Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand

Article pubs.acs.org/jmc

Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by Hybrid Approach Combining Small-Angle X‑ray Scattering and Molecular Dynamics Yasuaki Anami,† Nobutaka Shimizu,‡ Toru Ekimoto,§ Daichi Egawa,† Toshimasa Itoh,† Mitsunori Ikeguchi,§ and Keiko Yamamoto*,† †

Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan ‡ Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan § Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan S Supporting Information *

ABSTRACT: Vitamin D receptor (VDR) controls the expression of numerous genes through the conformational change caused by binding 1α,25-dihydroxyvitamin D3. Helix 12 in the ligand-binding domain (LBD) is key to regulating VDR activation. The structures of apo VDR-LBD and the VDR-LBD/antagonist complex are unclear. Here, we reveal their unprecedented structures in solution using a hybrid method combining small-angle X-ray scattering and molecular dynamics simulations. In apo rat VDRLBD, helix 12 is partially unraveled, and it is positioned around the canonical active position and fluctuates. Helix 11 greatly bends toward the outside at Q396, creating a kink. In the rat VDR-LBD/antagonist complex, helix 12 does not generate the activation function 2 surface, and loop 11−12 is remarkably flexible compared to that in the apo rat VDR-LBD. On the basis of these structural insights, we propose a “folding-door model” to describe the mechanism of agonism/antagonism of VDR-LBD.

■

INTRODUCTION Nuclear hormone receptors (NRs) are ligand-dependent transcriptional factors that regulate the expression of specific genes related to development, homeostasis, and metabolism,1,2 and consequently there have been intense efforts to discover pharmaceutical drugs targeting NRs.3−5 Transcriptional regulation is conducted by sequential molecular events: ligand binding, dimerization with a partner receptor, recruitment of coregulators (coactivators/corepressors), and binding to DNA.6 NRs have a highly conserved DNA-binding domain and a moderately conserved ligand-binding domain (LBD). Transactivation is initiated by conformational change of the LBD induced by ligand binding. X-ray crystal structures and other experimental results indicate that transactivation occurs through a local conformational change of helix 12 in the LBD, as follows (Figure 1).7 When an agonist binds to a NR, the loop between helices 10 and 11 converts to a helix to form © 2016 American Chemical Society

sequential helix 10/11, then helix 12 sequentially folds back and creates the activation function 2 (AF-2) surface (Figure 1a). The NR/agonist complex then recruits coactivator, and target gene expression occurs. In the case of antagonist binding, several hypotheses describing the mechanism have been suggested, depending on the antagonist structure. Most hypotheses are based on inhibition of coactivator binding. Upon antagonist binding, the conformation of helix 12 is modulated and helix 12 localizes at a position unsuitable for creating the AF-2 surface, resulting in failure of coactivator recruitment (Figure 1b).8−12 Several crystal structures of apo NR proteins have been reported and can be classified into three forms in terms of the conformation of helix 12: first form has an extended helix 12 (A form in Figure 1c),13 second form has a Received: May 4, 2016 Published: August 18, 2016 7888

DOI: 10.1021/acs.jmedchem.6b00682 J. Med. Chem. 2016, 59, 7888−7900

Journal of Medicinal Chemistry

Article

Figure 2. Active vitamin D3 (hormone) and synthetic analogues.

complex shown in Figure 1. The solution structure of VDRLBD bound to the natural hormone 1 was analyzed by smallangle X-ray scattering (SAXS) by Rochel et al.28 The obtained SAXS profile was consistent with the SAXS profile calculated from the crystal structure,28 indicating that the solution structure of the VDR-LBD/agonist complex is identical to the crystal structure and that the structure of the VDR-LBD/ agonist complex is not affected by crystal packing. On the other hand, no crystal structure of apo VDR-LBD has been reported, likely due to the disordered conformation of the C-terminus part, especially helices 10, 11, and 12. These helices are distinctly different among the A and B forms and the active form observed in crystal structures of apo NR-LBD, making the structures too unstable to crystallize. Nuclear magnetic resonance (NMR) and hydrogen−deuterium exchange mass spectrometry (HDX-MS) experiments suggest that the region around helix 12 is disordered in the apo form and changes from disordered structure to an ordered conformation upon agonist binding.29,30 However, the exact conformation of the apo form at atomic-level resolution remains unknown. Furthermore, only four crystal structures of the VDR-LBD/antagonist complex have been reported: the VDR-LBD/JB31 (Figure 2) complex (PDB code 2ZXM31), the VDR-LBD/TEI964732 complex (PDB code 3A2H33), the VDR-LBD/ADTT complex (PDB code 2ZMI34), and the VDR-LBD/ADMI4 complex (PDB code 2ZMJ34). Their main chains, including helix 12, are almost identical to those of the agonist-binding crystal structures. On the other hand, NMR experiments revealed that the Cterminus, including helices 11 and 12, is disordered in the VDR-LBD/antagonist complex, similar to the apo structure.29 Because the conformation of helix 12 plays a key role in regulating transcriptional activity, the crystal structures of the VDR-LBD/antagonist complex do not provide insights into the mechanism of antagonism. Consequently, understanding the apo and antagonist binding forms remains as an important issue for revealing the structural mechanism controlling the transactivation of VDR-LBD. In particular, the conformational change around helix 12 is key, and explicit information regarding this conformational change, is imperative for both

Figure 1. Conformational change of the ligand-binding domain induced by ligand binding. (a) When an agonist binds to the receptor, helix 12 (H12, magenta) is folded and the receptor recruits coactivator (red). The agonist complex is illustrated using PDB code 3ERD.8 (b) When an antagonist binds to the receptor, helix 12 localizes at the coactivator binding site. The antagonist complex is described using PDB code 3ERT.8 (c) Apo structures are classified into two forms: A form (PDB code 1LBD13) and B form (PDB code 3P0U15).

folded helix 12, similar to the antagonist binding structure (B form in Figure 1c),14−16 and third form has a folded helix 12, quite similar to the agonist binding structure (active form, Figure 1a).17,18 Interestingly, in A and B forms, a kink between helices 10 and 11 is observed as a common feature. Because of the loop structure in the kink, helix 11 bends toward helix 3 and covers the ligand-binding pocket (LBP) (Figure 1c). In addition, it has been suggested that the C-terminus, from the end of helix 11 to helix 12, is considerably disordered in the apo state.19−24 Taken together, it appears that the transcriptional activity of NRs is controlled by the local behavior of helix 12, and the explicit model for transactivation described above is called the “mouse-trap model” mechanism.25 Vitamin D receptor (VDR) belongs to the NR family and regulates the expression of genes related to calcium homeostasis, cell differentiation and proliferation, and immunomodulation by binding the hormone 1α,25-dihydroxyvitamin D3 (1, Figure 2).26 Many crystal structures of VDR-LBD complexed with agonist have been reported,27 and they are identical intrinsically to the crystal structure of the NR-LBD/agonist 7889



Article

Table 1. Data Collection and SAXS Statistics apo VDR-LBD data collection parameters instrument beam geometry photon flux [phs/s] wavelength [Å] camera length [mm] Q range [Å−1] exposure time [s] concentration range [mg/mL] temperature [K] structural parameters Rg [Å] from Guinier I(0) from Guinier molecular-mass determination molecular mass [kDa] from I(0) molecular mass [kDa] from sequence

VDR-LBD/3 (antagonist)

KEK PF BL-10C bent-cylindrical mirror + two slits (0.74 mm × 0.7 mm) 2.2 × 1010

KEK PF BL-10C bent-cylindrical mirror + two slits (0.74 mm × 0.7 mm) 2.2 × 1010

1.488 928

1.488 928

0.01−0.576 20 × 10 frame 0.8−4.1 (5 points)

0.01−0.576 20 × 10 frame 0.6−3.6 (5 points)

293

293

21.0 ± 0.6a

22.0 ± 0.9a

326.7 ± 1.2

331.7 ± 2.1

32.1c

32.6c

30.7

31.1

VDR-LBD/2 (agonist)

VDR-LBD/JB (antagonist)

ApoMD-open

AntagoMD-open

19.9b

19.4b

21.5b

21.9b

a

The Rg from Guinier was calculated from SAXS curve extrapolated to 0 mg/mL from plurality concentration data (Figures S2c and S3c). bThe statistics was calculated from the crystal structures by using CRYSOL.42 VDR-LBD/agonist 2 used PDB code 5B41, and VDR-LBD/JB used PDB code 2ZXM. cEstimated molecular weight was calculated from the I(0) values extrapolated to 0 mg/mL from plurality concentration data. Standard sample used ovalbumin which molecular mass is 44.3 kDa.

supply. Antagonist 3 is a surrogate of antagonist JB. Compound 3 and JB are antagonists having a butyl group at C-22 but not an original side chain terminus. Therefore, their structures are characteristic compared with typical antagonists in NR family members which have a large functional group to push out helix 12, not forming an AF-2 surface where coactivators are recruited. We reported that antagonistic activity of compound 3 and JB is caused by a combination of the extra cavity (butyl pocket) formation and insufficient hydrophobic interactions with helix 12.31,35 Thus, a butyl pocket is induced to accommodate the 22S-butyl group and the poor hydrophobic interactions are caused by a lack of the dimethyl group at the original side chain terminus of the ligand. Therefore, compound 3 and JB do not form an AF-2 surface, resulting in the failure of coactivator recruitment and heterodimerization with RXR. Consequently, compound 3 and JB show antagonistic activity against VDR. Because the A-ring of antagonist 3 is different from that of hormone 1 and antagonist JB, we conducted crystallographic analysis of the VDR-LBD/agonist 235 (Figure 2, PDB code 5B41) complex and investigated whether the difference in the A-ring affects the structure of VDR-LBD. Crystallization, diffraction data collection, and data analysis were conducted as previously reported.36 As described in the Supporting Information, the crystal structure of the VDR-LBD/agonist 2 complex was essentially identical to other crystal structures reported to date, indicating that the difference in the A-ring has no effect on the structure of VDR-LBD. As mentioned in the Introduction, the solution structure of the VDR-LBD/agonist complex is identical to that of the crystal structure and therefore the crystal structure of the VDR-LBD/agonist 2

understanding the mechanism and allowing modulation of the activity of the complex by structure-based drug design. The likely disordered structure around helix 12 limits the possibility of crystallographic analyses. Consequently, we analyzed the apo form and the VDR-LBD/antagonist complex by SAXS and investigated the conformational changes arising from ligand binding. SAXS analyses can reveal the overall shape of macromolecular structures under nearly physiological aqueous conditions and can capture both the flexible structure in the apo form and the conformational change in response to antagonist binding. We obtained SAXS profiles of apo rat VDRLBD and the rat VDR-LBD/antagonist complexes, but the profiles were different from the theoretical profiles calculated from crystal structures. To clarify these solution structures at atomic resolution, we conducted molecular dynamics (MD) simulations and collected each structural ensemble. Here, we obtained the structures of apo rat VDR-LBD and the rat VDRLBD/antagonist complex by comparing the SAXS profiles and the MD simulation results and report their structures in solution for the first time. Apo VDR-LBD has a wide entrance leading to the LBP, and the VDR-LBD/antagonist complex structure shows that the position of helix 12 is unsuitable for creating an AF-2 surface. On the basis of these results, we propose a mechanism for the agonism/antagonism of VDR by ligand binding that is an alternative to the mouse-trap model.

■

RESULTS AND DISCUSSION Selection of Ligands. To analyze the rat VDR-LBD/ antagonist complex using SAXS, we selected antagonist 335 (Figure 2) due to its stability, ease of synthesis, and ready 7890



Article

Figure 3. Relationship between structure and χ value. (a,d) Experimental X-ray scattering curves of apo VDR-LBD (a) and the VDR-LBD/ antagonist 3 complex (d) are shown as black dots. Theoretical scattering curves of ApoMD-open (continuous cyan line, χ = 0.29), agonist 2 complex (continuous green line, χ = 0.89), AntagoMD-open (continuous orange line, χ = 0.29), and the antagonist JB complex (continuous yellow−green line, χ = 0.69) calculated using CRYSOL are fitted against experimental curves. (b,e) ApoMD-open (cyan) and AntagoMD-open (orange) are superimposed on the VDR-LBD/agonist 2 complex (green) (b) and the VDR-LBD/antagonist JB complex (yellow−green) (e), respectively. Helix 11 and loop 11−12 are shown in yellow, and helix 12 of the MD and crystal structures are indicated by magenta and gray, respectively. (c,f) Distribution of χ values for snapshots of ApoMD sampled structures (c) and AntagoMD sampled structures (f) (see Supporting Information) with the SAXS experimental profile of apo VDR-LBD (c) or the VDR-LBD/antagonist 3 complex (f) on a distance map. A point represents a snapshot. Colors show the corresponding χ value calculated by CRYSOL.

value of the VDR-LBD/antagonist complex was 22.0 ± 0.9 Å, which is significantly larger than those of apo VDR-LBD and the crystal structure of the VDR-LBD/antagonist JB complex (PDB code 2ZXM) (Table 1). Fitting the experimental scattering curves of apo VDR-LBD and the VDR-LBD/ antagonist complex against the theoretical curves of the crystal structures of VDR-LBD/agonist 2 and the antagonist JB complex, respectively, provided discrepancies between the experimental and theoretical curves of χ = 0.89 and χ = 0.69 (Figure 3a,d and Figures S2c and S3c), showing that the theoretical curves appear to be different from the experimental curves. Notably, both curves deviate in the middle angle region by ∼0.15 (Figure 3a,d and Figures S2c and S3c). All the reported crystal structures of VDR-LBD listed in Table S2 were also fit to each experimental curve: their χ values were 0.54− 0.87 in apo VDR-LBD and 0.48−0.76 in the VDR-LBD/ antagonist 3 complex. Although the deviation in χ = 0.54 for apo VDR-LBD and χ = 0.48 for the antagonist complex

complex can be regarded as being identical to the solution structure of the VDR-LBD/agonist complex (Table S1 and Figure S1). SAXS Experiments. We performed SAXS experiments on apo VDR-LBD and the VDR-LBD/antagonist complex using rat VDR-LBD (residues 116−423, Δ165−211) with 10 extra residues at the N-terminus; this is the same construct used for crystallization. Each scattering data set in the small-angle region (QRg ≤ 1.3) could be fit with a linear function in the Guinier plots37 (Figures S2a,b and S3a,b). These plots show that the scattering intensities under the conditions used (10 mM TrisHCl, pH 7.0; 20 mM NaCl; 10 mM DTT) have no effect on aggregation and interparticle interference. The molecular weight estimated from I(0) was similar to that calculated from the amino acid sequence. The Rg value of apo VDR-LBD was 21.0 ± 0.6 Å, which is slightly larger than that of the crystal structure of the VDRLBD/agonist 2 complex (Table 1). On the other hand, the Rg 7891



Article

Figure 4. Structural characteristics for apo VDR-LBD in solution. The color used is the same as in Figure 3b,e. (a) Superimposition of ApoMD-open and the VDR-LBD/agonist 2 complex. In ApoMD-open, helix 11 bends to the outside and helix 12 is put around the canonical active helix 12. (b) Magnification of the structure from helix 10 to the end of helix 12. Hydrogen bonds and hydrophobic interactions are shown by red dotted lines and yellow dotted lines, respectively. (c) Hydrogen bonds (red dotted line) between amino acid residues in the C-terminal part and hydrophobic interactions (yellow dotted lines) between helices 3, 4, and 12.

structure showed a low χ value (χ = 0.29), and its theoretical curve was in good agreement with the experimental profile (Figure 3a,b). This structure is hereafter termed “ApoMDopen”. Comparison with the crystal structure of the VDRLBD/agonist 2 complex showed particular improvement of the ApoMD-open profile at the middle angle region (Q > 0.15) (Figure 3a,b), and agreement at the small-angle region was also improved: the Rg value of ApoMD-open (Rg = 21.5 Å) calculated by CRYSOL42 was identical to the experimental data (Rg = 21.0 ± 0.6 Å) within the error range, whereas the Rg value of the crystal structure of the VDR-LBD/agonist 2 complex (Rg = 19.9 Å, PDB code 5B41) was smaller than that of the experimental data (Table 1). These results indicate that the theoretical curve of ApoMD-open is in good agreement with the experimental SAXS profile in all Q ranges. The ApoMD-open structure is essentially identical to the crystal structure up to helix 10, whereas the portion from the end of helix 11 to the beginning of helix 12 is disordered. Helix 12 is partially unraveled (Glu416, Val417, Phe418, Gly419), and it is positioned around the canonical active position (Figures 3b and 4a and Figure S12) and fluctuates. Figure 3b shows the average conformation of VDR-LBD. Helix 11 bends to the outside of the LBP by a kink-centered hinge-bending motion, resulting in the formation of a wide entrance leading to the LBP (Figure 3b). In the crystal structure of the VDR-LBD/ agonist, the corresponding entrance is closed because helix 12 forms a lid over the LBP, indicating that the agreement of the SAXS profile of the ApoMD-open structure with the

decreased, both curves still deviated from the experimental profiles in the middle angle region by ∼0.15 (Figures S2d and S3d). These results show that agreement with the experimental profiles in this middle angle region is necessary to obtain the true molecular shape and thus the exact structures in solution. Furthermore, these comparisons indicate that the solution structures of apo VDR-LBD and the VDR-LBD/antagonist complex are different from the crystal structures of the VDRLBD/ligand complex reported previously, suggesting that the solution structures obtained here reflect inactive states. To understand their solution structures at atomic-level resolution, we first generated a structural ensemble in solution by using MD simulations and then calculated the theoretical profile of each structure (see Supporting Information). Comparison of each theoretical profile with the experimental profile provided accurate structures of apo VDR-LBD and the VDR-LBD/ antagonist complex, as described below. In MD simulations reported so far,38−41 solution structures near the crystal structure were investigated, so obtained structures were essentially identical to the crystal’s. In the present investigation, to improve the generation of a structural ensemble by MD simulations, we used various initial models with folded and unfolded helix 12 and performed multiple MD simulations from the initial models. Structural Investigation of Apo VDR-LBD by MD Simulations. The results of MD simulations (see Supporting Information) provided a solution structure for VDR-LBD consistent with the SAXS experimental profile. The VDR-LBD 7892



Article

Figure 5. Structural characteristics of the VDR-LBD/antagonist 3 complex in solution. The color is the same as in Figure 3b,e. (a) Superimposition of AntagoMD-open and the VDR-LBD/agonist 2 complex. Helix 12 in AntagoMD-open is positioned in the same space as coactivator in the VDRLBD/agonist 2 complex. (b) Magnification of the structure from helix 10 to the end of helix 12. Hydrogen bonds and hydrophobic interactions are shown by red dotted lines and yellow dotted lines, respectively. (c) Interactions between amino acid residues in helices 3 and 12 are indicated by red dotted lines.

(Figure 3d,e). In addition, the Rg value of AntagoMD-open (Rg = 21.9 Å) calculated by CRYSOL42 was identical to the experimental data (Rg = 22.0 ± 0.9 Å) within the error range, whereas the Rg value of the crystal structure of the VDR-LBD/ antagonist JB complex (Rg = 19.4 Å, PDB code 2ZXM29) was smaller than that of the experimental data (Table 1). These results demonstrate that the theoretical curve of AntagoMDopen is more accurate than that of the VDR-LBD/antagonist JB complex. Compared with the crystal structure, the AntagoMD-open structure has the wide and flexible loop between helices 11 and 12 (loop 11−12, Figures 3e and 5a). Helix 12 in AntagoMDopen is also partially unraveled (Pro412, Leu413) and is positioned around canonical coactivator binding region, resulting in the inhibition of formation of the AF-2 surface (Figures 3e and 5a). The relationship between the location of loop 11−12 and the χ values was determined by analyzing the width of the entrance to the LBP in the same manner as for ApoMD-open (Figure 3e,f). The two distances fluctuate in the range 12−28 Å (Leu220−Glu405) and 12−26 Å (Ala227− Leu410), and these distances are larger than those of the crystal structure (11.1 and 5.5 Å) of the VDR-LBD/antagonist JB complex. In particular, the distance of Ala227−Leu410 negatively correlates with the χ value, and thus the solution structure obtained by SAXS for VDR-LBD/antagonist 3 complex has a wide entrance to the LBP and C-terminus after helix 11 fluctuates near that of the AntagoMD-open structure. Interestingly, the fluctuation in the distance of Leu220−Glu405 in the VDR-LBD/antagonist 3 complex was larger than that for apo VDR-LBD.

experimental profile is due to the wide entrance to the LBP. To confirm the relationship between the width of the entrance to the LBP and the χ values, sampled MD structures were analyzed in terms of the extension of the LBP as follows. The entrance of the LBP was characterized by the distances between two pairs of α-carbon atoms of a specific residue in helix 12 and a specific residue in helix 3 appropriate for comparison of the entrance width: Leu220−Glu405 and Ala227−Leu410 (Figure 3b). As shown in the distribution map of χ values (Figure 3c), the distances between Leu220 and Glu405 and between Ala227 and Leu410 fluctuate in the range 7−25 Å and 9−23 Å, respectively, and these distances are larger than those of the crystal structure (10.8 and 5.5 Å) of the VDR-LBD/agonist 2 complex. The distance of Leu220−Glu405 is more extended and that of Ala227−Leu410 is moderately extended. The distance between Leu220 and Glu405 means the distance between helices 3 and 12 at the outermost part of the LBP, and the distance negatively correlates with the χ value. This indicates that the solution structure obtained by SAXS for apo VDR-LBD mainly fluctuates near the ApoMD-open structure with the wide entrance of the LBP. Structural Investigation of the VDR-LBD/Antagonist 3 Complex by MD Simulations. The MD simulations (see Supporting Information) provided a structure satisfying the SAXS experimental profile and showed a low χ value (χ = 0.29). This theoretical profile was in good agreement with the experimental profile and is termed “AntagoMD-open” (Figure 3d). Comparison with the crystal structure of the VDR-LBD/ antagonist JB complex showed remarkable agreement of the AntagoMD-open profile in the middle angle region (Q > 0.2) 7893



Article

centered hinge-bending motion observed in the present study may provide a model that satisfies the HDX-MS results. Interestingly, unlike the crystal structure of apo RXRα-LBD (Figure 1c, A form), the partially unraveled helix 12 in the ApoMD-open structure is positioned around the canonical active position (Figure 4a). Helix 12 in ApoMD-open, which is an average conformation of apo-form in solution, rotates approximately 90° toward the coactivator binding site (Figure 4a), and helix 12 interacts with helices 3 and 4 (Figure 4c). In addition, the NH of Ile422 in the C-terminus of helix 12 forms a hydrogen bond with Ser252 and the carboxylate of Ser423 forms a salt bridge with Arg248 (Figure 4c). The C-terminal section from the end of helix 11 to the beginning of helix 12, in addition, has many hydrophilic amino acid residues and most are exposed to the outside. In contrast, the hydrophobic amino acid residues tend to be buried (Figure S12). In addition, it appears that the localization of helix 12 near the receptor surface is more appropriate in terms of hydrophobicity than to be exposed to the solvent, as is the case with apo RXRα-LBD in the A form, shown in Figure 1c. Thus, the ApoMD-open structure is a conformation that may exist even in living cells. These results indicate that its structure is more stable in solution than the A form shown in Figure 1c. Our results demonstrate that structural investigation by all-atom MD simulations is a promising approach for obtaining plausible structures under nearly physiological aqueous conditions. Relationship between Structural Characteristics and Antagonism in the VDR-LBD/Antagonist 3 Complex. More detailed structural characteristics for antagonistic activity were elucidated by analyzing the AntagoMD-open structure at the amino acid residue level. Antagonistic activity is achieved due to flexible loop 11−12, which prevents formation of the active form of helix 12 and the AF-2 surface (Figure 5a). Alanine scanning mutational analysis revealed that Tyr397 (Tyr401 in human VDR) is an important amino acid residue for transcriptional activation.44−46 In the crystal structure of the VDR-LBD/agonist 2 complex, Tyr397 forms hydrophobic interactions with the methyl group of agonist 2 and the phenolic −OH of Tyr397 forms a hydrogen bond with the carbonyl of Gly419 on helix 12 (Figure 5b). In the solution structure of the VDR-LBD/antagonist 3 complex, in contrast, Tyr397 does not form hydrophobic interactions with antagonist 3 due to the lack of dimethyl groups in 3. The 22S-butyl group of antagonist 3 forces helices 7 and 10/11 to separate from their positions in the crystal structure of the VDR-LBD/agonist 2 complex, thus weakening the interactions between these helices compared to those in the agonist complex (Figure 5b and Figure S13). These weak interactions induced by the antagonist influence the stability of the C-terminal portion, resulting in a flexible structure at the end of helix 11. In the AntagoMD-open structure, helix 11 is one turn shorter than in the crystal structure of the VDR-LBD/agonist 2 complex: helix 11 ends at Arg398 in the antagonist 3 complex and at Phe402 in the agonist 2 complex (Figure 5b). This structural characteristic was compared with the crystal structures of other NRs8,11 (estrogen receptor α-LBD (ERα-LBD), retinoic acid receptor α-LBD (RARα-LBD), and mRXRα-LBD (F318A)) in an effort to better understand the mechanism controlling antagonism. This comparison showed that in all compared structures, helix 11 is one turn shorter than in the agonist complex and that the characteristic structure of the VDR-LBD/antagonist 3 complex is thus conserved in the NRs. NMR experiments of the VDR-LBD/antagonist complex29

The apo and antagonist binding structures in solution both possess a wide entrance to the LBP, but the longer distance of Ala227−Leu410 in the AntagoMD-open structure reflecting the motions of loop 11−12 is important for structural investigation of the VDR-LBD/antagonist 3 complex (Figure 3f). The validity of the relationship between the χ values and the structural characteristics revealed by MD simulations was examined by cross validation analysis (see Supporting Information) and showed distinctively different behavior in the fluctuation around the LBP in each apo and antagonistbinding form. The outward bend of helix 11 was necessary for the apo structure to be consistent with the experimental SAXS profile. The structure around the LBP for the AntagoMD sampled structures, including AntagoMD-open, was essential for retaining ligand-binding because the LBP in the ApoMD sampled structure was smaller, and therefore such ApoMD sampled structures are not suitable as antagonist binding structures. From these results, each structural characteristic of the apo or antagonist-binding form is distinguishable from each other, and thus we conclude that the ApoMD-open and AntagoMD-open structures are plausible solution structures that satisfy the SAXS experimental profiles. Relationship between Structural Characteristics and the Function in Apo VDR-LBD. More detailed structural characteristics were unveiled by analyzing the ApoMD-open structure at the amino acid residue level (Figure 4) and showed a remarkable feature in the C-terminus: helix 11 bends to the outside and the partially unraveled helix 12 is positioned around the canonical active position (Figure 4a and Figure S12). A helix break occurs at Gln396 on helix 10/11 due to disappearance of the hydrogen bond between the carbonyl of Glu392 and the NH of Gln396. The break at Gln396 creates a kink on helix 10/11, and the kink-centered hinge-bending motion of helix 11 leads to the wide entrance to the LBP (Figure 4b). The conformational change of helix 11 was essential for the theoretical SAXS curve to satisfy the experimental profile. This kink structure has not been observed in any of the crystal structures of VDR-LBD reported to date. To clarify the role of this kink in the apo state, apo crystal structures of other NRs possessing this kinked point (retinoid X receptor α-LBD; RXRα-LBD (PDB code 1LBD13), photoreceptor cell-specific nuclear receptor-LBD, PNR-LBD (PDB code 4LOG14), testicular receptor 4-LBD; TR4-LBD (PDB code 3P0U15), chicken ovalbumin upstream promoter-transcription factor II-LBD, and COUP TFII-LBD (PDB code 3CJW16)) were compared by superimposition. The results showed that the kinked point between helices 10 and 11 is structurally conserved, and the bent helix 11 covers the LBP. These structures are unfavorable for ligand incorporation (Figure 1). In ApoMD-open, in contrast, helix 11 bends outward at the kinked point, and the resulting wide entrance is favorable for easy access of the ligand to the LBP. Therefore, the characteristic structure of ApoMD-open is reasonable from the viewpoint of the function of the apo state. The disorder from the end of helix 11 to helix 12, furthermore, is consistent with the other experimental results. HDX-MS experiments of human and rat VDR-LBDs showed a difference in structural fluctuations between the apo and agonist-binding states in the region from helix 11 to helix 12.30,43 NMR experiments demonstrated that the structure of VDR-LBD including helices 11 and 12 was globally stabilized by agonist binding.29 These results indicate that this region changes from a disordered state to an ordered state upon agonist binding.29,30 The kink7894



Article

showed that the C-terminal part of VDR-LBD (Lys395− Glu421) is disordered. Analysis of the B-factors of our recently reported crystal structure shows that the C-terminal end of helix 11 is unstable.36 Taken together, these results suggest that the C-terminal part of the LBD may adopt a flexible conformation and that this flexibility results in helix 12 being in an unsuitable position for formation of the AF-2 surface. The solution structure of VDR-LBD/antagonist 3 is a reasonable structure for supporting antagonism. Function of the VDR requires that the VDR-LBD forms the active conformation of helix 12, recruits the coactivator, and dimerizes with RXR. In the AntagoMD-open structure, the position of partially unraveled helix 12 is unsuitable for coactivator binding because the amino acid residues involved in the charge clamp, Lys242 on helix 3 and Glu416 on helix 12,47 form interactions with other residues and helix 12 covers the coactivator binding site. Interestingly, Lys242 and Glu416 form hydrogen bonds with Ile422 and Gln235, respectively (Figure 5c). In addition, Asn420 forms a hydrogen bond with Gln235 and Glu421 forms a salt bridge with Lys236 (Figure 5c). Structural stabilization arising from these interactions causes helix 12 to put at this position where the coactivator is located in crystal structures. The crystal structure of ERα-LBD/4-hydroxy tamoxifen complex (PDB code 3ERT8) may shed light on the mechanism underlying antagonism: Met543 and Leu544 on helix 12 form hydrogen bonds with Lys362 on helix 3 involved in the charge clamp (Figure S14). This characteristic behavior is consistent with that of the AntagoMD-open structure. These interactions of amino acid residues may fix the position of helix 12 to the side of helix 3 and may help inhibit the canonical active conformation of helix 12. Consequently, the solution structure obtained in the present study prevents formation of the canonical active conformation of helix 12 and thus prevents coactivator binding. The structure of VDR-LBD/antagonist 3 well explains the antagonistic activity of VDR-LBD in solution. Thus, we revealed solution structure of VDR-LBD complexed with antagonist 3 which has a 22-butyl group. VDR antagonist without 22-butyl group are likely to induce different conformation of VDR-LBD.43 The unsuitable position of helix 12 for forming the AF-2 surface may lead to difficulty in crystallizing the complex. Human VDR-LBD was crystallized by packing helix 3n to mimic coactivator peptide under conditions normally used.48 Rat and zebrafish VDR-LBD were crystallized by the addition of coactivator peptide, indicating that the AF-2 surface is needed for crystallization.47,49 Because the C-terminal part of VDR-LBD is flexible in the antagonist complex, it may be difficult to pack symmetrically and thus difficult to crystallize the VDR-LBD without coactivator. Model for the Conformational Change of VDR-LBD by Ligand Binding. The use of a SAXS-MD hybrid approach revealed the structures of apo VDR-LBD and the VDR-LBD/ antagonist complex. Thus, now the apo, agonist-binding, and antagonist-binding structures are disclosed. Integration of the structural information obtained from these structures allowed us to propose a mechanism for agonist/antagonist activity controlled by ligand binding called the “folding-door model” (Figure 6). In the apo state, the kinked point at Gln396 plays an important role because the kink allows the kink-centered hinge-bending motion of helix 11, and helix 11 acts as a door for ligand-binding. When agonist 2 binds to the receptor, helix 12 repositions and closes the door (helix 11) by forming various interactions (e.g., canonical pincer-type hydrogen bonds

Figure 6. Folding-door model for the conformational change of VDRLBD upon ligand binding. Helices 3 (H3, gray), 10 (H10, gray), 11 (H11, yellow), and 12 (H12, magenta) and loop (black) structures are represented by cylinders and lines, respectively. Amino acid residues important for the conformational change are shown as black dots. Red and blue dashed lines show hydrogen bonds and hydrophobic interactions, respectively.

and hydrophobic interaction with Tyr397) (Figures 6 and 4b). Unlike the mouse-trap model, which is explained only by the open/close behavior of helix 12, in the folding-door model helix 12 works cooperatively with helix 11. This behavior has advantages in terms of the conformational change from the apo to the agonist-binding states: the folding-door allows ligands easier access to the LBP and easier formation of the active conformation of helix 12 by agonist binding compared to the mouse-trap model because helix 12 is positioned around the receptor surface. On the other hand, when antagonist 3 binds to the receptor, helix 12 is moved to a position unsuitable for forming the AF-2 surface due to flexible loop 11−12 (Figures 6 and 5b). From this viewpoint as well, the folding-door model is reasonable for explaining how VDR-LBD achieves agonism/ antagonism by ligand binding. In a biological system, the binding of the C-terminus of VDR to itself is competing with the C-terminus of RXR and/or with corepressors. Current study indicated that helix 12 in apo- and antagonist binding VDRs is disordered. Therefore, in living cells, the C-terminus of RXR and/or corepressor might interact with apo- and antagonist-binding VDR more strongly than agonist-binding VDR.

■

CONCLUSIONS The mechanism of agonism/antagonism of VDR by ligand binding was not understood clearly prior to the present study due to the lack of structures of the apo state, and all the crystal structures of VDR-LBD are almost identical regardless of agonist/antagonist binding. Here, we provided a reliable structure for the apo and antagonist binding forms of VDRLBD using a “SAXS-MD hybrid approach”. In apo VDR-LBD, helix 12 is partially unraveled and fluctuates around the canonical position and rotates about 90° toward the coactivator binding site. Helix 11 bends outward by a kink-centered hingebending motion, resulting in opening of the entrance into the LBP. In the VDR-LBD/antagonist complex 3, helix 12 is 7895



Article

collected with the exposure time of 20 s, and the image data in which the effect is not seen were averaged to improve S/N ratio. The twodimensional data were azimuthally averaged to convert into the onedimensional scattering intensity data using the macro program Nika55 developed for the software Igor Pro (WaveMetrics). Data processing including the subtraction of the intensity of a buffer was performed using the program PRIMUS.56 The final scattering curves extrapolated to zero concentration were calculated using the program PRIMUS QT.57 The radius of gyration, Rg, and the scattering intensity at the zero angle, I(0), were obtained by Guinier analysis in the small-angle region (QRg ≤ 1.3).37 The scattering intensity (I(Q)) is represented as I(Q) ≈ I(0) exp(−Q2Rg2/3), where Q = 4π sin θ/λ is the scattering vector for a scattering angle of 2θ and λ is the wavelength. All the analysis conditions and the resulting values of SAXS are summarized in Table 1. Modeling Process of Apo VDR-LBD and VDR-LBD/Antagonist 3 Complex for Molecular Dynamics Simulation. To carry out molecular dynamics (MD) simulations, initial structures of apo VDR-LBD and the VDR-LBD/antagonist 3 complex were prepared by modeling processes. Because no crystal structures of apo VDR-LBD have been reported to date, the VDR-LBD/1 (PDB code 2ZLC58) and the VDR-LBD/antagonist JB (PDB code 2ZXM31) crystal structures were employed as the initial structures with their ligands removed. Missing residues of both structures were 116−122 residues in the Nterminal, 160−217 (Δ165−211) residues, and 421−423 residues in the C-terminal. All the missing residues were modeled by MODELLER.59 To conform to the same structure used in SAXS experiments, extra sequences (GSHMGSPNSP) were added to the top of the N-terminal. Hereafter, the missing residues including deletion (160−217) were termed the 11-residue region. In the modeling processes for the VDR-LBD/antagonist 3 complex, the VDR-LBD/ antagonist JB crystal structure (PDB code 2ZXM) was employed as the initial structure and the antagonist JB was exchanged for the antagonist 3: the position of a carbon atom in the A-ring was changed (Figure 2). In both the apo VDR-LBD and the VDR-LBD/antagonist 3 complex, the modeling of the 11-residue region was done in two ways and an unfolded structure of helix 12 was modeled, as follows. Two conformations of the 11-residue region were prepared so that the conformations were allowed different flexibilities. One was the fully random loop created with no reference structures in modeling processes. The other was the loop partially forming a helical structure created by the homology modeling with the human VDR-LBD/1 crystal structure (PDB code 1DB148) as a reference. According to a sequence alignment between rat and human VDR-LBDs by TCOFFEE,60 the 11-residue region of rat VDR-LBD is assigned to 160−217 residues of human VDR-LBD. In the present calculation, 154−225 residues of human VDR-LBD were used so that the region includes the whole helical structure of 217−224 residues and both ends smoothly connect to the pre-existent structure. The modeled region of rat VDR-LBD was therefore 154−221 (Δ165−211) residues including the 11-residue region and allowances at both ends (154−159 and 218−221 residues). An unfolded conformation of helix 12 was created by the homology modeling. As a reference structure, the PPARα-LBD/antagonist crystal structure (PDB code 1KKQ61) was employed. In the complex, the helix 12 region (446−468 residues) is unfolded and is extended due to the interaction with the corepressor. According to a sequence alignment by T-COFFEE,60 the unfolded part of the PPARα-LBD was assigned to 399−423 residues of rat VDR-LBD corresponding to the region from the end of the helix 11 to helix 12. In structural investigations of apo VDR-LBD, eight MD simulations were carried out. The simulations were started from eight different modeled structures. The four models in the eight were prepared by the VDR-LBD/1 crystal structure (PDB code 2ZLC), and the other four models were prepared by VDR-LBD/antagonist JB crystal structure (PDB code 2ZXM). Each four model corresponds to four combinations of the modeled conformations of the 11-residue region and helix 12. Hereafter, the four models prepared by the VDR-LBD/1 crystal structure are respectively called as Apo1-H-H12, Apo1-Hunfold, Apo1-R-H12, and Apo1-R-unfold models, where “H” and “R”

partially unraveled and is put at a position unsuitable for formation of the AF-2 surface, thus preventing coactivator binding for transactivation. Because this antagonist-binding structure was obtained by using antagonist bearing butyl substituent at 22-position, distinct conformation is likely to be obtained when different structural antagonist is used. Therefore, this conformation is a representative of one class but not all of antagonist-binding structures. On the basis of the structures of all states of VDR-LBD (apo, agonist-binding, and antagonist-binding), we propose the “folding-door model” instead of the “mouse-trap model” to explain activity based on the open/close motion of helix 12. The “folding-door model” for agonism/antagonism observed here is likely applicable to other NRs.

■

EXPERIMENTAL SECTION

Protein Expression and Purification. Expression and purification of rat VDR-LBD (residues 116−423, Δ165−211) were done by the procedure reported previously.36,50 Purified rat VDR-LBD for crystallization was concentrated in buffer (10 mM Tris-HCl, pH 7.0; 2 mM NaN3; 2 mM TCEP) to 7.0 mg/mL, which was estimated by UV absorbance at 280 nm, and for small-angle X-ray scattering experiments was replaced in buffer (10 mM Tris-HCl, pH 7.0; 20 mM NaCl; 10 mM DTT). Crystallization. To a mixture of rat VDR-LBD and an agonist 2 (5 equiv) was added coactivator peptide (H2N-KNHPMLMNLLKDNCONH2) derived from DRIP205, including receptor interacting domain 2 in buffer (25 mM Tris-HCl, pH 8.0; 50 mM NaCl; 10 mM DTT, 2 mM NaN3). The mixture of VDR-LBD/ligand/peptide was allowed to crystallize by the vapor diffusion method using a series of precipitant solutions containing 0.1 M MOPS-Na (pH 7.0), 0.1 M sodium formate, 12% (w/v) PEG4000, and 5% ethylene glycol. Droplets for crystallization were prepared by mixing 0.75 μL of complex solution and 1 μL of precipitant solution, and droplets were equilibrated against 300 μL of precipitant solution at 20 °C. The mixture was stored at 20 °C, and crystals appeared after a few days. X-ray Crystallographic Analysis. Prior to diffraction data collection, crystals were soaked in a cryoprotectant solution containing 0.1 M MOPS-Na (pH 7.0), 0.15 M sodium formate, 18% (w/v) PEG4000, and 20% ethylene glycol. Diffraction data sets were collected at 100 K in a stream of nitrogen gas at beamline ARNW12A at the Photon Factory (PF) of the High Energy Accelerator Research Organization (KEK) (Tsukuba, Japan). Reflections were recorded with an oscillation range per image of 1.0°. Diffraction data were indexed, integrated, and scaled using the program iMOSFLM.51,52 The structures of ternary complex were solved by the procedure reported previously.36,53 The coordinate data for the structures were deposited in Protein Data Bank with accession numbers 5B41 (VDR-LBD/agonist 2 complex). Small-Angle X-ray Scattering. SAXS measurements of an apo VDR-LBD and a VDR-LBD/antagonist 3 complex were carried out at BL-10C54 at PF of KEK. PILATUS3 300K-W (Dectris) was used as a detector. The wavelength of X-rays was 1.488 Å, and the camera distance was 928 mm. The sample temperature at the cell was maintained at 293 K. The apo VDR-LBD was dissolved to a buffer, 10 mM Tris-HCl (pH 7.0), 20 mM NaCl, 10 mM DTT, for SAXS measurement. The VDR-LBD/antagonist 3 complex was also dissolved to the same buffer adding 0.25 mM antagonist 3 after incubating it under 0.25 mM antagonist 3 existence overnight. These samples were centrifuged at 21500g for over 10 min to eliminate the artificial aggregation, and their supernatant was applied to measurement. The data were collected at the concentration conditions of 0.8, 1.8, 2.5, 3.3, and 4.1 mg/mL for apo VDR-LBD and of 0.6, 1.4, 2.1, 3.1, and 3.6 mg/mL for VDR-LBD/antagonist 3 complex, respectively. Ovalbumin was prepared under the same buffer condition as a standard sample for the estimation of the molecular weight, and the data were collected at the concentration conditions of 1.1, 2.9, and 5.3 mg/mL. To evaluate the effect of a radiation damage, 10 images were 7896



Article

discrepancy between theoretical and experimental curves, χ and the radius of gyration, Rg, for the snapshots were estimated by CRYSOL.42

represent the helical form and random loop of the 11-residue region, and “H12” and “unfold” represent the canonical form and unfolded conformation of helix 12. The canonical form of helix 12 is the same structure of its crystal structure. The four models prepared by the VDR-LBD/antagonist JB crystal structure are also called as Apo2-HH12, Apo2-H-unfold, Apo2-R-H12, and Apo2-R-unfold models, respectively. In structural investigations of VDR-LBD/antagonist 3 complex, four MD simulations were carried out. The simulations were started from four different models corresponding to four combinations of conformations in the 11-residue region and helix 12, as is so for apo VDR-LBD. Hereafter, the models are called as Antago-H-H12, Antago-H-unfold, Antago-R-H12, and Antago-R-unfold models, respectively. Molecular Dynamics Simulation. A solution system was prepared by placing a modeled VDR-LBD structure at the center of a cuboid cell and adding a layer of water molecules. The thickness of the layer was 15 Å from the long axis of VDR-LBD, and the MD unit cells for modeled structures are summarized in Table S3. An example of the solution system is illustrated in Figure S4. The number of water molecules in the cell was set so that the water density was near 0.91 g/ cm3. Seven crystal water molecules are contained in the PDB structure of the VDR-LBD/antagonist JB crystal structure (PDB code 2ZXM) and were used in the MD simulation. They were observed to move freely between the PDB sites and the protein outside, and thus their initial positions had no effect on the results presented below. In the PDB structure of the VDR-LBD/1 crystal structure (PDB code 2ZLC), 144 crystal water molecules are contained and were used in the MD simulation. Their behavior in the simulation was similar to those of VDR-LBD/antagonist complex, and thus their initial positions have no effect on results. In addition to the water molecules, sodium and chloride ions were added so that ion intensity is 20 mM, and their initial positions were set to random. The missing hydrogen atoms were inserted in accordance with topology in CHARMM force field, and the N- and C-termini were set to NH3+ and COO−, respectively. The protonation states of 11 histidines were determined by the pKa values calculated by the program H++.62−65 According to the pKa calculation, the Nδ atom in H301 was protonated in the apo state, then the protonation was employed in simulations of apo VDR-LBD, however, in simulations of the VDR-LBD/antagonist 3 complex, the Nε atom was protonated to keep the hydrogen bond between the histidine and the antagonist. All the MD simulations were performed using the MD program package MARBLE66 with CHARMM36 force field67,68 and TIP3P water model.69 The CHARMM force field of the ligand was generated by the CHARMM General Force Field (CGenFF)70,71 (CGenFF version 2b8) created by ParamChem72−74 (version 0.9.7.1 beta). The isothermal−isobaric (NPT) ensemble was adopted with periodic boundary conditions. The temperature and pressure were set at 293 K and 1 atm. The rigid body simulation in the NPT ensemble was done by the extended-system approach66,75 with the symplectic integrator66,76 at 2 fs time step. The Lennard-Jones interaction was smoothly truncated by the switching function with the switching range of 8−10 Å.77 The electrostatic interaction was handled by the PME method.78 The real-space cutoff, spline order, and the Ewald tolerance were 10 Å, 4, and 10−6, respectively, and the reciprocal-space mesh size is summarized in Table S3. Counterions were added to keep the system charge-neutral. Calculations of the SAXS Profile from MD Simulations. The SAXS profile was calculated from a series of snapshots generated by MD simulations. Before the production run, a 2000-step minimization (steepest descent method), a heating simulation from 10 to 293 K over 250 ps with the solute restrained by a 1 kcal/mol/Å2 position harmonic potential, a 500 ps simulation for gradually removing the restraint, and a 1.25 ns equilibration were consecutively executed. The production run was done over 100 ns, and a snapshot was saved every 50 ps (2000 snapshots in total). At each snapshot, a scattering profile was calculated by CRYSOL.42 In the present study, the experimental profile within 0.02−0.36 Å−1 for the apo VDR/LBD or 0.015−0.36 Å−1 for the VDR-LBD/antagonist 3 complex was employed. The

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00682. Crystal structure of VDR-LBD/agonist 2 complex. SAXS data of apo VDR-LBD. SAXS data of VDR-LBD/ antagonist 3 complex. Initial solution structure of the Apo2-H-unfold model. Simulation time dependence of χ value and radius of gyration Rg for ApoMD models calculated by CRYSOL. Simulation time dependence of χ value and radius of gyration Rg for VDR-LBD/antagonist 3 complex models calculated by CRYSOL. Distribution of χ value on a distance map. Experimental and theoretical profiles for ApoMD-open (χ = 0.29) and AntagoMD-sample (χ = 0.34) structures. Distribution of χ value on a distance map. Experimental and theoretical profiles for AntagoMD-open (χ = 0.29) and ApoMDsample (χ = 0.3) structures. Minimum distances between the antagonist 3 and H393 or H301. The composition and orientation of amino acid residues in C-terminus part of ApoMD-open. The interactions between helices 7 and 10/11. The interactions of amino acid residues between helices 3 and 12 in the crystal structure of ERα-LBD/4hydroxy tamoxifen complex (PDB code 3ERT). Summary of data collection statistics and refinement of crystal structure. The results of χ values calculated by CRYSOL. Cell size and PME mesh size. Percentage of χ value for Apo models. Percentage of Rg for Apo models inside and outside of the experimental value (21.0 ± 0.6 Å) and averaged value over the last 50 ns. Percentage of χ value for VDR-LBD/antagonist complex models. Percentage of Rg for Apo models inside and outside of the experimental value (22.0 ± 0.9 Å) and averaged value over the last 50 ns. Crystal structure of VDR-LBD/ agonist 2 complex, structural investigation of apo VDRLBD by MD simulations, structural investigation of the VDR-LBD/antagonist 3 complex by MD simulations, cross validation analysis of apo structure and antagonist 3 complex (PDF) Accession Codes

PDB code: 5B41 Authors will release the atomic coordinates and experimental data upon article publication.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +81 42 721 1580. Fax: +81 42 721 1580. E-mail: [email protected]. Author Contributions

Y.A., N.S., M.I., and K.Y. designed the research. K.Y. supervised the overall project. Y.A., N.S., D.E.. and T.I. performed the SAXS experiments and analyzed the data. T.E. and M.I. performed MD simulations and analyzed the data. Y.A., T.I., and K.Y. performed X-ray crystal structure experiment and analyzed the data. Y.A., N.S., T.E., M.I., and K.Y. wrote the paper. All authors discussed the results and contributed to the final version of the manuscript. Notes

The authors declare no competing financial interest. 7897



■

Article

(13) Bourguet, W.; Ruff, M.; Chambon, P.; Gronemeyer, H.; Moras, D. Crystal Structure of the Ligand-Binding Domain of the Human Nuclear Receptor RXR-Alpha. Nature 1995, 375 (6530), 377−382. (14) Tan, M. H. E.; Zhou, X. E.; Soon, F. F.; Li, X.; Li, J.; Yong, E. L.; Melcher, K.; Xu, H. E. The Crystal Structure of the Orphan Nuclear Receptor NR2E3/PNR Ligand Binding Domain Reveals a Dimeric Auto-Repressed Conformation. PLoS One 2013, 8 (9), e74359. (15) Zhou, X. E.; Suino-Powell, K. M.; Xu, Y.; Chan, C.-W.; Tanabe, O.; Kruse, S. W.; Reynolds, R.; Engel, J. D.; Xu, H. E. The Orphan Nuclear Receptor TR4 Is a Vitamin A-Activated Nuclear Receptor. J. Biol. Chem. 2011, 286 (4), 2877−2885. (16) Kruse, S. W.; Suino-Powell, K.; Zhou, X. E.; Kretschman, J. E.; Reynolds, R.; Vonrhein, C.; Xu, Y.; Wang, L.; Tsai, S. Y.; Tsai, M.-J.; Xu, H. E. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor. PLoS Biol. 2008, 6 (9), e227. (17) Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.; Rosenfeld, M. G.; Willson, T. M.; Glass, C. K.; Milburn, M. V. Ligand Binding and Co-Activator Assembly of the Peroxisome Proliferator-Activated Receptor-γ. Nature 1998, 395 (6698), 137−143. (18) Uppenberg, J.; Svensson, C.; Jaki, M.; Bertilsson, G.; Jendeberg, L.; Berkenstam, A. Crystal Structure of the Ligand Binding Domain of the Human Nuclear Receptor PPAR. J. Biol. Chem. 1998, 273 (47), 31108−31112. (19) Rastinejad, F.; Huang, P.; Chandra, V.; Khorasanizadeh, S. Understanding Nuclear Receptor Form and Function Using Structural Biology. J. Mol. Endocrinol. 2013, 51 (3), T1−T21. (20) Rastinejad, F.; Ollendorff, V.; Polikarpov, I. Nuclear Receptor Full-Length Architectures: Confronting Myth and Illusion with High Resolution. Trends Biochem. Sci. 2015, 40 (1), 16−24. (21) Kallenberger, B. C.; Love, J. D.; Chatterjee, V. K. K.; Schwabe, J. W. R. A Dynamic Mechanism of Nuclear Receptor Activation and Its Perturbation in a Human Disease. Nat. Struct. Biol. 2003, 10 (2), 136− 140. (22) Johnson, B. A.; Wilson, E. M.; Li, Y.; Moller, D. E.; Smith, R. G.; Zhou, G. Ligand-Induced Stabilization of PPARγ Monitored by NMR Spectroscopy: Implications for Nuclear Receptor Activation. J. Mol. Biol. 2000, 298 (2), 187−194. (23) Berger, J. P.; Petro, A. E.; Macnaul, K. L.; Kelly, L. J.; Zhang, B. B.; Richards, K.; Elbrecht, A.; Johnson, B. a.; Zhou, G.; Doebber, T. W.; Biswas, C.; Parikh, M.; Sharma, N.; Tanen, M. R.; Thompson, G. M.; Ventre, J.; Adams, A. D.; Mosley, R.; Surwit, R. S.; Moller, D. E. Distinct Properties and Advantages of a Novel Peroxisome Proliferator-Activated Protein γ Selective Modulator. Mol. Endocrinol. 2003, 17 (4), 662−676. (24) Lu, J.; Cistola, D. P.; Li, E. Analysis of Ligand Binding and Protein Dynamics of Human Retinoid X Receptor Alpha LigandBinding Domain by Nuclear Magnetic Resonance. Biochemistry 2006, 45 (6), 1629−1639. (25) Moras, D.; Gronemeyer, H. The Nuclear Receptor LigandBinding Domain: Structure and Function. Curr. Opin. Cell Biol. 1998, 10 (3), 384−391. (26) Pike, J. W.; Meyer, M. B.; Bishop, K. A. Regulation of Target Gene Expression by the Vitamin D Receptor - an Update on Mechanisms. Rev. Endocr. Metab. Disord. 2012, 13 (1), 45−55. (27) Molnár, F. Structural Considerations of Vitamin D Signaling. Front. Physiol. 2014, 5, 191. (28) Rochel, N.; Tocchini-Valentini, G.; Egea, P. F.; Juntunen, K.; Garnier, J. M.; Vihko, P.; Moras, D. Functional and Structural Characterization of the Insertion Region in the Ligand Binding Domain of the Vitamin D Nuclear Receptor. Eur. J. Biochem. 2001, 268 (4), 971−979. (29) Singarapu, K. K.; Zhu, J.; Tonelli, M.; Rao, H.; Assadi-Porter, F. M.; Westler, W. M.; DeLuca, H. F.; Markley, J. L. Ligand-Specific Structural Changes in the Vitamin D Receptor in Solution. Biochemistry 2011, 50 (51), 11025−11033. (30) Zhang, J.; Chalmers, M. J.; Stayrook, K. R.; Burris, L. L.; GarciaOrdonez, R. D.; Pascal, B. D.; Burris, T. P.; Dodge, J. A.; Griffin, P. R. Hydrogen/Deuterium Exchange Reveals Distinct Agonist/Partial

ACKNOWLEDGMENTS This work was financially supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Agency for Medical Research and Development (AMED) to K.Y., N.S., and M.I., a Grant-in-Aid for Scientific Research (no. 26460155) from MEXT to K.Y., and innovative drug discovery infrastructure through functional control of biomolecular systems, priority issue 1 in post-K supercomputer development (project ID: hp150269 and hp160223) from MEXT to M.I. Synchrotron radiation experiments were performed at the Photon Factory (proposal no. 2013G656, 2013R-03, 2013R-30, 2014R-57), and we are grateful for the assistance provided by the beamline scientists at the Photon Factory. We thank Dr. Yuichi Kokabu of Yokohama City University for valuable discussions and comments.

■

ABBREVIATIONS USED VDR, vitamin D receptor; LBD, ligand-binding domain; NR, nuclear hormone receptors; SAXS, small-angle X-ray scattering; HDX-MS, hydrogen−deuterium exchange mass spectrometry; LBP, ligand-binding pocket; RXR, retinoid X receptor; ERα, estrogen receptor α

■

REFERENCES

(1) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schütz, G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.; Evans, R. M. The Nuclear Receptor Superfamily: The Second Decade. Cell 1995, 83 (6), 835−839. (2) Olefsky, J. M. Nuclear Receptor Minireview Series. J. Biol. Chem. 2001, 276 (40), 36863−36864. (3) Ottow, E.; Weinmann, H., Eds. Nuclear Receptors as Drug Targets; Wiley-VCH: Weinheim, Germany, 2008. (4) Gronemeyer, H.; Gustafsson, J.-Å.; Laudet, V. Principles for Modulation of the Nuclear Receptor Superfamily. Nat. Rev. Drug Discovery 2004, 3 (11), 950−964. (5) de Lera, A. R.; Bourguet, W.; Altucci, L.; Gronemeyer, H. Design of Selective Nuclear Receptor Modulators: RAR and RXR as a Case Study. Nat. Rev. Drug Discovery 2007, 6 (10), 811−820. (6) Mangelsdorf, D. J.; Evans, R. M. The RXR Heterodimers and Orphan Receptors. Cell 1995, 83 (6), 841−850. (7) Kojetin, D. J.; Burris, T. P. Small Molecule Modulation of Nuclear Receptor Conformational Dynamics: Implications for Function and Drug Discovery. Mol. Pharmacol. 2013, 83 (1), 1−8. (8) Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.; Agard, D. a.; Greene, G. L. The Structural Basis of Estrogen Receptor/ coactivator Recognition and the Antagonism of This Interaction by Tamoxifen. Cell 1998, 95 (7), 927−937. (9) Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engström, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.; Carlquist, M. Molecular Basis of Agonism and Antagonism in the Oestrogen Receptor. Nature 1997, 389 (6652), 753−758. (10) Pike, A. C. W.; Brzozowski, A. M.; Hubbard, R. E.; Bonn, T.; Thorsell, A.-G.; Engström, O.; Ljunggren, J.; Gustafsson, J.-A.; Carlquist, M. Structure of the Ligand-Binding Domain of Oestrogen Receptor Beta in the Presence of a Partial Agonist and a Full Antagonist. EMBO J. 1999, 18 (17), 4608−4618. (11) Bourguet, W.; Vivat, V.; Wurtz, J. M.; Chambon, P.; Gronemeyer, H.; Moras, D. Crystal Structure of a Heterodimeric Complex of RAR and RXR Ligand-Binding Domains. Mol. Cell 2000, 5 (2), 289−298. (12) Pike, A. C. W.; Brzozowski, A. M.; Walton, J.; Hubbard, R. E.; Bonn, T.; Gustafsson, J.-A.; Carlquist, M. Structural Aspects of Agonism and Antagonism in the Oestrogen Receptor. Biochem. Soc. Trans. 2000, 28 (4), 396−400. 7898



Article

Agonist Receptor Dynamics within Vitamin D Receptor/Retinoid X Receptor Heterodimer. Structure 2010, 18 (10), 1332−1341. (31) Inaba, Y.; Yoshimoto, N.; Sakamaki, Y.; Nakabayashi, M.; Ikura, T.; Tamamura, H.; Ito, N.; Shimizu, M.; Yamamoto, K. A New Class of Vitamin D Analogues That Induce Structural Rearrangement of the Ligand-Binding Pocket of the Receptor. J. Med. Chem. 2009, 52 (5), 1438−1449. (32) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman, A. W.; Ishizuka, S. Antagonistic Action of Novel 1, 25-Dihydroxyvitamin D3−26,23-Lactone Analogs on Differentiation of Human Leukemia Cells (HL-60) Induced by 1, 25-Dihydroxyvitamin D3. J. Biol. Chem. 1999, 274 (23), 16392−16399. (33) Kakuda, S.; Ishizuka, S.; Eguchi, H.; Mizwicki, M. T.; Norman, A. W.; Takimoto-Kamimura, M. Structural Basis of the HistidineMediated Vitamin D Receptor Agonistic and Antagonistic Mechanisms of (23S)-25-Dehydro-1α-Hydroxyvitamin D3-26,23-Lactone. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 918−926. (34) Nakabayashi, M.; Yamada, S.; Yoshimoto, N.; Tanaka, T.; Igarashi, M.; Ikura, T.; Ito, N.; Makishima, M.; Tokiwa, H.; DeLuca, H. F.; Shimizu, M. Crystal Structures of Rat Vitamin D Receptor Bound to Adamantyl Vitamin D Analogs: Structural Basis for Vitamin D Receptor Antagonism and Partial Agonism. J. Med. Chem. 2008, 51 (17), 5320−5329. (35) Sakamaki, Y.; Inaba, Y.; Yoshimoto, N.; Yamamoto, K. Potent Antagonist for the Vitamin D Receptor: Vitamin D Analogues with Simple Side Chain Structure. J. Med. Chem. 2010, 53 (15), 5813− 5826. (36) Anami, Y.; Itoh, T.; Egawa, D.; Yoshimoto, N.; Yamamoto, K. A Mixed Population of Antagonist and Agonist Binding Conformers in a Single Crystal Explains Partial Agonism against Vitamin D Receptor: Active Vitamin D Analogues with 22R-Alkyl Group. J. Med. Chem. 2014, 57 (10), 4351−4367. (37) Guinier, A.; Fournet, G. Small Angle Scattering of X-Rays; Wiley: New York, 1955. (38) Peräkylä, M.; Molnár, F.; Carlberg, C. A Structural Basis for the Species-Specific Antagonism of 26,23-Lactones on Vitamin D Signaling. Chem. Biol. 2004, 11 (8), 1147−1156. (39) Väisänen, S.; Peräkylä, M.; Kärkkäinen, J. I.; Steinmeyer, A.; Carlberg, C. Critical Role of Helix 12 of the Vitamin D3 Receptor for the Partial Agonism of Carboxylic Ester Antagonists. J. Mol. Biol. 2002, 315 (2), 229−238. (40) Lempiäinen, H.; Molnár, F.; Macias Gonzalez, M.; Peräkylä, M.; Carlberg, C. Antagonist- and Inverse Agonist-Driven Interactions of the Vitamin D Receptor and the Constitutive Androstane Receptor with Corepressor Protein. Mol. Endocrinol. 2005, 19 (9), 2258−2272. (41) Yaghmaei, S.; Roberts, C.; Ai, R.; Mizwicki, M. T.; Chang, C. A. Agonist and Antagonist Binding to the Nuclear Vitamin D Receptor: Dynamics, Mutation Effects and Functional Implications. Silico Pharmacol. 2013, 1 (1), 2. (42) Svergun, D.; Barberato, C.; Koch, M. H. CRYSOL - A Program to Evaluate X-Ray Solution Scattering of Biological Macromolecules from Atomic Coordinates. J. Appl. Crystallogr. 1995, 28 (6), 768−773. (43) Kato, A.; Itoh, T.; Anami, Y.; Egawa, D.; Yamamoto, K. Helix12Stabilization Antagonist of Vitamin D Receptor. Bioconjugate Chem. 2016, 27 (7), 1750−1761. (44) Choi, M.; Yamamoto, K.; Itoh, T.; Makishima, M.; Mangelsdorf, D. J.; Moras, D.; DeLuca, H. F.; Yamada, S. Interaction between Vitamin D Receptor and Vitamin D Ligands. Chem. Biol. 2003, 10 (3), 261−270. (45) Yamamoto, K.; Abe, D.; Yoshimoto, N.; Choi, M.; Yamagishi, K.; Tokiwa, H.; Shimizu, M.; Makishima, M.; Yamada, S. Vitamin D Receptor: Ligand Recognition and Allosteric Network. J. Med. Chem. 2006, 49 (4), 1313−1324. (46) Yamamoto, K.; Anami, Y.; Itoh, T. Development of Vitamin D Analogs Modulating the Pocket Structure of Vitamin D Receptor. Curr. Top. Med. Chem. 2014, 14 (21), 2378−2387. (47) Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J. W.; DeLuca, H. F. Molecular Structure of the Rat Vitamin D Receptor Ligand Binding Domain Complexed with 2-Carbon-Substituted

Vitamin D3 Hormone Analogues and a LXXLL-Containing Coactivator Peptide. Biochemistry 2004, 43 (14), 4101−4110. (48) Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. The Crystal Structure of the Nuclear Receptor for Vitamin D Bound to Its Natural Ligand. Mol. Cell 2000, 5 (1), 173−179. (49) Rochel, N.; Hourai, S.; Pérez-García, X.; Rumbo, A.; Mourino, A.; Moras, D. Crystal Structure of the Vitamin D Nuclear Receptor Ligand Binding Domain in Complex with a Locked Side Chain Analog of Calcitriol. Arch. Biochem. Biophys. 2007, 460 (2), 172−176. (50) Yoshimoto, N.; Sakamaki, Y.; Haeta, M.; Kato, A.; Inaba, Y.; Itoh, T.; Nakabayashi, M.; Ito, N.; Yamamoto, K. Butyl Pocket Formation in the Vitamin D Receptor Strongly Affects the Agonistic or Antagonistic Behavior of Ligands. J. Med. Chem. 2012, 55 (9), 4373−4381. (51) Battye, T. G. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.; Leslie, A. G. W. iMOSFLM: A New Graphical Interface for Diffraction-Image Processing with MOSFLM. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 271−281. (52) Leslie, A. G. W.; Powell, H. R. Processing Diffraction Data with Mosflm. In Evolving Methods for Macromolecular Crystallography; NATO Science Series II: Mathematics, Physics and Chemistry; Read, R. J., Sussman, J. L., Eds.; Springer Netherlands: Dordrecht, 2007; Vol. 245, pp 41−51. (53) Anami, Y.; Sakamaki, Y.; Itoh, T.; Inaba, Y.; Nakabayashi, M.; Ikura, T.; Ito, N.; Yamamoto, K. Fine Tuning of Agonistic/antagonistic Activity for Vitamin D Receptor by 22-Alkyl Chain Length of Ligands: 22S-Hexyl Compound Unexpectedly Restored Agonistic Activity. Bioorg. Med. Chem. 2015, 23 (22), 7274−7281. (54) Igarashi, N.; Watanabe, Y.; Shinohara, Y.; Inoko, Y.; Matsuba, G.; Okuda, H.; Mori, T.; Ito, K. Upgrade of the Small Angle X-Ray Scattering Beamlines at the Photon Factory. J. Phys. Conf. Ser. 2011, 272, 012026. (55) Ilavsky, J. Nika: Software for Two-Dimensional Data Reduction. J. Appl. Crystallogr. 2012, 45 (2), 324−328. (56) Konarev, P. V.; Volkov, V. V.; Sokolova, A. V.; Koch, M. H. J.; Svergun, D. I. PRIMUS: A Windows PC-Based System for SmallAngle Scattering Data Analysis. J. Appl. Crystallogr. 2003, 36 (5), 1277−1282. (57) Petoukhov, M. V.; Franke, D.; Shkumatov, A. V.; Tria, G.; Kikhney, A. G.; Gajda, M.; Gorba, C.; Mertens, H. D. T.; Konarev, P. V.; Svergun, D. I. New Developments in the ATSAS Program Package for Small-Angle Scattering Data Analysis. J. Appl. Crystallogr. 2012, 45 (2), 342−350. (58) Shimizu, M.; Miyamoto, Y.; Takaku, H.; Matsuo, M.; Nakabayashi, M.; Masuno, H.; Udagawa, N.; DeLuca, H. F.; Ikura, T.; Ito, N. 2-Substituted-16-Ene-22-Thia-1α,25-Dihydroxy-26,27Dimethyl-19-Norvitamin D3 Analogs: Synthesis, Biological Evaluation, and Crystal Structure. Bioorg. Med. Chem. 2008, 16 (14), 6949−6964. (59) Šali, A.; Blundell, T. L. Comparative Protein Modelling by Satisfaction of Spatial Restraints. J. Mol. Biol. 1993, 234 (3), 779−815. (60) Notredame, C.; Higgins, D. G.; Heringa, J. T-Coffee: A Novel Method for Fast and Accurate Multiple Sequence Alignment. J. Mol. Biol. 2000, 302 (1), 205−217. (61) Xu, H. E.; Stanley, T. B.; Montana, V. G.; Lambert, M. H.; Shearer, B. G.; Cobb, J. E.; McKee, D. D.; Galardi, C. M.; Plunket, K. D.; Nolte, R. T.; Parks, D. J.; Moore, J. T.; Kliewer, S. a; Willson, T. M.; Stimmel, J. B. Structural Basis for Antagonist-Mediated Recruitment of Nuclear Co-Repressors by PPARα. Nature 2002, 415 (6873), 813−817. (62) H++; http://biophysics.cs.vt.edu/H++ (accessed October 31, 2014). (63) Anandakrishnan, R.; Aguilar, B.; Onufriev, A. V. H++ 3.0: Automating pK Prediction and the Preparation of Biomolecular Structures for Atomistic Molecular Modeling and Simulations. Nucleic Acids Res. 2012, 40 (W1), W537−W541. (64) Myers, J.; Grothaus, G.; Narayanan, S.; Onufriev, A. A Simple Clustering Algorithm Can Be Accurate Enough for Use in Calculations of pKs in Macromolecules. Proteins: Struct., Funct., Genet. 2006, 63 (4), 928−938. 7899



Article

(65) Gordon, J. C.; Myers, J. B.; Folta, T.; Shoja, V.; Heath, L. S.; Onufriev, A. H++: A Server for Estimating pKas and Adding Missing Hydrogens to Macromolecules. Nucleic Acids Res. 2005, 33, W368− W371. (66) Ikeguchi, M. Partial Rigid-Body Dynamics in NPT, NPAT and NPγT Ensembles for Proteins and Membranes. J. Comput. Chem. 2004, 25 (4), 529−541. (67) Mackerell, A. D.; Feig, M.; Brooks, C. L. Extending the Treatment of Backbone Energetics in Protein Force Fields: Limitations of Gas-Phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations. J. Comput. Chem. 2004, 25 (11), 1400−1415. (68) MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wiórkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins †. J. Phys. Chem. B 1998, 102 (18), 3586−3616. (69) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79 (2), 926. (70) Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D. CHARMM General Force Field: A Force Field for Drug-like Molecules Compatible with the CHARMM All-Atom Additive Biological Force Fields. J. Comput. Chem. 2010, 31 (4), 671−690. (71) Yu, W.; He, X.; Vanommeslaeghe, K.; MacKerell, A. D. Extension of the CHARMM General Force Field to SulfonylContaining Compounds and Its Utility in Biomolecular Simulations. J. Comput. Chem. 2012, 33 (31), 2451−2468. (72) ParamChem Interface; https://cgenff.paramchem.org (accessed November 28, 2014). (73) Vanommeslaeghe, K.; MacKerell, A. D. Automation of the CHARMM General Force Field (CGenFF) I: Bond Perception and Atom Typing. J. Chem. Inf. Model. 2012, 52 (12), 3144−3154. (74) Vanommeslaeghe, K.; Raman, E. P.; MacKerell, A. D. Automation of the CHARMM General Force Field (CGenFF) II: Assignment of Bonded Parameters and Partial Atomic Charges. J. Chem. Inf. Model. 2012, 52 (12), 3155−3168. (75) Martyna, G. J.; Tobias, D. J.; Klein, M. L. Constant Pressure Molecular Dynamics Algorithms. J. Chem. Phys. 1994, 101 (5), 4177. (76) Miller, T. F.; Eleftheriou, M.; Pattnaik, P.; Ndirango, A.; Newns, D.; Martyna, G. J. Symplectic Quaternion for Biophysical Molecular Dynamics. J. Chem. Phys. 2002, 116 (20), 8649. (77) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. CHARMM: A Program for Macromolecular Energy, Minimization, and Dynamics Calculations. J. Comput. Chem. 1983, 4 (2), 187−217. (78) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103 (19), 8577.

7900


Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand

Recommend Documents