Charge Clamps of Lysines and Hydrogen Bonds Play Key Roles in the

Article pubs.acs.org/JPCB

Charge Clamps of Lysines and Hydrogen Bonds Play Key Roles in the Mechanism to Fix Helix 12 in the Agonist and Antagonist Positions of Estrogen Receptor α: Intramolecular Interactions Studied by the Ab Initio Fragment Molecular Orbital Method Chiduru Watanabe,*,†,⊥ Kaori Fukuzawa,‡,§ Shigenori Tanaka,∥ and Sachiko Aida-Hyugaji† †

Information and Communication Technology Education Center, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan ‡ Mizuho Information & Research Institute, Inc., 2-3 Kanda Nishiki-cho, Chiyoda-ku, Tokyo 101-8443, Japan § Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan ∥ Graduate School of System Informatics, Department of Computational Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyo̅go 657-8501, Japan S Supporting Information *

ABSTRACT: The mechanism to fix helix 12 (H12) in the agonist/antagonist position, which is involved in controlling transcriptional activation, of the human estrogen receptor α ligand binding domain (hERαLBD) is studied by using fragment molecular orbital calculations at the Møller−Plesset second-order perturbation levels to analyze inter-fragment interaction energies (IFIEs), electrostatic potentials (ESPs), and atomic charges. The mutually attractive and complementary relationships between H12 and highly conserved Lys529/Lys362 are shown through the IFIEs and ESPs. The highly conserved Lys529 and Lys362 are found to have strong attractive interactions with the anionic residues of H12 in the agonist and antagonist positions, respectively, thus playing roles of charge clamps to fix H12. Additionally, intramolecular interactions between the neutral residues of H12 including the LXXML motif and the other part of hERα are strengthened by the hydrogen bonds and polarization. It is noted that the highly conserved Asp351 forms a hydrogen bond with Leu540 of H12 in the hERα−agonist complex, while it is also involved in stabilization of ligand binding in the hERα−antagonist complex. The charges of residues at the interface between H12 and the other part of hERα approach approximately neutral upon forming the agonist/antagonist binding conformation so as to relax the electrostatic repulsion caused by the negative charges of H12 and the other part of hERα. Our observations would thus provide useful information to control the H12 position for regulation of transcription in hERα and other nuclear receptors. following steps at the molecular level.6,7 First, the ligand docks with the hNR and subsequently the position of helix 12 (H12), which is located in the C-terminal side of the LBD, changes. Second, the hNRs bind with each other, making homo or hetero dimers. The hNRs then recognize specific sequences of the genomic DNA with the binding of various kinds of

1. INTRODUCTION Human estrogen receptor α (hERα) belongs to the superfamily of human nuclear receptors (hNRs), which regulates the expression of various genes as a transcription factor. hNRs are known to be related to cancers, metabolic syndrome, osteoporosis, and other diseases,1−5 and thus are important drug targets. hNRs are composed of five domains: N-terminal domain, DNA binding domain (DBD), hinge region, ligand binding domain (LBD), and C-terminal domain (Figure 1). The transcriptional mechanism of the hNRs is composed of the © 2014 American Chemical Society

Received: November 26, 2013 Revised: April 11, 2014 Published: April 11, 2014 4993

dx.doi.org/10.1021/jp411627y | J. Phys. Chem. B 2014, 118, 4993−5008

The Journal of Physical Chemistry B

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Figure 1. Alignment of amino acid sequences corresponding to H3, H4/H5, H12, and the loop between H11 and H12 in the LBD of human NRs (ERα, ERβ, ERR, AR, PR, PPARγ, RXRα, and VDR). Conserved residues in NRs are marked by asterisks. In H3, H4/H5, and the loop between H11 and H12, the blue residues and yellow residues play roles in fixation of the H12 position by charge clamps and hydrogen bond interactions, respectively, from IFIE and ESP analyses for ERα. In other NRs, the residues that correspond to those of ERα are similarly colored. H12 is enclosed by a red broken line as an anionic domain. In H12, the negatively and positively charged residues are shown in red and blue, respectively.

Figure 2. (a) Superposition of the apo state of ERαLBD (PDB-ID: 1A52) and the complex of 17β-estradiol (EST), coactivator, and ERαLBD (PDBID: 1GWR); (b) superposition of the apo state of ERαLBD and the complex of 4-hydroxytamoxifen (OHT) and ERαLBD (PDB-ID: 3ERT). In parts a and b, atoms of the ligand molecules and a water molecule are represented by ball and stick models, and the whole structures of ERαLBD are depicted in green, while the H12 parts are in yellow (apo state), red (agonist-bound state), and blue (antagonist-bound state). The coactivator in the agonist-bound state is shown in purple. The structural formulas of the ligands (EST and OHT) are drawn in black lines.

ligand-induced conformational changes are called a “mouse trap”, from the structure observed by X-ray crystallography.8−12 The position of H12 depends on the ligand type (either agonist or antagonist) and controls transcriptional activation. When an agonist is bound to hERα, H12 lies in the “agonist position”, which is located close to the ligand binding pocket and is an active conformation (colored red in Figure 2a). Since this conformation forms a coactivator binding surface and can proceed to coactivator binding, transcriptional activity is enhanced. The coactivators of hNRs (colored purple in Figure 2a) contain three highly conserved LXXLL motifs (L represents Leu; X represents any amino acid) and increase gene expression.7,9,10,13,14 In the hNR and agonist complex, highly conserved residues of Lys and Glu (e.g., Lys362 and Glu542 in hERα, as shown in Figure 1) create negatively and positively

coactivators or corepressors, which are related to transcriptional activation/repression, to mediate the transcription of various target genes. X-ray crystallographic studies of NRs7 have suggested that whether the transcriptional mechanism proceeds to the coactivator binding step will be determined by the position of H12. Thus, a conformational change of H12 is important for transcriptional activation. Parts a and b of Figure 2 show conformations of hERα, which is related to breast cancer and osteoporosis, complexed with agonist, 17β-estradiol (EST), and antagonist, 4-hydroxytamoxifen (OHT), respectively. In Figure 2a and b, H12 is positioned far from the ligand binding pocket of hERα in the apo state (yellow).8 When a ligand is bound to hERα, H12 is folded and the positions of H12 differ in the agonist- and antagonist-bound states.9,10 Such remarkable 4994



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cost in terms of parallel computation, and as a result, it realizes reliable and accurate quantum analysis of biomacromolecules. In particular, by using the inter-fragment interaction energies (IFIEs) obtained by the FMO calculations, we can identify key residues for intermolecular and intramolecular interactions in the ligand-binding or folded structure. Several computational studies were performed by the FMO method for understanding the molecular mechanism of the NRs’ functions: ligand binding, DNA binding, and coactivator binding.31−38 First, Fukuzawa et al.31 calculated relative binding energies between the hERα and its ligands, and the result was in good correlation with experimental relative binding affinities. Moreover, IFIEs between EST and each amino acid residue of hERα indicated that certain residues (e.g., Glu353, Arg394, His524, and Thr347) and a water molecule play key roles in EST binding.30 Next, Watanabe et al.35 analyzed the IFIEs and electrostatic potentials (ESPs) for the complex of hERα and DNA. From ESP analysis, it was clarified that the dimer of hERα and DNA attract each other more strongly than do the monomer of hERα and DNA; the dimerization of hERα thus contributes to a stabilization of the hERα−DNA complex. Finally, Ito et al.36−38 performed the FMO calculations for a complex of the retinoid X receptor (RXR), an agonist and a coactivator, and analyzed the IFIEs between the RXR and its coactivator. They demonstrated that conserved residues of H12 in RXR (e.g., Phe450 and Glu453) and the LXXLL motif of the coactivator play essential roles in the complex formation by Coulomb interaction, dispersion interaction, and charge transfer (CT). For these studies, it is clear that the FMO calculation is a powerful tool to comprehensively analyze the biochemical functions of hNRs and also other proteins; nevertheless, the residues that are the key to fixing the agonist/antagonist position of H12 have not yet been identified by using the FMO method. Clarification of the mechanism involved in transcription via hNRs will provide important clues for drug discovery. As our present study on the elucidation of a transcriptional mechanism, we here focus on the mechanism to fix H12 in the agonist/antagonist position after the agonist/antagonist binding. To specify the key residues in this mechanism, we performed comprehensive FMO calculations for complexes of hERαLBD and the agonist/antagonist under gas phase conditions, and analyzed the IFIEs, ESPs, and atomic charges.

charged areas and work as a charge clamp of the coactivator by electrostatic potentials (ESPs). Moreover, the LXXLL motif of the coactivator forms hydrogen bonds with the highly conserved residues of hNR. On the other hand, when an antagonist is bound to hERα, H12 is located in the “antagonist position” (colored blue in Figure 2b), which differs from the “agonist position”. In the antagonist-bound state, the Leu-rich helical structure of H12 in the hNR (e.g., LXXML motif of H12 in hERα, as shown in Figure 1) mimics the LXXLL motif of the coactivator which interacts with the NR−agonist complex. Consequently, the position of H12 in the NR−antagonist complex interferes with the ensuing coactivator binding,10,15 and then transcription is suppressed. Thus, control of the H12 position is important for regulating transcriptional activation, and elucidation of its mechanism will be helpful in the discovery of drugs for diseases associated with NRs. In the case of hERα, previous studies related to the control of the H12 position have been reported involving many mutation experiments16−23 and molecular dynamics (MD) simulations.24,25 Various experiments have already been performed on point mutations in hERα.16 In the mutation experiments that changed the position of H12, results of a Cys to Ser triple mutant hERα (C381S + C417S + C530S) by Gangloff et al.17 are of particular interest. They reported that transcriptional activity of the triple mutant hERα was decreased, and its H12 was located in the antagonist position as observed by X-ray crystal structure analysis (PDB ID: 1QKT), although the triple mutant hERα was bound to an agonist. Thus, it is clear that the position of H12 is one of the keys to regulate the transcription. Moreover, other mutation experiments (e.g., Asp351, Lys529, Lys531, and so on)18−22 with respect to the control of transcription were also reported. It was noted that Asp351 in H3 would play roles in either regulating the position of H12 or the antagonist binding by mutation experiments and X-ray crystal analysis.19−23 Taken together, the various residues probably play crucial roles to control the position of H12 related to transcriptional activation. Further, to study the relationship between ligand binding and structural changes in hERαLBD, Celik et al.24 performed MD simulations for different conformations such as the apo, agonist, and antagonist positions of H12 in Figure 2, and those for different kinds of complexes such as binding structures with ligands and/or coactivator. A tendency for H12 to move upward toward the ligand binding pocket was reported, where H12 was located in the extended state (apo position) and the agonist was located in the ligand binding pocket at the beginning of the simulation. In our previous study,25 we investigated the relationship between the structural fluctuation of hERα in the apo state and its structural change upon ligand binding by using MD simulation and the linear response theory.26,27 We have concluded that H12 of the hERα essentially has a tendency to swing up from the extended position toward the ligand binding pocket, and such a preexisting swing-up motion is triggered by the ligand binding. Hence, the “mouse trap” mechanism, which changes the position of H12 induced by ligand binding, is elucidated by these studies with MD simulations. Various factors (e.g., the type of ligands) may be involved in this structural change; however, such factors involved in determining the agonist/antagonist position of H12 are yet unclear. Recently, Kitaura et al.28−30 developed the fragment molecular orbital (FMO) method, which enables large-scale molecular orbital calculations by reducing the computational

2. COMPUTATIONAL METHOD 2.1. Construction of Structures of ERαLBD−Ligand− Water Complexes. As the initial structures of complexes between hERαLBD and its agonist (EST: 17β-estradiol) or antagonist (OHT: 4-hydroxytamoxifen), we used the coordinates available in the Protein Data Bank (PDB) entry 1GWR or 3ERT, respectively. The entire ERαLBD (residue nos. 307− 547), its ligand, and a water molecule, which is involved in the ligand binding network, were used for the calculation. Basic and acidic amino acid residues (Arg, Lys, Asp, and Glu) of hERαLBD were treated as positively or negatively charged residues under the experimental conditions at pH 7.8 (PDB ID: 1GWR) and 7.0 (PDB ID: 3ERT). In addition, we treated the histidines in ERαLBD as the neutral ε-tautomer, so as not to break the surrounding hydrogen bond network from His524 to Glu339 via Glu419 and Lys531 and other hydrogen bonds from His524 to the ligand (EST).17,24 It has been suggested that the agonist position of H12 was partially stabilized by the hydrogen bond network from His524 to Glu339 in the N-terminal of H3 4995



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Table 1. IFIEs of Residues in the H12 and Ligand with Residues in the Other Part of ERα in (a) the ERαLBD−EST (Agonist) Complex and (b) the ERαLBD−OHT (Antagonist) Complexa (a) fragment of ER-main in the ERαLBD−EST (agonist) complex

IFIEs of H12 (kcal/mol)

IFIEs of ligand (kcal/mol) IFIE-sums of H12 (kcal/mol)

IFIEs of H12 (kcal/mol)

IFIEs of ligand (kcal/mol) IFIE-sums of H12 (kcal/mol)

Lys362 (+)

Asn348 (neutral)

Leu536 (neutral) Tyr537 (neutral) Asp538 (−) Leu539 (neutral) Leu540 (neutral) Leu541 (neutral) Glu542 (−) Met543 (neutral) Leu544 (neutral) Asp545 (−) Ala546 (neutral) EST (neutral) H12-all residuesb H12-anionic residuesc H12-neutral residuesd

−1.01 −12.73 1.46 −0.37 −1.31 −1.17 1.15 −0.25 −0.38 0.00 −0.10 −0.16 −14.70 2.61 −17.32

fragment of ER-H12 (charge)

Asn348 (neutral)

Asp351 (−)

Lys362 (+)

Glu380 (−)

Trp383 (neutral)

Lys529 (+)

OHT (neutral)

Leu536 (neutral) Tyr537 (neutral) Asp538 (−) Leu539 (neutral) Leu540 (neutral) Leu541 (neutral) Glu542 (−) Met543 (neutral) Leu544 (neutral) Asp545 (−) Ala546 (neutral) OHT (neutral) H12-all residuesb H12-anionic residuesc H12-neutral residuesd

0.07 0.12 0.38 0.01 0.13 −0.02 0.60 0.14 0.00 0.42 −0.01 −2.44 1.84 1.39 0.45

0.49 −0.09 21.06 −0.12 0.27 −1.45 21.10 0.52 −1.21 15.07 0.11 −9.12 55.75 57.23 −1.48

−0.96 −2.37 −23.60 −1.54 −3.90 −7.47 −38.99 −2.93 −37.56 −48.70 1.87 1.03 −166.17 −111.30 −54.87

−5.24 −23.64 30.04 −4.66 −5.31 −4.27 17.89 −2.48 −2.20 18.08 −0.45 −1.39 17.75 66.01 −48.27

−1.06 0.29 −1.91 0.33 −0.04 0.30 −0.51 0.37 0.20 −0.97 0.12 −2.29 −2.88 −3.38 0.50

4.82 4.51 −26.45 2.10 2.08 1.65 −14.97 1.26 1.15 −13.52 0.07 0.79 −37.31 −54.94 17.63

−0.20 0.02 −0.98 0.01 0.00 0.16 −0.96 −0.01 0.11 −0.55 −0.01

Asp351 (−)

Glu380 (−)

0.56 −0.14 −13.99 0.42 54.01 −18.31 −13.54 −0.59 −21.68 −0.14 −11.91 0.77 29.51 −25.05 −4.30 −0.61 −3.60 0.80 23.18 −14.55 −1.90 0.50 0.00 0.05 36.35 −56.90 106.70 −57.92 −70.35 1.02 (b) fragment of ER-main in the

Trp383 (neutral)

Lys529 (+)

fragment of ER-H12 (charge)

0.09 0.14 −2.11 −0.31 −0.18 −0.45 22.58 −0.33 −28.98 1.96 −0.41 −1.36 3.96 −1.61 −0.58 1.34 −0.90 −6.26 46.87 −4.04 −27.32 5.49 −4.64 0.79 0.32 −10.06 −0.66 30.90 −3.58 −76.65 −1.61 0.55 2.97 −0.03 −0.25 0.30 111.60 −25.05 −140.60 100.36 −7.94 −132.94 11.24 −17.11 −7.66 ERαLBD−OHT (antagonist) complex

EST (neutral) −0.03 −0.02 −0.48 0.07 0.04 0.14 −0.18 0.07 0.07 −0.19 0.01 −0.51 −0.85 0.33

−2.40 −2.48 0.09

a IFIEs between each ER-H12 residue and each ER-main residue were obtained from FMO calculations at the MP2/6-31G* level. The summations of the IFIEs (IFIE-sums) were also calculated over all (H12-all), three anionic (H12-anionic), and eight neutral (H12-neutral) residues of H12. Anionic and cationic residues are indicated in bold and italic characters, respectively. bIFIE-sums between the H12-all residues (residues nos. 536− 546) and each other residue. cIFIE-sums between the H12-anionic residues (Asp536, Glu542, and Asp545) and each other residue. dIFIE-sums between the H12-neutral residues (other residues except for the H12-anionic residues among the H12-all residues) and each other residue.

via Glu419 in the omega-loop and to Lys531 in the C-terminal of H11.17,24 Both N- and C-terminal residues (Ala307 and His547) of the peptide chains were capped with hydrogen atoms, and the missing hydrogen atoms and residues in the PDB files were complemented manually by using AMBER10 software.39 The missing residues of two loops (residue nos. 332−334 and 462−464) in the agonist-bound conformation (PDB-ID: 1GWR) were modeled by using the coordinates of another hERα structure (PDB-ID: 1A52). Thus, a complete structure of ERαLBD (residue nos. 307−547), the ligand (EST or OHT), and a water molecule (ER-all model) consists of about 4000 atoms without counterions. Structural optimizations were carried out through the following two steps using the AMBER10 software39 with the AMBER99SB force field. In the first step, all atoms of the missing residues added in the previous procedure were optimized under AMBER10, and the other residues were fixed at the positions given in the PDB data. In the second step,

the optimization was performed only for the added hydrogen atoms with all heavy atoms fixed at the positions obtained in the first step. Here, the potential (force field) parameters of EST and OHT were determined by using the general AMBER force field (GAFF) and atomic charges fitted as the restrained electrostatic potential (RESP) charges, where the RESP charges were produced using electrostatic potentials; the electrostatic potentials were obtained by ab initio MO calculations at the Hartree-Fock (HF) level with the 6-31G* basis set (HF/631G*) after geometry optimizations were carried out at HF/631G by using the Gaussian 03 program. We described force field parameter files for EST and OHT in the Supporting Information, sections C and D, respectively. To compare interactions between H12 and all of the other amino acid residues in the agonist complex with those in the antagonist complex, the H12s were defined as a common sequence. According to the PDB files (PDB-ID: 1GWR, 1ERE, 3ERD, 1ERR, and 3ERT), H12s were commonly located within 4996



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Figure 3. IFIE-sums between the residues of H12 and each other residue in (a) the ERαLBD−EST (agonist) complex and (b) the ERαLBD−OHT (antagonist) complex, calculated at FMO-MP2/6-31G*. In parts a and b, the IFIE-sums for the H12-all residues (residue nos. 536−546), the H12anionic residues (Asp538, Glu542, and Asp545), and the H12-neutral residues (other residues except for the H12-anionic residues in the H12-all residues) are depicted by red or blue (top), black (middle), and white (bottom) bars; the gray-colored areas represent H12-all regions (residue nos. 536−546).

residue nos. 538−546 in the agonist-bound state and residue nos. 536−544 in the antagonist-bound state. In this study, we defined the H12 regions of ERαLBD (ER-H12) as residue nos. 536−546 for both the agonist- and antagonist-bound states. Using the inter-fragment interaction energy (IFIE), electrostatic potential (ESP), and fragment charge analyses, we examined interactions between H12 (ER-H12) and the other part of ERαLBD (ER-main) to understand the mechanisms involved in fixing the H12 position. The structural details of the ER-H12 and ER-main models differed in the ESP and atomic charge analyses, and they will be described in sections 3.2 and 3.3. The ER-all and ER-H12 models have total charges of −7e and −3e, respectively. The electrostatic interactions might be important in determining the H12 position, because the

negative charge of H12 (−3e) is roughly half that of the entire ERαLBD−ligand−water complex. 2.2. FMO Calculations. To briefly describe the ab initio FMO method, a molecule or a molecular cluster is first divided into small fragments, and then the MO calculations on each fragment monomer and each fragment dimer are performed to obtain the properties of the whole system.28−38 The many-body effects arising from surrounding fragments are considered through the environmental electrostatic potentials. The total energy of the whole system Etotal is calculated by Etotal =

∑ Ei + (N − 2) ∑ Eij i

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i>j

(1)



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Figure 4. Visualizations of the summations of IFIEs (IFIE-sums) between the residues of H12 and each other residue. IFIE-sums of (a) the H12anionic residues (Asp538, Glu542, and Asp545) and (b) the H12-neutral residues (other residues of H12 except for the H12-anionic residues) in the ERαLBD−EST (agonist) complex. (c) The H12-anionic residues and (d) the H12-neutral residues in the ERαLBD−OHT (antagonist) complex are shown with colors indicating attractive (red) and repulsive (blue) interactions. The H12-anionic and H12-neutral residues are colored yellow in each figure.

where Ei and Eij are energies of the ith fragment monomer and ijth fragment dimer and N is the number of fragments in a given system. IFIE is defined as follows: ΔEij = (Eij′ − Ei′ − Ej′) + Tr(ΔPijV ij)

(2)

Ex′ = Ex − Tr(ΔP x V x)

(3)

(x = i , ij)

fractional point charge (esp-ptc), when the distance between the closest contact atoms in the two fragments exceeded 0.0 van der Waals (vdW) and 2.0 vdW radii, respectively.30 The Coulomb interaction approximation for the fragment pair calculations (dimer-es) was also applied in the case of two fragments with the separation exceeding 2.0 vdW radius. To obtain IFIEs and Mulliken charges, FMO calculations at the MP2/6-31G* level were performed on SGI Altix4700 6.4GFLOPS (128 CPUs) and on 12 quad AMD Opteron 2.4-GHz clusters (48 CPUs). Most of the FMO calculations were performed on the quad AMD Opteron clusters with 32 CPUs, where the elapsed times were ∼730 000 s (over 8 days) for biomolecular systems comprising about 4000 atoms. The ESP calculations were carried out at the HF/6-31G* level by using the cpf2den program in the ABINIT-MP, and the isosurfaces of ESP were generated at an electron density of 0.001e/au3. The results were visualized by using BioStation Viewer.40,41

where ΔPx is a difference density matrix and Vx is an environmental electrostatic potential for fragment x (x = i (monomer) or ij (dimer)) from other fragments. Ei′ and Eij′ are energies of the ith fragment monomer and ijth fragment dimer without the environmental electrostatic potential. In this study, each amino acid residue of ERαLBD, the ligand molecule (EST/OHT), and a water molecule was treated as a single fragment. There were 243 fragments in the ERαLBD− ligand−water complex. We carried out ab initio FMO calculations by using the ABINIT-MP program40,41 under gas-phase conditions at the Møller−Plesset second-order perturbation (MP2) levels with the 6-31G* basis set (FMO-MP2/6-31G*) for the complex of ERαLBD, the agonist/antagonist, and a water molecule. To save the computational time without losing significant accuracy, we used approximations of electrostatic potentials, described in terms of the Mulliken orbital charge (esp-aoc) and the

3. RESULTS To elucidate the determination mechanism to fix the position of H12 in ERαLBD, we investigated IFIEs, ESPs, and atomic charges in sections 3.1, 3.2, and 3.3, respectively. 3.1. IFIE Analysis between H12 and the Other Part of ERα. The IFIEs for H12 in the ERαLBD−EST/OHT complex 4998



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Figure 5. Positions of the H12-neutral residues and the residues in other parts of ERαLBD that contribute to hydrogen bonds in (a) the ERαLBD− EST (agonist) complex and (b) the ERαLBD−OHT (antagonist) complex. In parts a and b, the side chain atoms of residues in H12 and ER-main are represented by stick and ball-and-stick models, respectively, with the colors of white (hydrogen), gray (carbon), red (oxygen), and blue (nitrogen). The yellow and green lines represent the Cα backbones of H12 and other parts of ERαLBD, respectively. The distances are shown by dotted blue lines.

were obtained by the FMO calculations at the MP2/6-31G* level, as shown in Table 1 and Figures 3 and 4. We discuss the interactions arising from the entire structure of H12 in section 3.1.1, and further discuss the contributions from the amino acid residues of H12 in section 3.1.2. 3.1.1. Intramolecular Interactions between the Entire H12 and Each Residue of ER-Main. The IFIEs of residues in the H12 and ligand with residues in other parts of ERα were obtained, and the summations of these IFIEs (IFIE-sums) over all residues of H12 (residue nos. 536−546 (H12-all)) were calculated and shown in Figure 3. The value obtained for IFIEsum represents the interaction energy between each residue of ER-main and all residues of ER-H12. To analyze the contributions from both electrostatic interactions and other interactions, the IFIE-sums of only the anionic residues Asp538, Glu537, and Asp545 (H12-anionic) and only the neutral residues Leu536, Tyr537, Leu539, Leu540, Leu541, Met543, Leu544, and Ala546 (H12-neutral) are also shown in Figure 3. Figure 4 depicts the positions of residues having large IFIE-sum values among the H12-anionic and H12-neutral residues in the agonist- and antagonist-bound states, with the color deepness indicating magnitudes of the IFIE-sums. In Figure 5, the positions of some key residues and their atoms located at the interface between ER-main and ER-H12 are presented. In the agonist-bound state, from the IFIE-sums of H12-all residues shown in Figure 3a, it is confirmed that the residue most attractive to H12 was the cationic Lys529, and its IFIEsum value is −140.60 kcal/mol in vacuo, as seen in Table 1a. It is obvious that Lys529 is a key residue for determining the position of H12 with a negative charge. Although Lys531 is a neighbor of Lys529 and is also a cationic residue, its IFIE-sum value was only about one-third of that of Lys529 (−54.44 kcal/ mol), which is comparable with those of other cationic residues distant from Lys529. Thus, Lys531 seems not to be a key residue to fix the H12 position. In contrast, Glu380 is the residue with the most repulsive interaction to H12 and is considered to partially weaken the stabilizing energy of the agonist-bound conformation. According to the IFIE-sums of the H12-anionic residues, these residues, as well as the H12-all residues, have strong attractive interactions with Lys529 (−132.94 kcal/mol). In addition, Figure 4a shows that Lys529, the most attractive

residue to H12, is located close to H12. In contrast, the IFIEsums of the H12-anionic residues all have strongly repulsive interactions with Asp351 and Glu380 (+106.70 and +100.36 kcal/mol, respectively) owing to electrostatic force. On the other hand, the results for the H12-neutral residues show that they have large attractive interactions with Asp351, Asn348, and Trp383 (−70.35, −17.32, and −17.11 kcal/mol, respectively) and have the strongest repulsive energy against Glu380 (+11.24 kcal/mol). From Figures 4b and 5a, it is confirmed that Asn348/Asp351/Trp383 is accessible to the start/middle/end points of H12. Thus, it is clarified that H12 was fixed to the agonist position by some key residues of ERmain at three points: the start, middle, and end of H12. It is remarked that this finding can be obtained only through the IFIE analysis. In the antagonist-bound state, the results of IFIE-sums shown in Figure 3b indicated that Lys362 (a cationic residue) was the most attractive to H12-all having a negative charge by electrostatic force, and its IFIE-sum value was −166.17 kcal/ mol in vacuo, as seen in Table 1b. Although Arg363 is a neighboring cationic residue of Lys362, its IFIE-sum value (−55.75 kcal/mol) was only about one-third of that of Lys362 and was comparable with the other cationic residues distant from Lys362. Thus, it is suggested that Lys362 plays a crucial role to fix the H12 position, while Arg363 does not. From the IFIE-sums of the H12-anionic residues in Figure 3b, Lys362 was revealed to be the most attractive (−111.30 kcal/mol) to the H12-anionic residues owing to electrostatic force, as well as to the H12-all residues. Additionally, Figures 4c and 5b show that Lys362, the most attractive residue to H12, is located close to H12. In contrast, the IFIE-sums of the H12anionic residues all have strongly repulsive interactions with Glu380 (+66.01 kcal/mol) derived from electrostatic force. On the other hand, the H12-neutral residues have large attractive interactions with Lys362 and Glu380 (−54.87 and −48.27 kcal/mol, respectively). These attractive interactions between Glu380 and the H12-neutral residues are counteracted by the electrostatic repulsive interactions between Glu380, an anionic residue, and the H12-anionic residues. Although Glu380 seems to not be a key residue, because its IFIE-sum value to H12-all residues is positive, this residue plays a key role in fixing the H12 position, as discussed in section 3.1.2. It is 4999



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has the role of a charge clamp of H12 (−3e charged region) by the electrostatic interaction. We investigated the strongly attractive interactions between Glu380/Lys362, which are attractive with the H12-neutral residues (in Figure 3b), and each H12-neutral residue. It is obvious that Glu380 has a strong attractive interaction with Tyr537 located at the start of H12, where the IFIE was −23.64 kcal/mol. Figure 5b shows that interatomic distances between two oxygen atoms in a carboxylate group of Glu380 and two hydrogen atoms in the peptide bond and an indole ring of Tyr537 are short (r4 = 3.01 Å and r5 = 2.25 Å in Figure 5b), and the oxygen atom of Glu380 will form a hydrogen bond with the hydrogen atom of an indole ring of Tyr537. Moreover, an interatomic distance between Hη in the side chain of Lys362, which has a large IFIE value (−37.56 kcal/mol) to the Leu544 fragment, and an oxygen atom in the peptide bond of Met543, a residue neighboring Leu544, is 2.26 Å (r5 in Figure 5b). Similarly, as in the agonist-bound state, CO atoms in a peptide bond of Met543 were assigned to the Leu544 fragment, and then Lys362 seems to interact with Leu544 instead of Met543. For these results, we considered that H12 is fixed by hydrogen bonds at the two binding points of its start (Glu380Tyr537) and end (Lys362-Met543). Here, we describe the roles of the ligands (EST and OHT). The agonist (EST) does not interact with any key residues above, as seen in Table 1a. In contrast, the antagonist (OHT) has some attractive interactions with the key residues, especially with Asp351 (−9.12 kcal/mol), as seen Table 1b. Since EST is much smaller than OHT and located distant from Asp351 in the ligand binding pocket, Asp351 is free from EST binding. Thus, it is possible for a hydrogen bond to form between Asp351 and Leu540 of H12, and their IFIE value is the largest among IFIE values of the three key residues in the agonistbound state. Asp351 also has a role as a ligand binding residue in the antagonist-bound state, which is entirely different from its role as an H12-binding residue in the agonist-bound state. By comparing the agonist- and antagonist-bound states, it is considered that the position of H12 was fixed by the following two mechanisms, i.e., the strong electrostatic interactions by charge clamps of lysines and the interactions by some hydrogen bonds and polarization between ER-H12 and ER-main. First, H12 was attracted by the electrostatic interactions between Lys529/Lys362 and the H12-anionic residues, in the agonist/ antagonist-bound state. Second, the H12 position was more firmly fixed by the hydrogen bonds and polarization between the interfacial neutral residues of H12 and ER-main at two or three points. In addition, we speculate the role of Asp351 for both agonist and antagonist positions of H12. Asp351 fixes the position of H12 in the agonist-bound state; however, Asp351 stabilizes the antagonist (OHT) in the ligand binding pocket of ERαLBD in the antagonist-bound state. 3.2. Electrostatic Potentials of ER-Main and ER-H12. The ESPs were calculated to analyze the electrostatic interaction between H12 and the other part of ERαLBD in both the agonist- and antagonist-bound states. To reveal ESPs at the interface between H12 and the other part of ERαLBD, the optimized structure of ER-all (as described in the previous section) was divided into two parts, the ER-H12 and ER-main models. The ER-H12 model comprises 13 residues (residue nos. 535−547) of ERαLBD; the ER-main model comprises 229 residues (residue nos. 307−535) of ERαLBD, a ligand (EST/ OHT), and one water molecule. The terminal residue Pro535 was truncated at Cα and was capped with hydrogen atoms in

noted that the value of the repulsive IFIE-sum of the H12-all residues with Glu380 in the antagonist-bound state (+17.75 kcal/mol) was much smaller than that in the agonist-bound state (+111.60 kcal/mol). From Figures 4c,d and 5b, it is confirmed that Glu380 and Lys362 are accessible to the start and end points of H12, respectively, and fix H12 to the antagonist position at the two points of the start and the end of H12. 3.1.2. Details of Intramolecular Interactions of Each Residue to Fix the H12 Position. We summarized in Table 1 the results of some IFIEs that were involved in the highly attractive interactions of residues in H12 and ligand with residues in other parts of ERα in agonist- and antagonist-bound states. Parts a and b of Figure 5 show the positions of selected residues that sustain attractive interactions between the H12neutral and ER-main residues in the ERαLBD−EST and ERαLBD−OHT complexes, respectively. In the agonist-bound state, Table 1a reveals that the attractive interaction of Lys529 with Asp545 (−76.65 kcal/mol) is stronger than those interactions with the other H12-anionic residues Asp538 (−28.98 kcal/mol) and Glu542 (−27.32 kcal/ mol). Here, the distance from Lys529 to Asp545, which is located at the end of H12, is 3.41 Å, whereas the distances from Lys529 to Asp538 and to Glu542 are more than 7 Å. The obtained IFIEs are reasonable, since these interactions are dominated by electrostatic forces depending on the distances from Lys529. Thus, it is considered that Lys529 (a cationic residue) has the role of a charge clamp of H12 (−3e charged region) by the electrostatic interaction. In Table 1a, we also confirmed some strong interactions of Asn348/Asp351/Trp383 that interact with H12-neutral residues, as seen in Figure 3a for each H12-neutral residue. Asn348 and Asp351, located at the interface of ER-main, mainly interact with Tyr537 (−12.73 kcal/mol) and Leu540 (−21.68 kcal/ mol) at the interface of ER-H12, respectively. Additionally, the interatomic distances of Hδ2 (Asn348)Oη (Tyr537) and the Oδ (Asp351)H atom in a peptide bond of Leu540 are 2.11 Å (r1 in Figure 5a) and 2.31 Å (r2 in Figure 5a), respectively. They are short enough to form hydrogen bonds. Moreover, in Table 1a, Trp383 seems to be strongly attractive to Leu544 (−10.06 kcal/mol). The Hε1 of Trp383, however, is close enough to interact with an oxygen atom in the peptide bond of Met543 (r3 = 2.88 Å in Figure 5a) as compared with atoms of Leu544. Here, CO in the peptide bond of Met543 was assigned to the Leu544 fragment through fragmentation in FMO procedures, because proteins have to be divided into fragments at localized orbitals (sp3) between Cα and CO of the main chain in the amino acid residue. Thus, the IFIE value of Trp383 with Leu544 was large compared to that with Met543. Since the distance r3 is longer than a hydrogen bond, the interaction between Trp383 and Leu544 fragments may be derived by polarization of CO in the main chain of Met543. From these results, we confirmed that H12 has three binding points by two hydrogen bonds and polarization at the start (Asn348-Tyr537), middle (Asp351-Leu540), and end points of H12 (Trp383-Met543), respectively. In the antagonist-bound state, as shown in Table 1b, Lys362 was strongly attractive with Asp538, Glu542, and Asp545 among the H12-anionic residues, where the IFIEs were −23.60, −38.99, and −48.70 kcal/mol, respectively. Here, Asp538, Glu542, and Asp545 are separated from the nearest-neighbor atoms of Lys362 at distances of more than 4 Å and do not face Lys362. Thus, it is considered that Lys362 (a cationic residue) 5000



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Figure 6. ESPs of the ERαLBD−EST (agonist) complex (top) and the ERαLBD−OHT (antagonist) complex (bottom) calculated at the FMO-HF/ 6-31G* level. For the agonist-bound state, the isosurfaces of ESPs are shown for (a) ER-main (ERαLBD except for the H12 region, the ligand (EST: agonist), and a water molecule) on the H12-binding side and (b) ER-H12 on the Lys529-binding side. For the antagonist-bound state, the isosurfaces of ESPs are shown for (c) ER-main (ERαLBD except for the H12 region, the ligand (OHT: antagonist), and a water molecule) on the H12-binding side and (d) ER-H12 on the Lys362-binding side. The ESP map is represented on an isosurface at the electron density of 0.001e/au3. The negative and positive ESPs are shown in red and blue, respectively. In parts a and c, the structures of H12 in ERαLBD are depicted by a yellow solid ribbon.

both models. Electric charges of the ER-H12 and ER-main models were −4e and −3e, respectively. Optimizations only for the added hydrogen atoms of Pro535 in the ER-H12 and ERmain models were performed by using AMBER10 with the AMBER99SB force field, where the other atoms were fixed at the positions given in the previously optimized ER-all model (section 2.1). The ESPs of each optimized structure of the ERH12 and ER-main models were calculated at the FMO-HF/631G* level. The isosurfaces of ESP of the ER-main and ERH12 models were generated at the electron density of 0.001e/ au3 and are shown in Figure 6, where H12 was depicted as a yellow solid ribbon. It was found that some strongly positive and negative ESP spots commonly exist on the H12-binding sides of ER-main in both agonist- and antagonist-bound states (Figure 6a and c). On the other hand, the ESPs of the ER-H12 models are shown as being entirely negative in both states (Figure 6b and d). In the agonist-bound state, there was a strongly positive ESP spot at Lys529 located close to H12 (Figure 6a). It is suggested that Lys529 having a strong positive potential plays a crucial role in fixing the position of negatively charged H12 by the electrostatic interaction (Figure 6b). In fact, the IFIE-sum value indicated the presence of a highly attractive interaction between H12 and Lys529 in ER-main of the ERαLBD−EST complex in Figure 3a and Table 1, as mentioned in section 3.1.

Furthermore, the strongly negative ESP spot of Asp351 in ER-main was located close to the start of H12, as shown in Figure 6a. Since H12 showed an entirely negative ESP, intuitively it would seem to induce a repulsive interaction between Asp351 and H12. In reality, however, it is suggested that stabilization by the hydrogen bond formation between Asp351 and Leu540 located in H12 occurs prior to the electrostatic repulsion between Asp351 and H12, as seen in section 3.1.2. In the antagonist-bound state, as seen in Figure 6c, there was a strongly positive ESP spot at Lys362 located close to H12. It is supposed to be an essential determinant to fix the position of negatively charged H12 by electrostatic interaction (Figure 6d). The IFIE-sum value shows the highly attractive relationship between H12 and Lys362 in ER-main of the ERαLBD−OHT complex in Figure 3b and Table 1. On the other hand, Figure 6c shows that the strongly negative ESP spot of Glu380 in ERmain is close to the start of H12, which would be intuitively predicted to induce a repulsive interaction between Glu380 and H12. Although IFIE-sums of H12 also indicate repulsion between Glu380 and H12 anionic residues, Glu380 is able to be located close to Tyr537 in H12 to form the hydrogen bond, as described in section 3.1.2. In both the agonist- and antagonist-bound states, the surface of H12 consists of several areas with negative ESP, some of which 5001



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Figure 7. The Mulliken fragment charge qi, calculated by the FMO-MP2/6-31G* method, and the charge difference Δqi: Δqi = qi(ER-all) − qi(ERmain) for residue nos. 307−532 of ER-main, the ligand, and a water molecule; Δqi = qi(ER-all) − qi(ER-H12) for residue nos. 533−547 of ER-H12. (a) For the ERαLBD−EST (agonist) complex, qi (top) and Δqi (bottom) are represented by black and red bars, respectively. (b) For the ERαLBD− OHT (antagonist) complex, qi and Δqi are represented by black and blue bars, respectively. In parts a and b, the gray-colored areas represent the H12 regions (residue nos. 536−546).

3.3. Charge Differences through Conformational Change of H12. H12 in the apo state is extended from the ligand binding pocket of the ERαLBD, and its position moves to the agonist or antagonist position by ligand binding. To assess whether the electric charge on H12 was influenced by such a conformational change, we analyzed the Mulliken charge of each residue fragment in ERαLBD by the FMO method. We confirmed that fragment charges given by Mulliken atomic charges were not qualitatively different from those of natural population atomic net charges42,43 for the model system including a ligand and 125 amino acid resides surrounding the ligand, H12, and the charge clamp lysines. (See the validation in the Supporting Information, section B.) The FMO calculations for ERαLBD−ligand complexes were performed using structures of the ER-all model as the agonist/antagonist conformations and structures of the ER-main and ER-H12 models as hypothetical structures of the apo state (H12extended conformation).

are strong and the others are weak. The strong negative ESP side of H12 faces toward Lys529/Lys362 (in the agonist/ antagonist conformation), which has strongly positive ESP, and both strongly interact with each other by the electrostatic force. In this interaction, Lys529 and Lys362 both play crucial roles as “charge clamps” to fix the positions of H12 in the agonist- and antagonist-bound states, respectively. Although the other side of H12 also has a negative potential, it faces toward Asp351/ Glu380 (in the agonist/antagonist conformation) having strongly negative ESP, since the potential of this side of H12 is weakly negative ESP. Moreover, as seen in section 3.1.2, Asp351/Glu380 (in the agonist/antagonist conformation) forms a hydrogen bond with the Leu540/Tyr537 of H12, which also contributes to fix the position of H12. It is suggested that the hydrogen bond formation between Asp351/Glu380 and Leu540/Tyr537 occurs prior to the electrostatic repulsion between the strongly negative potential spot of Asp351/Glu380 and the weakly negative potential side of H12, in the agonist/ antagonist-bound state. 5002



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Figure 8. Visualizations of the charge differences through the conformational changes of H12 (Δqi) of (a) the ERαLBD−EST (agonist) complex and (b) the ERαLBD−OHT (antagonist) complex. In parts a and b, the positive and negative charge differences are shown in blue and red, respectively. The Cα backbones are represented by solid ribbons; the ligands and water molecules are shown by ball-and-stick models.

indicating positive values for the charge difference. When these regions are located close together, the interfacial sides are preferably positive. We supposed that the electronic polarization would be changeable so as to relax the electrostatic repulsion caused by the negatively charged ER-main and ERH12. In fact, residue fragments on the interfacial sides of H3, H4, H11, and H12 indicated positive charge differences. These include Asp351 (+0.11e) and Glu380 (+0.09e) of H3; Cys381 (+0.05e) of H4; Tyr526 (+0.06e) of H11; and Tyr537 (+0.03e), Leu540 (+0.02e), Met543 (+0.05e), and Ala546 (+0.06e) of H12, as seen in Figure 7a. In contrast, the opposite sides of the α-helical structures in such residues are colored in red, indicating negative charge differences, e.g., Asp358 (−0.02e) of H3 and Leu541 (−0.07e) and Asp545 (−0.04e) of H12. These results indicate that the residues on the interfacial sides donate negative charges to the residues on the opposite sides, and such changes will suppress the electrostatic repulsions derived from each negative charge of ER-main and ER-H12. We noticed that the value of the charge difference of Lys529 was almost zero (+0.002e), although Lys529 is located on the interfacial side in the agonist-bound state, as seen in Figures 7a and 8a. That is, Lys529 attracts the negatively charged H12 without losing its positive charge as well as in the apo state. The microscopic mechanism is then speculated as follows: The charge difference of Asp351 was the largest positive value (+0.11e) among all of the residues (Figure 7a). It is inferred that a negative charge (electron) is transferred from Asp351 to Lys529 through surrounding residues. For example, Leu541 in the vicinity of Asp351 is located adjacent to Lys529 and interacts with both Asp351 and Lys529 (−11.91 and −6.26 kcal/mol), as seen in the IFIE analysis (Table 1a). The negative charge difference of Leu541, the residue adjacent to Lys529, was larger (−0.07e) than that of the other residues that are accessible to Lys529. Since an absolute value for the charge difference of Leu541 was smaller than that of Asp351, the negative charge of Asp351 might be transferred to Lys529. The negative charge difference caused by a CT to Lys529 is counteracted by the positive charge difference of Lys529 located at the interface of the H12-binding site; therefore, Lys529 is characteristic of neither the residue at the interface nor the charge acceptor from Asp351. We speculate that

The construction and optimization procedures of the ERmain and ER-H12 models were described in section 3.2. Differently, however, the ER-main model comprises 229 residues (residue nos. 307−535) of ERαLBD, the ligand (EST/OHT), and a water molecule, whereas the ER-H12 model is composed of 17 residues (residue nos. 531−547) of ERαLBD. Here, the ER-main and ER-H12 models have five overlapping residues (residue nos. 531−535) on the cleavage site so as to reduce the influences of truncated structures on electric charges. The net charges and the charge differences arising through the conformational changes of H12 were then obtained by using these constructed model structures. To investigate the CT, which is caused by the conformational change of H12 from the extended position to the agonist/antagonist position, the Mulliken charge of each residue fragment (qi) was calculated at the FMO-MP2/6-31G* level, and the charge difference (Δqi) was calculated as follows: Δqi = qi(ER‐all) − [qi(ER‐main) + qi(ER‐H12)]

(4)

where qi(ER-all), qi(ER-main), and qi(ER-H12) are the Mulliken charges of the ith fragment/molecule in the whole ERαLBD−ligand complex (ER-all), the ERαLBD−ligand complex without the H12 (ER-main), and the only H12 (ERH12) of ERαLBD, respectively. Since there are five overlapping residues between the ER-main and ER-H12 models, the charge differences were estimated by Δqi = qi(ER-all) − qi(ER-main) for residue nos. 307−532 of ERαLBD, the ligand and water, and Δqi = qi(ER-all) − qi(ER-H12) for residue nos. 533−547 of ERαLBD. When Δqi shows a negative value, an electron is transferred from the surrounding residues to the ith residue fragment. On the contrary, a positive value of Δqi indicates that an electron is transferred from the ith residue fragment to the surrounding residues. Figure 7 shows the Mulliken fragment charges (qi) and the charge differences through conformational change (Δqi). In parts a and b of Figure 8, color is used to depict the positions of the residues having a large magnitude of charge differences (Δqi), with blue and red indicating positive and negative changes, respectively. In the agonist-bound state, Figure 8a shows the presence of blue interfacial regions between ER-main and ER-H12, 5003



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among hNRs.18−23 The previous studies about Lys529 have reported, for example, that a double mutant (K529E + K531E)18 increases affinities to antiestrogens (antagonist, e.g., OHT) and inhibitory activities to agonist transcription, whereas affinities to estrogens (agonist, e.g., EST) are reduced. These results suggest that Lys529 or Lys531 is certainly involved in discrimination between agonist and antagonist, and also in the change of H12 position. The results of this double mutant and our calculations lead to the speculation that the electrostatic interaction between Lys529 and H12 will change from attractive to repulsive by a mutation of Lys to Glu; then, H12 could not be tightly fixed in the agonist position and the transcriptional activity would be altered. It was also reported that a mutation of D351K or D351L19 in hERα causes the disappearance of a hydrogen bond between the side chain of Asp351 in H3 and the main chain of Leu540 in H12, and as a consequence would probably lead to the disorder of H12 in the agonist position. This mutation analysis suggested the importance of the hydrogen bond to fix H12 in the agonist position. Our results of the IFIE value between Asp351 and Leu540, which showed the attractive interaction, would provide evidence of this suggestion.19 In the antagonist-bound state, H12 might interfere with coactivator binding. As shown in Figure 2a, the transcriptional coactivator is bound to its binding surface of hERα after the agonist binding and enhances transcription. When an antagonist is bound to hERα, H12 covers the coactivator binding surface (Figure 2b), and the transcriptional coactivator is prevented from binding to hERα. As a result, transcription is suppressed. Actually, the X-ray structures in the agonist- and antagonistbound states show that the LXXML motif of H12 in place of the LXXLL motif of the coactivator is bound to the particular coactivator binding surface of hERα.13,14 We numerically confirmed the presence of a hydrogen bond between CO in the peptide bond of Met543 on the inner surface of H12 and a side chain of Lys362 in the coactivator binding site (H3) by the IFIE analysis of FMO calculations. This hydrogen bond causes the LXXML motif in H12 to mimic the NR binding motif LXXLL of the coactivator.7,9,13,14 Our results will thus support the results by X-ray crystal structural analysis. In addition, we also confirmed that Lys362 plays the role of a charge clamp and strongly interacts with H12; therefore, the position of H12 is stable and the LXXML motif of H12 will not be easily replaced by the LXXLL motif of the coactivator. As a result, transcription is suppressed by interfering with the coactivator binding. The antagonists of OHT and raloxifene, which have large volumes, are shown to interact with Asp351 by their X-ray crystal structures. The IFIEs of Asp351 quantitatively support these experimental results, suggesting that Asp351 has a role as an anchor of the antagonist.23 Asp351, which interacts with H12 in the agonist-bound state, strongly binds to the antiestrogenic side chain of the antagonist with a large volume, and thus it becomes impossible to interact with H12 in the antagonist-bound state. Moreover, protrusion of the antagonist causes steric hindrance with the beginning region of H12. Consequently, the released H12 is located in the antagonist position. On the other hand, it is reported that the D351Y or D351E mutant exhibits an estrogenic response to OHT. We favor the speculation described in previous studies that, in the D351Y/D351E mutant, Tyr351/Glu351 cannot interact with the antagonist and H12 may be stable in the agonist

Asp351 contributes to the charge differences of both Leu541 and Lys529 through a conformational change from the apo state to the agonist-bound state. In the antagonist-bound state, Figure 8b shows the presence of interfacial sides of ER-main and ER-H12 in blue, indicating positive charge differences. These include Val355 (+0.05e), Ile358 (+0.03e), and Asn359 (+0.03e) of H3; Leu372 (+0.04e), Val376 (+0.04e), and Glu380 (+0.08e) of H4; and Tyr537 (+0.05e), Leu540 (+0.03e), Met543 (+0.03e), and Asp545 (+0.04e) of H12, as seen in Figure 7b. The opposite sides of the α-helical structures were in red, and thus negatively charged, e.g., Trp360 (−0.03e) of H3 and Asp374 (−0.03e) of H4. These results indicate that negative charges of the residues on the interfacial sides were transferred to the opposite sides so as to relax the electrostatic repulsion between ER-main and ERH12. Nevertheless, Lys362 is located at the interface and its charge difference was negative (−0.04e), as seen in Figures 7b and 8b. Although the positive charge of Lys362 decreased from +0.89e to +0.85e, Lys362 still retains an attractive interaction with H12. This suggests that Lys362 accepts a negative charge from the surrounding residues such as Met543 through the hydrogen bond between these two residues described in section 3.1.2. Here, as mentioned in the same section, an oxygen atom in the peptide bond of Met543 that is a member of the hydrogen bond is treated as an atom of the Leu544 fragment owing to the fragmentation procedures employed in the FMO method. The charge difference of the Leu544 fragment was expected to be positive, but almost unchanged (+0.01e), because Leu544 is located at the interface of ER-H12 and is inferred to donate a negative charge to Lys362 through the oxygen atom originally in Met543. Instead of Leu544, therefore, the charge differences of Met543 and Asp545 were positive. From these results, it is speculated that negative charges of Met543 and Asp545 were transferred to Lys362 through the surrounding residues (e.g., Leu544).

4. DISCUSSION In this section, the importance of the key residues shown in section 3 will be discussed with respect to X-ray crystal structures7−15,17 and previous studies of mutations in hERα.17,16−23 Further, we will also provide insight into the roles of these residues in other hNRs based on sequence conservation. Among many hNRs, sequences, tertiary structures, and functions of their LBDs are similar to each other, and the structural difference of H12 in hERαLBD between agonist- and antagonist-bound states, as seen in Figure 2a and b, is also observed to be identical in other hNRs.7 Hence, it is possible to infer the key residues to fix the position of H12 in hNRs from that in hERα. Finally, we discuss effects of solvation and screening in a biological system. 4.1. Comparison with Experimental Data of hERα. The sequence alignments of H3, H4/H5, H12, and the loop between H11 and H12 of some hNRs (hERα, hERβ, hERRγ, hAR, hPR, hPPARγ, hRXRα, and hVDR) are shown in Figure 1. It is confirmed that the LBDs in hNRs have certain highly conserved residues, which correspond to key residues strongly interacting with H12 in hERαLBD from IFIE analysis. In fact, there are some previous studies17−23 suggesting that these key residues are important for transcriptional activation. In the agonist-bound state, transcriptional activation is lost by mutation of either Lys529 or Asp351, which is highly conserved 5004



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Figure 9. Positions of the H12 (yellow) and charge clamps of Lys362 and Lys529 (light blue) of the ERαLBD−EST complex from the MD snapshot are shown in part a. The ESP map of only the ERαLBD−EST complex in vacuo (b) and the explicit water model (c) is represented on an isosurface at an electron density of 0.001e/au3. The negative and positive ESPs are shown in red and blue, respectively.

position.20−22 Thus, this speculation supports our computational result, which suggests that Asp351 plays a key role in antagonist binding and may also be related to the position of H12. 4.2. Conservation Analysis for hNRs. It is strongly suggested that highly conserved residues in some hNRs are important to control transcriptional activity. Such evidence includes the relationship among the intramolecular interactions obtained by the FMO calculations of hERα, the change of transcriptional activation by the mutation analysis of hERα, and the similar positions of H12 confirmed by X-ray crystal structures in hERα and other hNRs. The highly conserved anionic residue in the H12s of hNRs, which have charges of −1e to −3e, is Glu (Glu542 in hERα). In addition, the Lys located in the loop between H11 and H12 (Lys529 in hERα) and the Lys in H3 (Lys362 in hERα) are also highly conserved. Lys529 and Lys362 were proposed to act as the “charge clamp” of H12 in the agonist- and antagonist-bound states, respectively, by our ESP analysis. It follows from these results that the highly conserved lysines interact with the highly conserved Glu in H12 and are critical to fixing each of the agonist and antagonist positions of H12, not only in hERα but also in other hNRs. In the antagonist-bound state, the hydrogen bond between the highly conserved Lys in H3 (Lys362 in hERα) and CO atoms in a peptide bond of amino acid residue in H12 (Met543 in hERα) strengthens the fixing of H12 in the antagonist position. Since this hydrogen bond is similar to that between the Lys (Lys284 in RXRα) and CO atoms in the backbone of the LXXLL motif of the coactivator (Leu636 in steroid receptor coactivator-1),37 the hydrogen bond derived from the Lys in H3 is important for the H12 position in the antagonist-bound state and the coactivator binding in the agonist-bound state. In addition, other highly conserved residues are Asp in H3 (Asp351 in hERα) and Leu at the middle of H12 (Leu540 in hERα). They form a hydrogen bond in hERα that plays a key role in fixing H12 in the agonist position. Thus, we predict that the interaction between Asp in H3 and Leu in H12 also contributes to fixing H12 in the agonist position in other hNRs. Furthermore, this Asp in H3 is involved in the stabilization of ligand binding in the hERα−antagonist complex. It is reasonable to speculate that this Asp might also contribute to the stabilization of the antagonist binding in other hNRs.

These experimental data are in good agreement with our results of FMO calculations. Therefore, our results support that Asp351, Lys362, Lys529, and Glu542 in hERα, which are highly conserved residues in hNRs, will play key roles in fixing H12 in the agonist/antagonist position and will also be important in the regulation of transcriptional activation in hNRs. 4.3. Effects of Solvation and Screening. In this paper, the results obtained from quantum calculations of only X-ray crystal structures in vacuo represent the first step toward the understanding of the mechanism to fix H12 in the agonist/ antagonist position. However, we have also recognized the importance of solvent, screening, and fluctuation effects for biomolecular systems.45−49 Preliminary calculations at the FMO-MP2/6-31G* levels were performed by using the explicit water model for the ERαLBD−EST complex immersed with a 4 Å thick water from a MD snapshot (Figure S1, Supporting Information) to discuss the solvent and screening effects. (See the Supporting Information, section A.) We have thus confirmed that the IFIE values between H12 and the key residues are almost unchanged in the absence or presence of solvent (Figure S2 and Table S1, Supporting Information). On the other hand, the Mulliken atomic charges are affected by explicit water molecules and the CTs from the ERαLBD−EST complex to water molecules occur (Figure S3, Supporting Information). Here, the positions of Lys362, Lys529, and H12 are shown in Figure 9a and the ESPs of the ERαLBD−EST complex from the 2 ns MD snapshot in vacuo and in the explicit water model are depicted in Figure 9b and c, respectively. The isosurfaces of ESP were generated at an electron density of 0.001e/au3 and are shown in Figure 9. It is clear that the ESPs of the ERαLBD−EST complex depend on its own total charges. Most ESPs in vacuo take negative values except for charge clamps of Lys363 and Lys529. As compared with the ESPs in vacuo, the ESPs in the explicit water model are shifted toward neutral (zero) values from the negative ones by the CTs from protein to water molecules and clearly reflect the character of each amino acid residue (e.g., Lys362 and Lys529). Therefore, the incorporation of the solvent effect by using the explicit water model may be important in order to discuss ESPs in a biomolecular system. We consider that there is room for improvement in evaluation of interaction energies, because the bare IFIE values tend to overestimate the electrostatic interactions between the electrically charged fragment pair. Such overestimated inter5005



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bonds with Tyr537 and Met543, respectively, where the LXXML motif of H12 mimics the interaction between a helical LXXLL motif of coactivator peptide and the hERα−agonist complex. These results support the previously reported results7−23 with respect to the stabilization of the agonist/ antagonist position of H12 by using X-ray crystal structure analysis and mutation experiments. For example, the mutation of Lys529 or Asp35118−23 in the ERαLBD−EST complex is inferred to diminish the attractive interaction between H12 and Lys529/Asp351, and consequently, H12 will not be located in the agonist position. Thus, these residues are important to regulate transcription and our results are in agreement with the mutation experiments. Furthermore, the highly conserved Asp351 forms a hydrogen bond in hERα that plays a key role in fixing H12 in the agonist position, while it is involved in the stabilization of ligand binding in the hER−antagonist complex. The electric charges of residue fragments at the interface between H12 and the other part of hERα approach approximately neutral upon the structural change from the apo conformation (in the extended state of H12) to the agonist/antagonist binding conformation to suppress the electrostatic repulsion between each part, having negative charges. The charge differences of Lys529 and Lys362 having positive charges, however, show a different tendency from that of other residues located at the interface. We have proposed that these results would be caused by CTs from Asp351 to Lys529 in the agonist-bound state, and from two residues, Met543 and Asp545, to Lys362 in the antagonist-bound state, while the attractive interaction with H12 is still retained. Here, we might also have to employ a configuration analysis for the fragment interaction (CAFI) method32,44 to analyze the CT interaction energies associated with the hydrogen bonds between residues of H12 and those of the other part of hERα, where the Mulliken atomic charges and their charge differences alone are insufficient to discuss the CTs in detail. In the present paper, we have shown the key residues that fix H12 in the agonist and antagonist positions by the IFIE, ESP, and atomic charge analyses using FMO calculations under the gas phase. These residues will be clearly essential to maintain the agonist/antagonist conformations in hERα. It is not clear, however, how the ligand directly regulates the agonist or antagonist positions of H12. We will consider that interactions between the ligand and H12 will reveal how the position of H12 is controlled by the type of the ligand in future works. Furthermore, the calculation using X-ray crystal structures discussed in this work may not perfectly represent the biological circumstances, because the X-ray crystal structure and the biomolecular structure with structural fluctuations are obtained under different conditions (e.g., with and without solvent water). We have examined and discussed the solvent and screening effects through preliminary calculations employing explicit and implicit water models. The details on the effects of solvation, screening, and structural fluctuation will be reported elsewhere. Furthermore, future work should examine IFIE analysis under the influence of structural fluctuation in the biological circumstance.45,46 In addition, we would also gain insight into the roles of the highly conserved residues in other hNRs based on sequence conservation and experimental data of hERα. Taken together, we propose that the charge clamp of highly conserved lysine in the loop between H11 and H12 plays key roles in the mechanism to fix H12 having highly conserved glutamic acid in the agonist position, while the conserved lysine

action energies would be reduced by considering the screening effect. To estimate interaction energies with the screening effect in the FMO method, analysis of the statistically corrected IFIE (SCIFIE)47 is useful in Table S2. (See the Supporting Information, section A.) In addition, we performed the FMO calculations at the MP2/6-31G level with the implicit solvent model based on the Poisson−Boltzmann (PB) equation48 for the 125-residue model of ERαLBD complexed with the ligand (Figure S4, Supporting Information), as seen in the Supporting Information, section A. Here, the IFIEs in solvation can be evaluated with or without solvent screening by induced surface charges around the fragment pair.49 The IFIE with solvent screening is electrostatically reduced as compared to that without solvent screening, which is not so different from the IFIE obtained in vacuo (Table S3, Supporting Information). For example, in the agonist-bound state (ERαLBD−EST complex), IFIE-sum of Lys529 with solvent screening is reduced by ca. 20% as compared to that without solvent screening. However, the behaviors of IFIEs with solvent screening are in qualitative agreement with those without solvent screening. Thus, we have concluded that the importance of the key residues (e.g., Lys362 and Lys529) is unchanged irrespective of the absence or presence of solvent. More detailed studies of the effects of solvation and screening will be reported elsewhere.

5. CONCLUSION The ab initio FMO calculations were performed for the hERαLBD and EST/OHT complexes with different positions of H12 to clarify the mechanism of fixing H12 in the agonist/ antagonist positions after the ligand binding. The intramolecular interactions between H12 and the other part of hERαLBD were revealed in detail by using the IFIE, ESP, and atomic charge analyses based on the FMO method. The results of IFIE and ESP analyses have revealed that the position of H12 is determined by the intramolecular interactions originating from strong electrostatic interactions and hydrogen bonds. The highly conserved Lys529 and Lys362 are found to have strong attractive interactions with the anionic residues (Asp538, highly conserved Glu542, and Asp545) of H12 in the agonist and antagonist positions, respectively. Moreover, the complementation of ESPs has also visually revealed that the highly conserved Lys529 and Lys362 play the roles of charge clamps to fix H12 in the agonist and antagonist positions, respectively. In hERα, the spots with strong positive ESPs (Lys362 and Lys529) and negative ones (Asp351 and Glu380) at the interface between H12 and the other part of hERα are common to both the agonist- and antagonist-bound states, whereas, in H12, the overall ESP surfaces (including Asp538, highly conserved Glu542, and Asp545) in both agonist and antagonist conformations have mostly negative values. Therefore, it is clear that highly conserved Lys529 and Lys362 electrostatically interact with H12 to stabilize the agonist and antagonist conformations. Additionally, the H12 position is more firmly fixed by intramolecular interactions between the neutral residues of H12 and the other part of hERα. In the agonist-bound state, Asp348 and highly conserved Asp351 form the hydrogen bonds with Tyr537 and highly conserved Leu540, respectively. Trp383 located at the interface between H12 and the other part of hERα takes part in anchoring H12 in the agonist position by the polarization of the main chain of Met543. In the antagonist-bound state, on the other hand, Glu380 and highly conserved Lys362 also fix H12 by hydrogen 5006



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in H3 plays the role of charge clamp to fix H12 in the antagonist position. In addition, the hydrogen bond between the neutral residue of H12 having the LXXML motif and the highly conserved aspartic acid in H3 strengthens the fixing of H12 in the agonist positions, whereas the neutral residue of H12 forms the hydrogen bond with the highly conserved lysine in the H3 to fix H12 in the antagonist position. In the antagonist-bound state, the highly conserved Asp351 without contributing to the fixing of H12 position is involved in the stabilization of antagonist binding. The observations obtained from these FMO calculations will facilitate our better understanding of the transcription mechanisms of hERα and hNRs and will provide useful information for controlling the H12 position for regulation of transcription and drug discovery for diseases associated with the hNRs.

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(3) Berkenstam, A.; Gustafsson, J.-Å. Nuclear Receptors and Their Relevance to Diseases Related to Lipid Metabolism. Curr. Opin. Pharmacol. 2005, 5, 171−176. (4) Moore, J. T.; Collins, J. L.; Pearce, K. H. The Nuclear Receptor Superfamily and Drug Discovery. Chem. Med. Chem. 2006, 1, 504− 523. (5) Kaminuma, T. Pathways and Networks of Nuclear Receptors and Modeling of Syndrome X. Chem-Bio Inf. J. 2003, 3, 130−156. (6) Aranda, A.; Pascual, A. Nuclear Hormone Receptors and Gene Expression. Physiol. Rev. 2001, 81, 1269−1304. (7) Bourguet, W.; Germain, P.; Gronemeyer, H. Nuclear Receptor Ligand-binding Domains: Three-Dimensional Structures, Molecular Interactions and Pharmacological Implications. Trends Pharmacol. Sci. 2000, 21, 381−388. (8) Tanenbaum, D. M.; Wsng, Y.; Williams, S. P.; Sigler, P. B. Crystallographic Comparison of the Estrogen and Progesterone Receptor’s Ligand Binding Domains. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5998−6003. (9) Wärnmark, A.; Treuter, E.; Gustafsson, J.-Å.; Hubbard, R. E.; Brzozowski, A. M.; Pike, A. C. W. Interaction of Transcriptional Intermediary Factor 2 Nuclear Receptor Box Peptides with the Coactivator Binding Site of Estrogen Receptor α. J. Biol. Chem. 2002, 277, 21862−21868. (10) 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, 927−937. (11) Renaud, J. P.; Rochel, N.; Ruff, M.; Vivat, V.; Chambon, P.; Gronemeyer, H.; Moras, D. Crystal Structure of the RAR-γ Ligandbinding Domain Bound to All-trans Retinoic Acid. Nature 1995, 378, 681−689. (12) Wagner, R. L.; Apriletti, J. W.; McGrath, M. E.; West, B. L.; Baxter, J. D.; Fletterick, R. J. A Atructural Role for Hormone in the Thyroid Hormone Receptor. Nature 1995, 378, 690−697. (13) Pike, A. C. W.; Brzozowski, A. M.; Hubbard, R. E. A Structural Biologist’s View of the Oestrogen Receptor. J. Steroid Biochem. Mol. Biol. 2000, 74, 261−268. (14) Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.; Rosenfeldk, 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, 137− 143. (15) Gampe, R. T.; Montana, V. G.; Lambert, M. H.; Miller, A. B.; Bledsoe, R. K.; Milburn, M. V.; Kliewer, S. A.; Willson, T. M.; Xu, H. E. Asymmetry in the PPARγ/RXRα Crystal Structure Reveals the Molecular Basis of Heterodimerization among Nuclear Receptors. Mol. Cell 2000, 5, 545−555. (16) Herynk, M. H.; Fuqua, S. A. W. Estrogen Receptor Mutations in Human Disease. Endocr. Rev. 2004, 25, 869−898. (17) Gangloff, M.; Ruff, M.; Eiler, S.; Duclaud, S.; Wurtz, J. M.; Moras, D. Crystal Structure of a Mutant hERα Ligand-binding Domain Reveals Key Structural Features for the Mechanism of Partial Agonism. J. Biol. Chem. 2001, 276, 15059−15065. (18) Pakdel, F.; Katzenellenbogen, B. S. Human Estrogen Receptor Mutants with Altered Estrogen and Antiestrogen Ligand Discrimination. J. Biol. Chem. 1992, 267, 3429−3437. (19) Kim, J. H.; Lee, M. H.; Kim, B. J.; Kim, J. H.; Han, S. J.; Kim, H. Y.; Stallcup, M. R. Role of Aspartate 351 in Transactivation and Active Conformation of Estrogen Receptor α. J. Mol. Endocrinol. 2005, 35, 449−464. (20) Levenson, A. S.; Jordan, V. C. The Key to the Antiestrogenic Mechanism of Raloxifene Is Amino Acid 351 (Aspartate) in the Estrogen Receptor. Cancer Res. 1998, 58, 1872−1875. (21) Webb, P.; Nguyen, P.; Valentine, C.; Weatherman, R. V.; Scanlan, T. S.; Kushner, P. J. An Antiestrogen-responsive Estrogen Receptor-a Mutant (D351Y) Shows Weak AF-2 Activity in the Presence of Tamoxifen. J. Biol. Chem. 2000, 275, 37552−37558. (22) Levenson, A. S.; MacGregor Schafer, J. I.; Bentrem, D. J.; Pease, K. M.; Jordan, V. C. Control of the Estrogen-like Actions of the

ASSOCIATED CONTENT

S Supporting Information *

Solvation and screening effects are examined and discussed in section A. A comparison between Mulliken and natural population atomic charges of the model system is shown in section B. Details on force field parameters of EST and OHT are described in sections C and D, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-45-503-9551. Fax: +81-45-503-9432. E-mail: [email protected]. Present Address ⊥

(C.W.) RIKEN Center for Life Science Technologies, Drug Discovery Computational Chemistry Platform Unit, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, 2300045, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors express their appreciation for technical assistance and useful comments by Dr. Yoshio Okiyama, Dr. Hirofumi Watanabe, Dr. Tatsuya Nakano, and Dr. Zhenxia Zhu. This research was performed within the “Supporting activities for female researchers” project supported by the Work-life Balance Office of Tokai University. A part of this research was carried out within the “Core Research for Evolutional Science and Technology” project of the Japan Science and Technology Agency (JST-CREST) and the “Research and Development of Innovative Simulation Software” project supported by Research and Development for Next-generation Information Technology of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The computations were partially performed at the Research Center for Computational Science, Okazaki, Japan.

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Charge Clamps of Lysines and Hydrogen Bonds Play Key Roles in the

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