Fragment Molecular Orbital Study of the Interaction between Sarco

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Fragment Molecular Orbital Study of the Interaction Between Sarco/Endoplasmic Reticulum Ca -ATPase and Its Inhibitor Thapsigargin Toward Anti-Malarial Development 2+

Takeshi Ishikawa, Satoshi Mizuta, Osamu Kaneko, and Kazuhide Yahata J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04509 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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The Journal of Physical Chemistry

Fragment Molecular Orbital Study of the Interaction between Sarco/Endoplasmic Reticulum Ca2+-ATPase and its Inhibitor Thapsigargin toward Anti-Malarial Development

Takeshi Ishikawa1,2,*, Satoshi Mizuta1, Osamu Kaneko2,3, and Kazuhide Yahata3

1

Department of Molecular Microbiology and Immunology, Graduate School of

Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 2

Leading Program, Graduate School of Biomedical Sciences, Nagasaki University,

1-12-4 Sakamoto, Nagasaki 852-8523, Japan 3

Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki

University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Plasmodium falciparum, the causative agent of malignant malaria, is insensitive to thapsigargin (TG), a well-known inhibitor of the human sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To understand the key factor causing the difference of the sensitivity, the molecular interaction of TG and each SERCA was analyzed by the fragment molecular orbital (FMO) method. While the major component of the interaction energy was the non-polar interaction, the major difference in the molecular interaction arose from the polar interaction, namely, the hydrogen bonding interaction with a hydroxyl group of TG. Additionally, we successfully confirmed these FMO calculation results by measuring the inhibitory activity of a synthesized TG derivative. Our calculations and experiments indicated that, by replacing the hydroxyl group of TG with another functional group, the sensitivities of TG to human and P. falciparum SERCAs can be reversed. This study provides important information to develop anti-malarial compounds targeting P. falciparum SERCA.

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1. INTRODUCTION Identification of a lead compound by screening a chemical compound library is one of the most important processes of drug discovery. Since for such screening it is essential to rapidly predict the binding free energy of small molecules with a target protein, docking simulation is routinely used in many drug discovery projects.1−3 Optimization of the lead compound is also important in the drug discovery process. However, the computational methods for such a process have not been well established compared with the screening process. As a result, the optimization process is generally carried out by trial and error by synthetic organic chemists in an expensive and time-consuming manner. The application of an appropriate strategy of molecular design using computational chemistry would make this optimization process more efficient. For the computational design of a drug molecule, accurate evaluation of the local interaction energy between the lead compound and the target protein is important as well as prediction of the total binding free energy. For example, information about the local interaction can clarify a site of the compound essential for binding to a protein. Ab initio quantum chemical calculation, in which the reliable electronic structure is obtained by solving the Schrödinger equation of the electrons, is suitable for evaluation of such local interaction energy. In particular, the fragment molecular orbital (FMO) method4−9 is one of the most promising approaches because quantum chemical calculations of large molecules (e.g., proteins) are available with less computational cost by dividing the target molecule into small fragments.10,11 Additionally, the FMO method can calculate the interaction energies of each amino acid residue or each part of the molecule.12 Such interaction energies are generally called inter fragment interaction energies (IFIEs) or pair interaction energies (PIEs). Some studies have reported the

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molecular design or optimization of a lead compound using the IFIE obtained from FMO calculations.13−17 For example, Sriwilaijaroen et al.14 reported the design of several novel inhibitors against influenza neuraminidase based on the existing drugs oseltamivir and zanamivir. However, such studies have been limited to only a few diseases. To clearly demonstrate the potential of the FMO method in the optimization process of the drug discovery, this method should be widely applied to various diseases. Malaria is one of the most serious infectious diseases caused by intracellular protozoan parasites, including the deadly species Plasmodium falciparum. Because of the lack of effective vaccines and the emergence of parasites resistant to the currently available drugs, there is an urgent need to develop new anti-malarial compounds. Recently, we reported a new technology to monitor the change of the cytosolic Ca2+ concentration using transgenic P. falciparum expressing the Ca2+ sensor yellow cameleon-Nano.18 In malaria parasites, Ca2+ acts as a secondary messenger for intracellular signaling, and its concentration is maintained at a low level in the cytosol.19 The endoplasmic reticulum, a putative intracellular Ca2+ storage compartment in P. falciparum,20

is

known

to

regulate

the

Ca2+

concentration

through

the

sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump.21,22 Because SERCA inhibitors can prevent the parasite growth, P. falciparum SERCA (PfSERCA) is considered to be a potential target of anti-malarial drug development. Several inhibitors of SERCA have already been discovered and used to elucidate the mechanism of Ca2+ transport.23 Thapsigargin (TG) is the inhibitor most extensively used as a pharmacological tool in basic research. TG is a sesquiterpene lactone found in the roots of Thapsia garganica,24 which induces the release of intracellularly stored Ca2+ without hydrolysis of inositol phospholipids via the inhibition of mammalian 4


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SERCAs, including that of humans (huSERCA).25 Many X-ray structures of the complex between SERCA and TG have been reported (almost all of them used rabbit SERCA (raSERCA)).26−35 However, our Ca2+ monitoring technology with the yellow cameleon-Nano sensor revealed that PfSERCA was insensitive to TG and the shape of its binding pocket for TG was different from that of huSERCA.18 Thus, we propose that the modification of TG would yield compounds with the opposite sensitivity, i.e., effective against P. falciparum and ineffective against humans. In this study, we aim to identify key factors responsible for the differential activity of TG on huSERCA and PfSERCA using the FMO method. Such information will enable us to design new anti-malarial compounds from TG. This study also illustrates the potential of the FMO method for the optimization process in general drug discovery.

2. MATERIALS AND METHODS 2.1 Theoretical background of the interaction analysis with the FMO method In the FMO method, a target molecule is divided into small fragments and the total property is approximately evaluated by monomer and dimer calculations of the fragments.4,5 For example, the total energy at the Hartree-Fock (HF) level of theory is calculated by the following equation: = ′ + ∆ ,

(1)

where and are the indices of the fragment, ′ is the internal energy of the fragment, and ∆ is the interaction energy between the two fragments.12 This

interaction energy is generally called IFIE. By additionally applying the second-order Møller-Plesset perturbation (MP2) theory, the IFIE including the electron correlation 5



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can be obtained from the following equation:36−38 ∆ = ∆ + ∆ ,

(2)

where the second term is the correlation contribution to the IFIE, which is obtained from the MP2 calculations of the monomer and dimer. The HF calculation mainly includes the electrostatic and charge-transfer interactions, resulting in the value of ∆ reflecting the polar interaction energy. On the other hand, the value of ∆ is considered to be the non-polar interaction

energy because the electron correlation is mainly caused by the dispersion interaction or van der Waals interaction. In typical FMO calculations of a protein, amino acid residues are treated as single fragments. Thus, we can separately calculate the polar and non-polar interaction energies of each amino acid residue, which can be used for detailed analysis of the molecular interaction between a protein and a small molecule. 2.2 Modeling of the complex structure Modeller9.1439 was used for the sequence alignment and structure modeling. Five structures were generated in each homology modeling, and one structure was selected with consideration of the structural reliability of the TG binding site. To perform the FMO calculations, four peptide chains around the TG binding site were picked up and their N- and C-terminals were capped with −COCH3 and −NHCH3, respectively. Finally, TG was placed at the binding site and classical energy minimization was performed using the AMBER99SB40 and GAFF41 force fields. 2.3 FMO calculation To analyze the molecular interaction, the FMO calculations at the MP2 level of theory were carried out employing the cc-pVDZ basis set.42 To reduce the computational cost, the resolution of the identity approximation43 was adopted, where 6


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the auxiliary basis set developed by Weigent et al.44 was used. Amino acid residues were treated as a single fragment, and TG was divided into four fragments (Frag A, B, C, and D) as shown in Figure 1. All the FMO calculations were performed in vacuum condition. In this study, PAICS45 was used for the FMO calculations. 2.4 Preparation of TG-acetate From the FMO calculations, we identified key factors causing the difference of TG sensitivity between huSERCA and PfSERCA: The major difference was the hydrogen bonding interaction with a hydroxyl group of TG. Therefore, we tentatively designed and synthesized a TG derivative with its hydroxyl group protected to examine the TG sensitivities to huSERCA and PfSERCA. This derivative is named TG-acetate. The reaction of TG with acetic anhydride in the presence of CuSO4 was performed, affording the corresponding product, TG-acetate in 77% yield. The structure was identified by NMR and HRMS spectroscopic analyses. Details of the organic synthesis are given in Scheme S1. 2.5 Cell growth inhibition assay in human cells and P. falciparum To measure the TG and TG-acetate activities against human cells and P. falciparum, embryonic kidney cells (FreeStyle 293 cells; Thermo Fisher Scientific, Waltham, MA, USA) and P. falciparum Dd2/YC-Nano50 parasites18 were used. Briefly, FreeStyle 293 cells were cultured in accordance with the manufacture’s instructions and plated at 2 × 104 cells per well in a 96-well plate (Corning, New York, NY, USA) a day before drug administration. TG and TG-acetate were diluted for dose-response at 1:5 serial dilutions, mixed with the cells, and incubated for 48 h. Cell viability was determined using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and the absorbance at 450 nm was measured with iMARK microplate absorbance reader 7



(Bio-Rad, Hercules, CA, USA). A P. falciparum Dd2/YC-Nano50 parasites were maintained with O+ human erythrocytes and plasma with RPMI-1640 medium containing 2.5 µg/mL BSD (InvivoGen, San Diego, CA, USA). P. falciparum culture at 0.1% parasitemia and 1% hematocrit was plated in a 96-well plate with 1:5 serially diluted TG or TG-acetate. After 72 h, the parasitemia was determined by SYBR Green I assay (Lonza Ltd., Basel, Switzerland) with an ARVO MX microplate reader (Perkin Elmer, MA, USA) at 485 and 530 nm. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 6 software (GraphPad Software, Inc., CA, USA). Human erythrocytes and plasma were obtained from the Nagasaki Red Cross Blood Center and their usage was approved by the ethical committee of the Institute of Tropical Medicine, Nagasaki University.

3. RESULTS AND DISCUSSION 3.1 Analysis of the X-ray structures First, we downloaded the 21 X-ray structures of the complex between raSERCA and TG from the Protein Data Bank (PDB), for which the distance between TG and the amino acid residues was examined (i.e., the distance between the two nearest atoms was examined). The PDB codes are listed in Table S1. The average distances over the 21 X-ray structures are given in Table 1 for the 17 residues located within 4.0 Å from TG. We noted that 13 residues were non-polar amino acids: F834, I829, V263, L260, A306, I765, V769, F256, L828, L253, M838, V772, and I267. Additionally, Y837 can undertake a large non-polar interaction because it has an aromatic ring. Thus, we can assume that a major component of the interaction energy between raSERCA and TG is from the non-polar interaction. On the other hand, the remaining three residues (Q259, 8


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E255, and N768) are polar amino acids, which mainly undertake polar interaction. In particular, E255 is assumed to have a large polar interaction because it is negatively charged and the average distance from TG is sufficiently small (3.43 Å). The small standard deviations indicate the similarity of the binding structures among the 21 X-ray structures. These 17 residues are completely conserved between human and rabbit SERCAs, indicating that the molecular interaction of TG with huSERCA is similar to that with raSERCA. In PfSERCA, on the other hand, the amino acid residues at the positions equivalent to F834 and E255 of raSERCA were leucine, and those at the positions equivalent to V263, V769, L253, and M838 of raSERCA were isoleucine. Thus, these residues are assumed to cause the difference in the molecular interaction. 3.2 Model of the complex structures Three-dimensional structures of huSERCA and PfSERCA have not been reported, so we performed homology modeling using the X-ray structure of raSERCA (PDB code, 2AGV29) as a template. The sequence alignment used for the modeling is shown in Figure S1. The sequence identity of raSERCA to huSERCA was very high (96.7%), as expected, whereas that to PfSERCA was 40.8%. The similarity between raSERCA and PfSERCA is low but sufficient for homology modeling. Thus, we performed the structure modeling and obtained the three-dimensional structures of huSERCA and PfSERCA. Next, 144 residues around the TG binding site were picked up, which formed four peptide chains: 254th−275th, 288th−327th, 740th−781st, and 823rd−856th. These peptide chains corresponded to the transmembrane helices46 M3, M4, M5, and M7, respectively. The sequence alignment of these 144 residues is shown in Figure 2-A. The sequence identities of these peptide chains with the template of the homology modeling were 9



99.3% for huSERCA and 68.1% for PfSERCA, indicating that the reliability of our models around the TG binding site is sufficiently high. Energy minimization was performed after placing TG at the binding site, and the model structures of the complex were obtained (Figure 2-B). Although a systematic evaluation of the reliability of the three-dimensional models obtained from the homology modelling is generally difficult, it is useful to show the root mean square deviation (RMSD) between the template and models. The RMSD values of the main-chain atoms of the models used in this study are given in Figure S2. The RMSD values were 0.334 and 0.440 Å for huSERCA and PfSERCA, respectively, indicating that the models and template were well overlapped. These models were used for the following FMO calculations. 3.3 Molecular interaction analysis between SERCA and TG The polar and non-polar interaction energies of each amino acid residue were separately calculated by the FMO calculations. Figure 3 shows the interaction energies between amino acid residues and TG, where the HF (∆ ) and MP2 (∆ )

interaction energies are given in blue and red, respectively. Thus, we can consider that the blue values are the polar interaction energy, and the red values are the sum of the polar and non-polar interaction energies. In our previous paper, similar FMO calculations were given (Figure S5 in reference 18), where only non-polar interaction energies were presented. However, our analysis of the 21 X-ray structures suggested that the polar interaction was also important to understand the difference between human and P. falciparum. Therefore, in this study, the polar interaction is given together with the non-polar interaction. The total interaction energies of the 144 amino acid residues are also given. The MP2 total interaction energies were −138.5 and −110.2 kcal/mol for huSERCA 10


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and PfSERCA, respectively, indicating that the binding affinity of TG with huSERCA is higher than that with PfSERCA (Figure 3). This is consistent with our previous report,18 indicating that huSERCA was inhibited by TG while PfSERCA was not. The non-polar interaction is the major component for both TG−SERCA interactions because the weight of the HF total interaction energy (−37.3 and −10.3 kcal/mol, respectively) is significantly smaller than the MP2 total interaction energy. This result is also consistent with the analysis of the 21 X-ray structures (i.e., the binding site of TG mainly consists of non-polar amino acid residues). We noted that the interaction energy of TG with huSERCA E255 is particularly large. The major component of this interaction is polar interaction because the HF energy (−25.6 kcal/mol) is 83.5% of the MP2 energy (−30.6 kcal/mol). On the other hand, the interaction energies of TG with PfSERCA L263, which is located at the position equivalent to huSERCA E255, is very small (−1.7 kcal/mol). This indicates that the polar interaction of these residues is very different between human and P. falciparum. The atomic structures around these residues are shown in Figure 4. E255 makes a hydrogen bonding interaction with a hydroxyl group of TG, resulting in large polar interaction energy. Furthermore, E255 makes an ionic interaction (or salt bridge) with K252, by which the hydrogen bond with TG can be strongly fixed. These polar interactions associated with E255 have important roles in stabilizing the complex. On the other hand, such polar interactions are extremely reduced in the TG−PfSERCA interaction because L263 cannot form such hydrogen bonding and ionic interactions. This is the most significant difference in the molecular interaction of TG with huSERCA and with PfSERCA. Figure 3 presents the other differences. For example, the non-polar interaction 11



energy (i.e., difference between blue and red values) of F834 in the TG−huSERCA complex was large (−9.0 kcal/mol), while the value of L1046 in the TG−PfSERCA complex was small (−3.3 kcal/mol). This also likely participates in the difference of TG sensitivity. We also noted that the non-polar interaction of F256 was particularly large, indicating that this residue was also important for the TG binding. Wootton et al.47 reported that the mutation of F256V resulted in a significant decrease of the TG sensitivity. Thus, the calculated large interaction energy of F256 is consistent with their experimental result. Interaction energies of Frag A, B, C, and D of TG (see Figure 1) with SERCAs are given in Table 2. Because the hydroxyl group that has a hydrogen bond with huSERCA E255 is located in Frag A, HF interaction energy of this fragment with huSERCA (−30.2 kcal/mol) was significantly large. We also noted that MP2 interaction energy of Frag C with PfSERCA (−20.6 kcal/mol) was larger than that with huSERCA (−14.9 kcal/mol), indicating that the non-polar interaction of this fragment is favorable to the binding with PfSERCA. In addition to P. falciparum, we performed structure modeling and FMO calculations for SERCAs of the other eight malaria parasite species (Figure S3). For all species, the non-polar interaction was the major component, and the total interaction energy was lower than that of huSERCA. The residues at the position equivalent to huSERCA E255 were serine (P. malariae, P. knowlesi, P. cynomolgi, P. berghei, and P. yoelii), leucine (P. ovale and P. reichenowi), and alanine (P. vivax). As a result, the calculated interaction energies of these residues were significantly small, suggesting that the hydrogen bonding interaction with the hydroxyl group of TG cannot be made. Thus, we propose that the overall mechanism of the molecular interaction of TG with 12


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the SERCAs of these malaria parasite species is similar to that of P. falciparum. 3.4 Molecular design according to the FMO calculations Our FMO calculations revealed that the major component of the interaction energy is the non-polar interaction, but at the same time the major difference between human and P. falciparum was found to arise from the polar interaction, namely, the hydrogen bonding interaction with the hydroxyl group of TG. To design anti-malarial compounds based on TG, the binding affinity with huSERCA should be decreased, and conversely that with PfSERCA should be increased. According to our FMO calculations, the most effective modification is to change the hydroxyl group of TG, by which the critical hydrogen bond with huSERCA is removed. To confirm this assumption, we synthesized a TG derivative without the hydroxyl group, termed TG-acetate in this study (Figure 5-A), and evaluated its inhibitory activity on human cells and P. falciparum (Figure 5-B). The inhibitory activity of TG-acetate against human cells was decreased 15-fold (TG, IC50 = 5.5 nM; TG-acetate, IC50 = 84 nM), suggesting that the hydroxyl group of TG played a critical role in the binding with huSERCA. On the other hand, the inhibitory activity of TG-acetate on P. falciparum was similar to that of TG (TG, IC50 = 4.36 µM; TG-acetate, IC50 = 4.08 µM). Finally, we performed FMO calculations for the complexes of TG-acetate with SERCAs using the same computational procedure (Figure 6). The interaction energy of huSERCA E255 and TG decreased from −30.6 to −14.9 kcal/mol (see also Figure 3), consistent with the results of the inhibitory activity of TG-acetate against human cells. On the other hand, the interaction energy of PfSERCA L263 and TG remained small. This is also consistent with the inhibitory activity against P. falciparum. Although we experimentally examined only TG-acetate in which its inhibitory 13



activity against human cells was still higher than that against P. falciparum, our computational and experimental results suggest that TG-derivatives with opposite sensitivity to huSERCA and PfSERCA can be designed by inserting more non-polar (or hydrophobic) functional groups. Through this strategy, the polar interaction with huSERCA E255 would be abolished, and, at the same time, the non-polar interaction with PfSERCA L263 would be enhanced. Such derivatives have the potential to be new anti-malarial compounds targeting PfSERCA. Additionally, we consider that compounds designed through this strategy would also be effective against other malaria parasite species because their mechanism of interaction with TG is basically similar to that of PfSERCA.

4. CONCLUSIONS In this study, interaction analysis was carried out using the FMO method to identify key factors causing the difference of TG sensitivity between huSERCA and PfSERCA. Our FMO calculations revealed that the major component of the interaction energy was a non-polar interaction in both SERCAs, which is consistent with the fact that the binding site of TG is mainly composed of non-polar amino acid residues. On the other hand, a significant difference in the molecular interaction was found in the polar interaction: huSERCA E255 had an especially large interaction energy associated with the hydrogen bonding interaction with the hydroxyl group of TG, while the interaction energy of the equivalent position of PfSERCA (L263) with TG was very small. Thus, we consider that the difference of TG sensitivity is mainly caused by the molecular interaction of TG with E255 and L263. To confirm this assumption, a TG derivative (TG-acetate) without the hydroxyl group forming this hydrogen bond was synthesized, 14


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and its inhibitory activity on huSERCA was significantly decreased, strongly supporting our assumption. Although our synthesized TG-acetate did not show the inhibitory activity on PfSERCA, this study indicates that an efficient molecular design of anti-malarial compounds can be achieved by replacing the hydroxyl group with a non-polar functional group. Furthermore, the FMO calculations conducted in this study suggest the potential for using the FMO method in the lead optimization process of general drug discovery.

SUPPORTING INFORMATION Organic synthesis of TG-acetate (Scheme S1). PDB codes used for the distance analysis (Table S1). Sequence alignment (Figure S1). RMSD values between the template and models (Figure S2). Interaction energies for eight species of malaria parasite (Figure S3).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Takeshi Ishikawa: 0000-0002-3187-1381 Satoshi Mizuta: 0000-0002-9023-7671 Osamu Kaneko: 0000-0003-0675-8296 Kazuhide Yahata: 0000-0003-4780-8878 Notes 15



The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (C) Grant No. 16K00397 (T.I.), a SUNBOR grant (S. M.), and the Takeda Science Foundation (K.Y.). This research was also supported by AMED under Grant Number JP18am0101088. Human erythrocytes and plasma were obtained from the Nagasaki Red Cross Blood Center.

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22


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Table 1. Average distances between TG and amino acid residues of rabbit SERCA. Standard deviations are also given. These values were calculated using 21 X-ray structures of the complex downloaded from PDB (Table S1).

average (Å)

standard deviation (Å)

F834

3.14

0.22

I829

3.19

0.18

V263

3.25

0.19

L260

3.40

0.16

Q259

3.42

0.18

E255

3.43

0.28

A306

3.43

0.29

I765

3.50

0.21

Y837

3.51

0.19

V769

3.59

0.17

F256

3.61

0.14

L828

3.61

0.22

L253

3.67

0.20

M838

3.71

0.40

N768

3.72

0.23

V772

3.75

0.20

I267

3.94

0.52

23



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Table 2. Interaction energies of the fragments of TG with SERCA. Definition of the fragmentation is given in Figure 1. HF interaction energies (∆ ) are mainly polar interaction, and MP2 interaction energies (∆ ) additionally include non-polar interaction. These energies are given in kcal/mol.

huSERCA

PfSERCA

∆

∆

Frag A

−30.2

−79.7

Frag B

−14.9

−33.8

Frag C

+4.0

−14.9

Frag D

+3.8

−10.1

Total

−37.3

−138.5

Frag A

−4.9

−48.0

Frag B

−13.0

−30.7

Frag C

+5.1

−20.6

Frag D

+2.4

−10.8

Total

−10.3

−110.2

24


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FIGURE LEGENDS

Figure 1. Fragmentation of TG used in the FMO calculations. TG was divided into four fragments, Frag A (blue), B (red), C (orange), and D (green). Two small circles indicate electron pairs.

Figure 2. Modeling of the three-dimensional structures of TG and SERCAs. (A) Sequence alignments of huSERCA and PfSERCA to raSERCA for the 144 residues around the TG binding site. The alignments of the whole sequence are given in Figure S1. (B) Structures of the complex used for FMO calculations in this study. The four peptide chains correspond to the transmembrane helices M3, M4, M5, and M7. TG is shown in red.

Figure 3. Interaction energies of amino acid residues of SERCA with TG. Blue bars indicate HF energy, which is mainly polar interaction energy. Red bars indicate MP2 energy, which additionally includes non-polar interaction energy. The total interaction energy is the sum of the interaction energies of the 144 residues.

Figure 4. Three-dimensional structures around huSERCA E255 and PfSERCA L263. In the complex with huSERCA, the hydroxyl group of TG (dotted circle) forms a hydrogen bond with E255, and K252 forms a salt bridge with E255. On the other hand, such hydrogen bond and salt bridge cannot be made with PfSERCA because the carboxylic acid does not exist in L263.

25



Figure 5. Organic synthesis of TG-acetate and its inhibition assay. (A) Chemical structure of TG-acetate, where the hydroxy group of TG was replaced with −OCOCH3. (B) The results of the inhibition assay of TG and TG-acetate for human cells and P. falciparum. The values are the average ± s.d. of triplicate experiments.

Figure 6. Interaction energies of amino acid residues of SERCA with TG-acetate. Blue bars indicate HF energy, which is mainly polar interaction energy. Red bars indicate MP2 energy, which additionally includes non-polar interaction energy. The total interaction energy is the sum of the interaction energies of the 144 residues.

26


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TOC Graphic

27



Figure 1. Fragmentation of TG used in the FMO calculations. TG was divided into four fragments, Frag A (blue), B (red), C (orange), and D (green). Two small circles indicate electron pairs. 92x65mm (300 x 300 DPI)


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Figure 2. Modeling of the three-dimensional structures of TG and SERCAs. (A) Sequence alignments of huSERCA and PfSERCA to raSERCA for the 144 residues around the TG binding site. The alignments of the whole sequence are given in Figure S1. (B) Structures of the complex used for FMO calculations in this study. The four peptide chains correspond to the transmembrane helices M3, M4, M5, and M7. TG is shown in red. 225x300mm (150 x 150 DPI)



Figure 3. Interaction energies of amino acid residues of SERCA with TG. Blue bars indicate HF energy, which is mainly polar interaction energy. Red bars indicate MP2 energy, which additionally includes non-polar interaction energy. The total interaction energy is the sum of the interaction energies of the 144 residues. 290x224mm (150 x 150 DPI)


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Figure 4. Three-dimensional structures around huSERCA E255 and PfSERCA L263. In the complex with huSERCA, the hydroxyl group of TG (dotted circle) forms a hydrogen bond with E255, and K252 forms a salt bridge with E255. On the other hand, such hydrogen bond and salt bridge cannot be made with PfSERCA because the carboxylic acid does not exist in L263. 333x172mm (150 x 150 DPI)



Figure 5. Organic synthesis of TG-acetate and its inhibition assay. (A) Chemical structure of TG-acetate, where the hydroxy group of TG was replaced with −OCOCH3. (B) The results of the inhibition assay of TG and TG-acetate for human cells and P. falciparum. The values are the average ± s.d. of triplicate experiments. 304x230mm (150 x 150 DPI)


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Figure 6. Interaction energies of amino acid residues of SERCA with TG-acetate. Blue bars indicate HF energy, which is mainly polar interaction energy. Red bars indicate MP2 energy, which additionally includes non-polar interaction energy. The total interaction energy is the sum of the interaction energies of the 144 residues. 290x224mm (150 x 150 DPI)


Fragment Molecular Orbital Study of the Interaction between Sarco

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