexible docking protocol used to generate this hypothesis is the first simulation that lacks bias in the conformation and placement of the ligand within the VDR LBP (7,30,32). Importantly, the average 1,25D/MK⅐hVDRwt ⌬G binding values (Table 3) qualitatively support the fact that a 10-fold excess of MK is required to be an efficient 1,25D-hVDR antagonist (Figs. 3 and 4A and  According to our 1,25D/MK⅐hVDRwt flexible docking results the MK⅐hVDRwt Region 2 complex is energetically favored ( Table 3) and represents a good antagonist pose within the hVDR LBP. This hypothesis is based on the observation that important vdW contacts with the Leu-414 and Val-418 helix-12 residues were absent when MK bound with Region 2 of the VDR LBP (Fig. 7D). So when bound to Region 2, MK was incapable of efficiently anchoring helix-12 in the transcriptionally active, closed conformation (Fig. 8).
This hypothesis is consistent with the antagonist potency of MK being increased by the helix-3/helix-12 interface and H305A mutants (Figs. 3, 4B, and 4C). All of these mutations  increase the free molecular volume of the VDR LBP and exacerbate the intrinsic mobility of helix-12. In the H305A LBP in particular, the MK side chain was shown to exclusively bind to Region 2 of the VDR LBP (Table 3 and Fig. 7F).
Furthermore, the MK⅐hVDRwt Region 2 complex is consistent with the formation of an MK⅐hVDRwt covalent adduct.
However, our results suggest the adduct was formed with His-305 ( Fig. 6) rather than Cys-403 or Cys-410, as previously proposed (see supplemental Fig. 6) (10). Importantly, the MK-His-305 adduct could only be formed when the MK side chain occupied Region 2 of the hVDR LBP; therefore, increasing the half-life of the antagonist pose of MK (Fig. 7D). Thus our comprehensive structure-function analyses provide a detailed molecular understanding underlying the augmented ability of the MK⅐hVDRwt complex to recruit nuclear co-activator (NCoA) proteins (32).
The flexible docking results also provide a rationale for how MK can function as a partial agonist (7,10) or superagonist (Fig. 4, F and G). When bound to the VDR LBP in Region 1, the C26 atom of MK formed vdW contacts with Leu-227, Leu-414, and Val-418. Therefore, MK makes vdW interactions with the helix-3/helix-12 interface residues in Region 1 and explains why MK is not a complete antagonist in hVDRwt (Figs. 2 and 4A) (7,10,16).
This hypothesis is consistent with the observation that in the H305F and H305F/H397F LBPs the MK side chain favors binding to Region 1 (Fig. 7G). Furthermore, in these VDR constructs, MK showed the ability to efficiently stabilize hVDR-c1 (Fig. 5). These two results, coupled with the calculations indicating MKs affinity is increased in the H305F and H305F/H397F constructs (Table 3) provide a rationale explanation for how MK is converted into a superagonist ligand (Fig. 4, F and G). Thus the models (Table 3 and Fig. 7) seem to be more consistent with the MK structure (PSA, Figs. 5 and 6) and function (EC 50 , Fig. 4) results than are the IC 50 values ( Table 2). This is likely due to the dynamics and complexity of ligand binding to the hVDR molecule being much more complex then that inferred by a traditional NR induced-fit model (19).
To our knowledge the switch of a known antagonist into a superagonist by the introduction of a conservative mutation is a novel finding in the NR field. Importantly, the measured MK EC 50 values in the His-305 and H305F/H397F mutants are par-  (25) and the lowest energy (⌬G binding ) flexible docking complex. The all atom root mean square deviation of 1,25D was 0.850 Å. Superimposition of the VDR C␣ atoms of the same two complexes produced a root mean square deviation of 0.618 Å; all VDR backbone atoms were 0.635 Å; all VDR side-chain atoms were 0.956 Å. B, molecular overlay of the top 10 MK⅐hVDR LBP flexible docking complexes, depicting the two distinct MK side-chain conformational isomers observed in these complexes (Region 1, green dashed circle; Region 2, red dashed circle). C, the intermolecular interactions made between the 25-OH group and the C26 and C27 atoms of 1,25D observed in the lowest energy flexible docking complex (⌬G binding ). The VDR helix 11/helix-12 residues (Cys-403, Leu-404, Cys-410, Leu-414, and Val-418) are shown in red, and the helix-3, Leu-227 residue is shown in yellow. The same color scheme is used in panel D. The closest van der Waals (vdW) distance between Leu-227 (3.27 Å), Cys-403 (11.96 Å), Leu-404 (5.25 Å), Cys-410 (7.96 Å), Leu-414 (4.70 Å), Val-418 (4.03 Å), and the C26 or C27 terminal methyl groups of the 1,25D side-chain atoms are indicated by solid lines. The overlay of the top 10 1,25D-hVDRwt flexible docking complexes is presented in supplemental Fig. 5. D, the MK⅐hVDR LBP Region 2 complex and the intermolecular vdW interactions formed between the lactone C26 atom (Fig. 1) and Leu-227 (3.28 Å), His-305 (3.32 Å), His-397 (2.93 Å, to lactone ring oxygen), Leu-404 (3.93 Å), Leu-414 (5.05 Å), and Val-418 (6.98 Å) residues. These contacts are highlighted by purple lines. Importantly, the C26 atom of MK (Fig. 1) lies perpendicular to the sp 2 -hybrid imidazole nitrogen atom of His-305 in this complex, a good molecular geometry for formation of a covalent adduct (supplemental Fig. 6). E, the top 10 MK rodent VDR (rVDR) LBP flexible docking complexes indicate an enhanced selectivity for the MK side chain to localize in Region 1. To view the vdW interactions formed between the lactone of MK and the two His residues in the rVDR complex see supplemental Fig. 7. F, overlay of the top 10 H305A LBP flexible docking complexes. In all of these complexes the MK side chain occupies Region 2 of LBP, indicated by the red dashed circle. G, overlay of the top 10 MK-H305F LBP flexible docking complexes, indicating the C26 atom of MK that exclusively occupies Region 1 (green circle). DECEMBER 25, 2009 • VOLUME 284 • NUMBER 52 allel or better to those reported for historic hVDRwt superagonists (11,33).

Physicochemical Control of 1,25D/MK-VDR Structure-function
We have previously demonstrated that the VDR molecule exists as an ensemble of conformational states, subject to a population distribution change, induced by ligand binding and/or mutation (Fig. 8) (18 -21). The apo-H305F/H397F PSA results demonstrated that the population distribution VDR ensemble members can be altered by subtle changes in the VDR primary sequence. The switch of Cys-403 and Cys-410 of the hVDR to Ser and Asn, respectively, in the rodent VDR (rVDR) has a similar effect, in that the intramolecular stability of the closed helix-12 conformation is enhanced (7, 20) (supplemental Fig.  9). Thus when coupled to the observation that the MK side chain favors Region 1 when flexibly docked to the rodent (rVDR) LBP (Fig. 7E), the inability of MK to form a covalent adduct with the rVDR (10) and MK's species specificity can be understood.
Viewing the VDR or helix-12 as a molecular ensemble (Fig. 8) (18 -21) also provides an explanation for how 1,25D can function to activate transcription as efficiently in the H305F/H397F construct as was observed in hVDRwt, despite a significant reduction in its binding affinity ( Table 2). The same intramolecular effect also provides an explanation for why MK showed greater potency in the H305F/H397F construct relative to H305F (Fig. 4, F and G). In closing, the evidence that the H305F/ H397F double mutation can dramatically alter the population distribution of helix-12 ensemble members indicates that it may be possible to engineer a constitutively active hVDR construct.