Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations.

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Investigation of Ligand Selectivity in CYP3A7 by Molecular Dynamics Simulations a

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Jing-Rong Fan , Qing-Chuan Zheng , Ying-Lu Cui , Wei-Kang Li & Hong-Xing Zhang

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International Joint Research Laboratory of Nano-Micro Architecture Chemistry, State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, People’s Republic of China b

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, Jilin, 130023, People’s Republic of China Accepted author version posted online: 12 Jun 2015.

Click for updates To cite this article: Jing-Rong Fan, Qing-Chuan Zheng, Ying-Lu Cui, Wei-Kang Li & Hong-Xing Zhang (2015): Investigation of Ligand Selectivity in CYP3A7 by Molecular Dynamics Simulations, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2015.1054884 To link to this article: http://dx.doi.org/10.1080/07391102.2015.1054884

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Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics DOI: http://dx.doi.org/10.1080/07391102.2015.1054884

Investigation of Ligand Selectivity in CYP3A7 by Molecular Dynamics Simulations Downloaded by [UQ Library] at 10:20 15 June 2015

Jing-Rong Fana, Qing-Chuan Zhenga,b*, Ying-Lu Cuia, Wei-Kang Lia, Hong-Xing Zhanga a

International Joint Research Laboratory of Nano-Micro Architecture Chemistry,

State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130023, People’s Republic of China b

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of

Education, Jilin University, Changchun, Jilin, 130023, People’s Republic of China Corresponding author: Qing-Chuan Zheng International Joint Research Laboratory of Nano-Micro Architecture Chemistry State Key Laboratory of Theoretical and Computational Chemistry Institute of Theoretical Chemistry Jilin University Changchun, 130023 1

People’s Republic of China Fax: (+86) 431-8849-8966 E-mail address: [email protected]

Abstract Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7-ligands complexes, three endogenous ligands were selected,

Downloaded by [UQ Library] at 10:20 15 June 2015

namely, dehydroepiandrosterone (DHEA), estrone and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue as the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. The present study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.

Keywords: cytochrome P450 3A7; homology modeling; molecular docking; 2

molecular dynamics simulation; MM-GB/SA calculation

1. Introduction Cytochrome P450s (CYPs) constitute a superfamily of heme-containing monooxygenases that are ubiquitious in both eukaryotes and prokaryotes. The CYP enzymes metabolize a wide variety of endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous chemicals including drugs, carcinogens and environmental pollutants (Cui et al., 2013; Cui et al., 2013; Denisov, Makris, Sligar,


& Schlichting, 2005; Li et al., 2008; Park, Lee, & Suh, 2005). The active site of CYPs contains a heme group which is deeply buried in the protein core and the iron atom is covalently bound to a conserved cysteine (Shen et al., 2012). In the human liver, CYP3A subfamilies are considered as the major CYP450 isoforms. Four functional members of the CYP3A subfamilies have been identified, including CYP3A4, CYP3A5, CYP3A7 and CYP3A43 (Smit et al., 2005; Williams et al., 2002). CYP3A7 is very active in the fetal liver, which accounts for 50% of the total hepatic CYP content in fetus and up to 87-100% of total fetal hepatic CYP3A content (Smit et al., 2005). Moreover, it is also detectable in low amounts in the placenta, in gynecologic malignancies and in some adult liver and intestine. (Lacroix, Sonnier, Moncion, Cheron, & Cresteil, 1997; Stevens et al., 2003). The catalytic properties of CYP3A7 exhibit striking functional differences in the metabolism of endogenous ligands, such as dehydroepiandrosterone (DHEA), estrone, and estradiol. DHEA, which is produced in the adrenal glands, gonads and brain, is an important endogenous steroid hormone and the most abundant circulating steroid in humans 3

(Lee, Conney, & Zhu, 2003; Nakamura et al., 2003; Smit et al., 2005). It acts as a predominantly metabolic intermediate in the biosynthesis of the androgen and estrogen sex steroids (Mo, Lu, & Simon, 2006). Estrone is an estrogenic hormone secreted by the ovary as well as the adipose tissue. It is a common carcinogen for females, which can cause breast tenderness, nausea, headache, hypertension, and leg cramps (Santner, Feil, & Santen, 1984). For males, estrone has been known to cause anorexic, nausea, vomiting and erectile disfunction (Jasuja et al., 2013). Estradiol,


likes other steroids (Rothman et al., 2011), is derived from cholesterol. There are receptors for estradiol in many other cells of the body of men and women including gonads, brain, precursor hormones and arterial walls (Yang, Owen, Ramsay, Xie, & Roberts, 2004). Therefore, the most representative ligands, namely, DHEA, estrone, and estradiol are chosen to investigate the structural features relevant to the ligand selectivity of CYP3A7. In this study, a series of methods have been adopted to study the CYP3A7 at the atomic level. However, up to now no report has been found about the three-dimensional (3D) structure of the CYP3A7, and thus theoretical studies on the binding modes of the CYP3A7 with its ligands are necessary to reveal the interaction occurring in the active site. First of all, the three dimensional (3D) model of CYP3A7 was built by homology modeling using the crystal structure of CYP3A4 as a template. Then the docking study was carried out using the optimized model with three typical ligands: DHEA, estrone and estradiol, respectively. Finally, through MD simulations along with binding free energy calculations and decomposing free energy calculations, 4

we have been able to explain the binding characteristics, including residue interactions and certain critical residues responsible for ligand binding, moreover, the dominant access channels for ligands access were identified. The obtained results can provide insights into the poorly understood structural features of CYP3A7. The analysis of the interactions between substrates and the residues in the active site could help to guide further drug design. Moreover, the binding free energy results reveal the most important residues responsible for the binding of ligands, and provides a


theoretical basis for the single point mutation and experimental protein engineering research.

2. Computational Methods 2.1 Homology modeling The primary sequence of CYP3A7 was obtained from the SwissProt database (accession number P24462). The crystal structure of CYP3A4 was extracted from the Protein DataBank (PDB ID: 3UA1) and used as the template (Sevrioukova & Poulos, 2012). The sequence alignment between the template CYP3A4 and CYP3A7 was performed by using the protein protocol of Discovery Studio 2.5 (Studio, 2009). The initial 3D structural model of CYP3A7 was generated by using the Protein Modeling protocol of Discovery Studio2.5 (Studio, 2009).

2.2 Molecular dynamics simulation for CYP3A7 MD simulations for CYP3A7 were generated by the xleap module in the AMBER software package (Case et al., 2010). First of all, the protein was solvated in a layer of TIP3P water box (Jorgensen, Chandrasekhar, Madura, Impey, & Klein, 1983) with 10 5

Å distance from any residue of CYP3A7 to the boundary. Then the Cl- was added into water to ensure the overall neutrality of the system. The whole system was minimized with a total of 5000 steps of steepest decent minimization and conjugate gradient minimization. After that, the system was minimized without restraints for 7000 steps. Subsequently, the system was gradually heated from 0 to 310 K for 300 ps and equilibrated for 500 ps in the NVT ensemble. Then, the MD simulation of 30 ns was performed at 310 K with the NPT ensemble. During the simulation, the non-bond


cutoff radius of 12 Å and the particle-mesh Ewald (PME) for long-range electrostatics were used (Tosaka, Yamamoto, Ohdomari, & Watanabe, 2010). SHAKE algorithm (Feng et al., 2012) was employed to constrain the covalent bonds to hydrogen atoms. The step time of MD simulation was 2 fs and the trajectory was stored every 1 ps for analysis. In the end, the structure was checked by using Profile-3D and Ramachandran plots (Laskowski, MacArthur, Moss, & Thornton, 1993) and used as subsequent molecular docking.

2.3 Docking and subsequent MD simulations for complex structures The initial structures of these ligands, DHEA (Accession Number: DB01708), estrone (Accession Number: DB00655) and estradiol (Accession Number: DB00783) were obtained from the DrugBank database (Wishart et al., 2006). The CDOCKER protocol of Discovery Studio 2.5 was employed to dock all three ligands into the active site of CYP3A7. The semiflexible approach was chosen, in which the protein was fixed and the ligands were free to move during docking simulation. In the process of the whole simulation, all other parameters were maintained at their default settings. After 6

docking, the highest score conformations were chosen and subsequent MD simulations of CYP3A7-ligand complex structures were carried out using AMBER software package and the ff99SB force field. The ligands were generated by Antechamber. The mopac quantum mechanics code and the AM1-BCC charge mode were used in Antechamber to calculate the atomic point charges. The CYP3A7-ligand complex minimizations were performed with steepest descent method for 2500 steps, followed by conjugated gradient method for 4000 steps. The complex structures were


heated from 0 to 310 K under the NVT ensemble, then the structures equilibrized for 500 ps. In the NPT conditions, a 20 ns MD simulations of CYP3A7-ligand complex was performed. All of the figures were created with PyMOL (DeLano, 2002).

2.4 Analysis of access channels in CYP3A7-ligands complex structures CAVER (Petrek et al., 2006) is well-known for its accurate identification of egress channels from the surface to the active site of the enzyme. In the channel searching, we have put the ligands above the heme group as the starting point. The CAVER algorithm divides three-dimensional space into a grid and calculations are based on grid-points. In the course of the calculations, grid spacing was set to 0.8 Å and other parameters were maintained at their default settings. Then the channels were visualized by using PyMOL.

2.5 Free Energy Calculations The binding free energies were calculated using the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) method implemented in AMBER 11 (Hou, Wang, Li, & Wang, 2010). For each complex, a total number of 100 snapshots 7

were taken from the last 10 ns on the MD trajectory. The binding free energy (∆Gbind) in MM-GB/SA between a ligand (L) and a receptor (R) to form a complex RL can be summarized as ∆Gbind = Gcomplex - （Greceptor + Gligand）

(1)

G = EMM + Gsol - TS

(2)

EMM = Eint + Eele + Evdw

(3)

Gsol = GGB + GSA

(4)


In Equation (2), the EMM, Gsol, and TS represent molecular mechanics component in gas phase, the stabilization energy due to solvation, and a vibrational entropy term, respectively. EMM is given as a sum of Eint, Eele, and Evdw, which are internal, Coulomb, and van der Waals interaction terms, respectively. Solvation energy (Gsol) is separated into an electrostatic solvation free energy (GGB) and a nonpolar solvation free energy (GSA). The former can be obtained from Generalized Born (GB) method. The latter is considered to be proportional to the molecular solvent accessible surface area (SASA) (Hou, Li, Li, & Wang, 2012; Hou, Zhang, Case, & Wang, 2008).

3. Results and Discussion 3.1 Homology modeling of CYP3A7 In homology modeling, a suitable template is crucial to success. The BLAST searching revealed that CYP3A7 had a high sequence identity with CYP3A4 (PDB ID: 3UA1), which can reach up to 81.5% (Figure S1). Thus, the crystal structure of CYP3A4 (PDB ID: 3UA1) was chosen as the template to build the initial model of CYP3A7. Then, the model was optimized by 30 ns MD simulations. After the MD 8

simulations, the root-mean-square deviations (RMSD) was calculated (Figure S2). From Figure S2, the RMSD of backbone atoms reached a plateau in the last 10 ns simulation time. The final stable structure with the lowest energy was obtained from the last 10 ns simulation trajectories. The overall structure with twelve α-helices and eight sheets is shown in Figure 1. A comparison of the secondary structures of CYP3A7 and the template CYP3A4 is listed in Table S1. The final structure of CYP3A7 was checked by Profile-3D (Figure S3). The self-compatibility score of


CYP3A7 was 214.18, which is higher than the low score 96.38 and close to the top score 218.61. Figure S3 shows that most of the residues are reasonable except for Arg233. Fortunately, Arg233 is far away from the active site of CYP3A7 and may not affect the following study. Furthermore, A Ramachandran plot was used to evaluate the protein structure (Figure S4). The statistical score of the Ramachandran plot shows that 92.8% are in the most favored regions, 5.7% in the additional allowed regions, and 1.5% in generously allowed regions. In summary, the results of quality assessment indicate that the CYP3A7 structure is reliable.

3.2 Docking study In order to gain insights into interactions of complex proteins and identify key residues responsible for the ligand specificity of CYP3A7, three ligands including DHEA, estrone, estradiol (Figure 2), were docked into the active site of CYP3A7 in an orientation conductive to their 16α–hydroxylation, which is closed to the heme group. DHEA is metabolized to androgens and estrogens in steroidogenic tissues (Miller et al., 2004) and plays an indirect biological role in the organism. In this study, 9

the metabolism of DHEA was completed by CYP3A7. Two major estrogens (Estrone and estradiol) are distributed widely in mammalian tissues. They have been shown to be oxidized at 16α-position by CYP3A7. Hydroxylation of estradiol by P450 enzymes at the 16α-position plays an important role in mammary carcinogenesis (Yamazaki, Shaw, Guengerich, & Shimada, 1998). Finally, the highest score conformations were chosen (Figure 3). From the docking result, the distances between the Fe atom and the 16α site of DHEA, estrone and estradiol were within 5Å, suggesting that the ligands


can be catalyzed in this range. Thus, the binding modes of CYP3A7-ligands were reasonable. In order to gain further insight into the stability of CYP3A7-ligands complex, 20 ns MD simulations were performed. The average RMSD values for CYP3A7-ligands complexes are shown in Figure 4. In the process of the whole simulation, the RMSD values remain within 2.8 Å for last 10 ns simulations in all simulated systems. Hydrogen bond interactions enhance the stability of the CYP3A-ligands. As shown in Figure 3, it is clear that Ser119 forms hydrogen bond with all ligands. This structural feature is consistent with the previous study of CYP3A4 (Lee et al., 2005). Estrone and estradiol formed only one hydrogen bond with CYP3A7 in approximately 92% and 49% respectively in the simulation trajectories (Figure 3B & 3C). Moreover, for the CYP3A7-DHEA, residues Ser119 and Arg372 are found to form hydrogen bonds with DHEA that are maintained for more than 85% frames in the process of MD simulations. The hydroxyl group of DHEA forms a hydrogen bond with the backbone oxygen atom of Arg372 (Figure 3A) and the ketone oxygen of DHEA forms 10

a hydrogen bond with the side chain hydroxyl group of Ser119. We consider that the DHEA may be fixed by hydrogen bonds at both ends of DHEA. The reason is that DHEA prefers turning into sheet β3, however, estrone and estradiol are located towards helix I. The hydrogen bonds have significant effects on the binding modes of CYP3A7. The above analysis elucidate that the hydrogen bond plays an important role in stabilizing the ligand in the active site of CYP3A7.

3.3 Analysis of access channels for CYP3A7-ligands complex structures Downloaded by [UQ Library] at 10:20 15 June 2015

Channel is relevant for the passage of the small ligand into the active site of the enzyme involved in the reaction. The function of the channels was essential for ligand ingress and egress from the active site to the surface of the protein. In the present study, the most accessible channels were found by the program CAVER (Figure 5). As shown in Figure 5, the way of the DHEA, estrone, estradiol through the protein were all contributed from S channel. The S channel, which runs between the E, F and I helices and β5 sheet, is proposed as an important secondary ligand/product egress route in other P450s (Cui et al., 2013).

3.4 Total interaction energy calculations To investigate the binding free energies between the protein and ligands, the MM-GB/SA method was employed. These results (shown in table 1) demonstrate that DHEA has the highest binding affinity, estrone takes the second place, and estradiol is the last one of all three ligands. Anthony and his coworkers also reported that the binding affinity of estrone is better than the estradiol’s (Lee et al., 2003). Our results are in agreement with the experimental conclusion. According to the further analysis 11

of the binding free energy, the nonpolar interaction was found to be the main driving force of binding affinity. It’s worth noting that the polar interaction has a much smaller but favorable contribution in the CYP3A7-DHEA complex. In comparison to the CYP3A7-DHEA complex, the polar interaction of CYP3A7-estrone and CYP3A7-estradiol complexes has an unfavorable contribution. Thus, we can speculate that the polar interaction might be one of the causes leading to such distinction.


Decomposing the binding free energies can offer us pivotal clues to identify the key residues in the binding process. The interaction energies between crucial residues and the ligand are listed in the Table S2 and these residues are considered to be essential for ligand binding. For CYP3A7-DHEA complex, the key residues that contribute to the binding of the ligand within the active site are listed as follow: Arg105, Phe108, Ser119, Ile301, Phe304, Ala370, Met371, Arg372, Leu373, Arg375, Gly481 and Leu482 (N. Torimoto et al., 2007). The representative conformation of CYP3A7-DHEA complex, which was extracted from the MD trajectory snapshots, is shown in the Figure 6A. As can be seen from Figure 6A, two hydrogen bonds have been formed between DHEA and two different residues on the CYP3A7, Ser119 and Arg372. Interestingly, Ser119 and Arg372 are located on the two ends of the DHEA molecule. Meanwhile, the ligand is surrounded by the hydrophobic residues, particularly Phe108, Ile301, Phe304, Ala370, Met371, Leu373 and Leu482, thereinto, Phe108, Phe304 and Leu482 are located on the upper of the DHEA and Leu373 is located below the ligand. They got together to 12

form a hydrophobic cavity and provide hydrophobic interactions to stabilize the ligand. Figure 6B shows a representative MD trajectory snapshot for estrone bound in the active site of CYP3A7. Several hydrophobic interactions were observed between estrone and side chains of residues Phe213, Ile301, Phe304 and Ala305. The 3D conformation of the estradiol bound with CYP3A7 is shown in Figure 6C. What’s more, several key residues play an important role in binding with estradiol. Residues Ile120, Phe213, Phe304, Ala305 and Val369 are found to form a hydrophobic cavity


and provide hydrophobic interactions to stabilize the ligand. The hydrophobic cavities of estrone and estradiol are smaller than that of DHEA. The methyl of DHEA may have some effects on the hydrophobic cavity. Phe residues are significant hydrophobic residues in the CYP3A family. In the CYP3A7-DHEA complex, DHEA has considerable hydrophobic interactions with Phe304 and Phe108 which is situated on the B-C loop. In the CYP3A7-estrone and CYP3A7-estradiol complexes, the ligands are stabilized by hydrophobic interaction with Phe304 and Phe213 situated on the F’-F loop. The results mentioned above demonstrate that hydrophobic residues make significant contribution to stabilize the ligands. It was previously reported that Ala370 plays an important role in stabilizing the progesterone in the active site of CYP3A4 (Park et al., 2005), and the rate of CYP3A4-catalyzed hydroxylation of progesterone would be decreased when Ala370 mutated. In this study, Ala370 also has enormous impact on the stabilization of ligand which contains DHEA and estrone. According to the further decomposing energy analysis, Arg105 plays a key role in binding to the ligands. It is clear that only two residues (Arg372 and Leu482) make 2 13

kcal/mol contributions to binding in the CYP3A7-DHEA complex. For Arg372, the contributions of electrostatic interaction to binding work favorable interactions, and van der waals interaction of Leu482 plays major role in binding. However, for other two ligands (estrone and estradiol), Arg212 and Phe304 make chief contribution to binding the ligand. In the CYP3A7-DHEA complex, the other five residues, including Ala370, Met371, Leu373, Arg375 and Gly481, work positive effect in binding the ligand. Unlike DHEA, outstanding residues are Phe213, Ala305, Thr309 and Val369


in the CYP3A7-estrone and CYP3A7-estradiol complexes.

4. Conclusion The development of the computer simulations of biomolecules is an unstoppable trend. Docking methods and molecular dynamics simulation (MD) play an important role in investigating the protein and ligands interactions (Conner, Woods, & Atkins, 2011). The absence of a crystal structure of the CYP3A7 influences computational investigation of protein-ligand interactions. Therefore, a 3D model of CYP3A7 has been constructed using human CYP3A4 as a template and refined by energy minimization and MD simulations. Afterwards, docking approaches were employed to dock three ligands (DHEA, estrone and estradiol) into the active site of the proposed 3D model. By means of MD simulations, the binding mode of the complex is in good agreement with the available experimental data. Through MD simulations along with the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) calculations, decomposing free energy calculations and access channel analysis, we have been able to describe the binding characteristics and 14

residue interactions. On the result of MM-GB/SA, DHEA was found to be the ligand with the highest binding affinity, followed by estrone and estradiol, respectively. Several key residues were identified to be responsible for the ligand selectivity. In these residues, Phe108, Ser119, Phe304, Ala370 and Leu482 are considered to play important roles in binding. They have strong interaction energy with both DHEA and estrone. The hydrophobic residues lying in the active site collectively form hydrophobic cavity and provide hydrophobic interactions to stabilize the ligand.


Furthermore, DHEA is stabilized in the active site by the formation of two hydrogen bonds with Ser119 and Arg372. Estrone and estradiol form only one hydrogen bond with Ser119. These hydrogen bonds make ligands more stable. The major access channel was discovered for the ligand ingress and egress from the active site to the surface of the protein. Our present study provides important insights into understanding the structural features of human CYP3A7 at the atomic level, and should contribute to further understanding of related protein structures and dynamics.

Acknowledgments This work is supported by Natural Science Foundation of China (Grant Nos.21273095).

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21


Figure 1. The overall structure of CYP3A7. The heme group is represented as a red stick. The side chain atoms are shown as sticks in the active site of CYP3A7.

22

Figure 2. Molecular structures of the three ligands of CYP3A7: DHEA, estrone,


estradiol.

23


Figure 3. The binding modes of (A) CYP3A7-DHEA, (B) CYP3A7-estrone, (C) CYP3A7-estradiol complexes in stereo mode. The complex structures are shown in transparent ribbon representation (helixes in red, beta strands in yellow, loops and turns in green). The heme group and ligands are represented by red and yellow sticks, respectively. The hydrogen bonds are shown in blue dashed lines.

24

5

DHEA estrone estradiol

RMSD (Å)

4

3

2

1

0 0

5

10

15

20


Time (ns)

Figure 4. RMSD of CYP3A7-ligands complexes for DHEA, estrone and estradiol during 20ns simulations.

25


Figure 5. Access channels identified from the average structures of CYP3A7-ligand complexes for (A) CYP3A7-DHEA complex, (B) CYP3A7-estrone complex, (C) CYP3A7-estradiol complex, respectively. Channels are shown as yellow spheres, the complex structures are shown in ribbon representation (DHEA in green, estrone in cyan, estradiol in pink), ligands and heme are shown in stick representations.

26


Figure 6. The key residues in the active site of CYP3A7-ligand complexes for (A) CYP3A7-DHEA complex, (B) CYP3A7-estrone complex, (C) CYP3A7-estradiol complex in stereo mode, respectively. All the molecules are shown in stick representations. The carbon atoms for heme, ligands and residues are colored in green, red and yellow, respectively.

27

Table 1. Binding free energies (kcalmol-1) for three ligands in the complexes. system

∆Eele

∆Evdw

∆GGB

∆GSASA

∆Gpola

∆Gnonpolb

∆GMM-GB/SAc

-T∆S

∆GTOTd

DHEA

-15.65

-41.69

13.64

-5.30

-2.01

-46.99

-49.00

16.52

-32.48

etrone

-8.53

-42.21

14.18

-4.84

5.65

-47.05

-41.40

22.60

-18.80

estradiol

-5.68

-37.97

13.23

-5.15

7.55

-43.12

-35.57

18.95

-16.62

a

∆Gpol = ∆Eele +∆GGB

b

∆GMM-GB/SA = ∆Eele + ∆Evdw + ∆GGB + ∆GSASA.


c

∆Gnonpol = ∆Evdw + ∆GSA

28

Supporting Information Investigation of Ligand Selectivity in CYP3A7 by Molecular Dynamics Simulations


Table S1. Comparison between CYP3A7 and the template CYP3A4. CYP3A4

CYP3A7

Secondary structure Residues

Secondary structure Residues

A -Helix A-Helix Sheet Sheet B-Helix

Leu32-Lys35 Ile50-Tyr68 Val71-Asp76 Gln79-Ile84 Pro87-Leu94

C-Helix D -Helix D-Helix Sheet E-Helix F -Helix F-Helix G -Helix G-Helix H-Helix I-Helix J -Helix J-Helix Sheet Sheet Sheet Sheet K -Helix K-Helix L -Helix L-Helix Sheet Sheet

Gly112-Ser116 Asp123-Phe137 Ser139-Glu165 Val170-Thr171 Leu172-Phe189 Pro202-Thr207 Pro218-Val225 Ile230-Val235 Arg243-Glu258 Phe271-Ser278 Ser291-His324 Pro325-Lys342 Tyr347-Leu366 Leu373-Val376 Val381-Ile383 Met386-Ile388 Val393-Ile396 Ser398-His402 Pro416-Phe419 Asn423-Ile427 Met445-Asn462 Phe463-Pro467 Val490-Ile396

A -Helix A-Helix Sheet Sheet B-Helix C -Helix C-Helix

Asn22-Arg27 Tyr30-Lys40 Val44-Asp49 Gln52-Ile57 Pro60-Val74 Phe86-Asn89 Glu97-Phe110

D-Helix

Ser112-Thr137

E-Helix F -Helix F-Helix

Leu145-Ser161 Pro175-Lys182 Pro191-Leu209

G-Helix H-Helix I-Helix J -Helix J-Helix Sheet Sheet Sheet Sheet K -Helix K-Helix L -Helix L-Helix Sheet Sheet

Arg216-Lys230 Phe245-Ile249 Asp265-Thr296 Pro298-Val311 Glu327-Leu339 Arg345-Lys350 Val354-Ile356 Met359-Ile361 Gly364-Pro370 Ser371-His376 Pro389-Phe391 Lys397-Asn399 Gly417-Asn435 Phe436-Lys439 Lys465-Ser468

29

Table S2. (A) Decomposition of binding free energy (kcal mol-1) on per-residue basis


for CYP3A7-DHEA complex Residue ∆Evdw

∆Eele

∆Gpol

∆Gnonpol ∆Gbind

Arg105

-1.65

0.15

0.08

-0.18

-1.61

Phe108

-1.88

-0.12

0.41

-0.29

-1.87

Ser119

-0.43

-3.41

3.46

-0.08

-0.47

Ile301

-0.43

-0.15

0.10

-0.02

-0.49

Phe304

-0.70

-0.25

0.50

-0.13

-0.58

Ala370

-0.89

0.19

-0.41

-0.07

-1.17

Met371

-0.18

0.08

-0.40

0.00

-0.50

Arg372

0.05

-3.90

1.36

-0.06

-2.55

Leu373

-0.72

-0.07

-0.09

-0.02

-0.90

Arg375

-0.12

0.41

0.79

0.00

-0.50

Gly481

-0.65

-0.85

1.00

-0.06

-0.57

Leu482

-2.01

0.14

-0.23

-0.32

-2.42

Table S2. (B) Decomposition of binding free energy (kcal mol-1) on per-residue basis for CYP3A7-estrone complex Residue ∆Evdw

∆Eele

∆Gpol

∆Gnonpol ∆Gbind

Arg105

-0.47

0.82

-1.25

-0.10

-1.01

Arg212

-2.18

-0.54

1.70

-0.25

-1.28

Phe213

-1.62

0.18

0.70

-0.21

-0.95

Ile301

-0.88

-0.42

0.58

-0.02

-0.74

Phe304

-1.73

-0.56

0.88

-0.09

-1.49

Ala305

-0.77

0.04

-0.02

-0.08

-0.83

Thr309

-0.54

0.07

-0.16

-0.05

-0.67

Val369

-0.51

-0.03

-0.24

-0.03

-0.81

Ala370

-0.48

-0.27

0.20

-0.10

-0.65

Leu482

-0.44

-0.14

0.00

-0.09

-0.67

30

Table S2. (C) Decomposition of binding free energy (kcal mol-1) on per-residue basis


for CYP3A7-estradiol complex Residue ∆Evdw

∆Eele

∆Gpol

∆Gnonpol ∆Gbind

Arg105

-1.51

1.00

-1.00

-0.13

-1.64

Ser119

-1.13

-1.41

1.94

-0.15

-0.75

Ile120

-0.69

-0.09

-0.11

-0.10

-0.99

Arg212

-1.02

-0.87

0.40

-0.16

-1.65

Phe213

-1.79

-0.08

1.03

-0.29

-1.12

Phe304

-1.45

-0.06

0.65

-0.21

-1.07

Ala305

-0.96

0.15

-0.01

-0.14

-0.96

Thr309

-0.54

-0.05

-0.09

-0.07

-0.74

Val369

-0.93

-0.33

0.70

-0.18

-0.74

Figure S1.Sequence alignment of CYP3A7 and CYP3A4. 31

5

RMSD (Å)

4

3

2


1

0 0

5

10

15

20

25

30

Time (ns)

Figure S2.Time-dependent rms deviation values from the starting structures along the simulation trajectory.

Figure S3. 3D-profile-verified results of the CYP3A7 model, residues with positive verify score are folded reasonably.

32


Figure S4.Ramachandran plot of CYP3A7 obtained by Discovery Studio 2.5.

33

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