Molecular Diversity
New Insights in the Activation of Human Cholesterol Esterase to
Design Potent Anti-cholesterol Drugs
Shalini John, Sundarapandian Thangapandian, Prettina Lazar, Minky Son, Chanin Park, and
Keun Woo Lee*
Division of Applied Life Science (BK21 Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant
Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science
(RINS), Gyeongsang National University (GNU), 501 Jinju-daero, Gazwa-dong, Jinju 660-701, Republic of
Korea.
*Corresponding author. Tel. +82-55-772-1360; Fax. +82-55-772-1359.
E-mail address: kwlee@gnu.ac.kr
Supporting Information
Text S1
1. Comparison of activation efficiency of different bile salts
1.1.
Stability analysis
In general the RMSD of the three systems were well converged throughout the simulation.
The average backbone RMSD values of hCEase_TCH, hCEase_CHA and hCEase_GCH
systems are 0.20, 0.24 and 0.19 nm respectively (Fig. S2A). The potential energy graph has
shown that the energy difference between the systems is very less and all the four systems
have maintained a stable average value between -12,202 to -12,187 kJ/mol. The Rg was
compared to measure the compactness of the structures of the proteins. The Rg value of
hCEase_TCH system was maintained at an average value of 2.29 nm. The hCEase_CHA and
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Molecular Diversity
hCEase_GCH systems have shown slightly decreased average values of 2.27 nm and 2.24 nm,
respectively.
1.2.
Fluctuation analysis
The Cα RMSF was measured for the three systems to monitor the fluctuation of each residue
in presence of different bile salts. From the RMSF graph it was observed that the areas
belonging to the secondary structure elements are more stable. The region in the graph with
highest fluctuation belongs to the loops and coils between helices and sheets (Fig. S2B). The
fluctuation was high in the loop regions of hCEase_TCH system when compared to the
hCEase_CHA that has shown more fluctuation than hCEase_GCH system. The catalytic triad
and oxyanion hole residues of hCEase_TCH system was very well stabilized than
hCEase_GCH but hCEase_CHA system has shown more fluctuation in this region (Fig. S2C).
The RMSF analysis revealed that TCH fluctuated more in the loop regions but stabilized well
in the catalytic site region. The hCEase_GCH had considerable stability but the fluctuation of
hCEase_CHA was very high in the catalytic site region.
1.3.
Structural changes and binding mode analysis
The movement of 120-loop, in hCEase_TCH, was in the open conformation than
hCEase_CHA, towards the closed conformation, whereas hCEase_GCH was near to the open
conformation. It has clearly been observed that the big steroid moiety of TCH was placed in
front of the 120-loop and the side chain was nuzzled in the gap between 120- and 450-loop
(Fig. 2A). But in case of CHA, due to lack of long side chain the interaction was lost thereby
the big steroid moiety cannot move the 120-loop enough to get the open conformation (Fig.
2C). In case of hCEase_GCH, though GCH has lengthy amide group as side chain, due to the
lack of SO3- group the strong interaction required to place the big steroid moiety exactly
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Molecular Diversity
opposite to the 120-loop was missing (Fig. 2E). Thereby the conformation of GCH was not in
an appropriate position. Hence the active site of hCEase_GCH was largely disturbed.
Likewise the remote TCH binds well in the cleft region compared to CHA and GCH as their
binding orientations are not favorable and the binding site was largely disturbed. The
analyses based on structural changes, H-bond interactions and binding modes revealed that
TCH binds the active site in a favorable manner and brought essential conformational
changes comparing to binding of CHA and GCH.
1.4.
Distance analysis
The distance between two key residues, one from the catalytic triad, His435, and another
from oxyanion hole, Gly107, was measured throughout the simulation. From the distance
graph (Fig. S4) it was observed that hCEase_TCH has maintained an average of 0.527 nm
whereas hCEase_CHA and hCEase_GCH has shown decreased and increased average values
of 0.487 nm and 0.650 nm, respectively.
1.5.
Interaction energy calculations
The energy of interaction between individual residues and ligands were calculated for the
three different enzyme-ligand complexes to get the comprehensive view of effects of the
important residues on the binding affinity of the three different systems. The interaction
energy between the residues and the ligand was calculated using the Calculate Interaction
Energy protocol available in DS with the default parameters. The important amino acids
showing interactions with proximal TCH are Asn117, Asn118, Phe119, Asn121, Asn122,
Tyr123, Leu124, Tyr125, Gln440, Lys445, Ala448, Thr449, Pro450, Thr451, Gly452, and
Tyr453. The 3α-hydroxyl group of proximal TCH has shown an H-bond interaction with
Ile124 and the 7α-hydroxyl group with Thr449. This result was similar to the important
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Molecular Diversity
interaction observed for bCEase. The important amino acids showing interaction with remote
TCH molecule are Leu224, Thr352, Thr523, Ile353, Ile399, Trp522, Phe351, Thr354, Tyr526,
and Pro396. The contribution of electrostatic and van der Waals interactions to the nonbonded interaction energies of different bile salts TCH, CHA, and GCH that forming direct
interactions with active site residues were calculated and summarized (Table S1 and S2).
From the result it was observed that the electrostatic energy of the residues Asn117, Phe119,
Asn121, Leu124, Lys445, Thr449, Pro450, and Thr451 have major contribution in the ligand
binding. The TCH has more satisfactory interaction energies with these important residues
comparing to GCH and CHA. Interestingly many of these residues have shown strong Hbond interaction with TCH than the other two ligands. This outcome enhances the importance
of 120-loop because the major bonding and non-bonding interacting residues are from the
120-loop. Though CHA and GCH have shown favorable interaction energies their
contribution to the electrostatic interaction energy was less than TCH. This favorable
interaction energy may be directly related to higher binding affinity of TCH towards hCEase.
The van der Waals interactions were the major weakening interactions in terms of CHA
molecules that showed a high positive value of 201.868 kcal mol-1 towards Leu124. This is
the main reason for its instability and the disturbed mode of binding at the active site. Though
the vdw interactions of TCH and GCH have destabilizing effect it was compensated by the
more favorable electrostatic contributions. In case of remote bile salt, Ser225, Thr523, Ile399,
Trp522, Phe351, Thr354, Tyr526, Pro396, Thr352, and Ile353 residues have shown
substantial contributions to the electrostatic interaction energy. The TCH has shown good
electrostatic energy when compared to CHA and GCH molecules. The vdw interaction has
shown destabilizing effect in CHA but not in TCH and GCH binding because of the
beneficial electrostatic interaction. Among all ligands the energy contribution of CHA is less
due to the Ile399 residue with 1244.36 kcal mol-1 of vdw interaction. The difference in their
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Molecular Diversity
interaction energy was well explained by the sum of the interaction energies of all residues
for TCH, CHA and GCH, -60.067, 257.625 and 3.195 in proximal and -16.123, 1185.293 and
72.822 in remote, respectively. This provides evidence that TCH has strong interaction
energy when compare to CHA and GCH.
Text S2
2. Importance of both the bile salt binding sites (proximal & remote)
2.1. Overall structural stability
In this part, analysis of non-ligated, double and single ligated systems were considered to
observe the importance of the presence of both the bile salts and how these bile salts are
involved in the activation of the hCEase. The backbone RMSD of these systems was
analyzed to scrutinize the behaviors of the fluctuation of each atom with respect to their
apoform (hCEase_Apo). The average RMSD values of each system, hCEase_Apo,
hCEase_TCH, hCEase_Prx, and hCEase_Rmt, were 0.250, 0.203, 0.204, and 0.205 nm,
respectively (Fig. S5A). The apoform of the protein has shown high deviation with an average
value of 0.250 nm when compared to the complexed systems. The RMSD results indicated
that the ligated systems were more stable than the apoform during the 5 ns MD simulations.
The structure of hCEase is mainly comprised of α/β secondary structures which are
connected by flexible loops. The flexibility of these loops is directly connected with the
activation of the enzyme. The Cα RMSF values of four systems were calculated and analyzed.
The overall RMSF distribution was similar for all the four systems. The variable loop
residues have shown high flexibility over the residues forming secondary structures (Fig.
S5B). Interestingly, the double ligated system, hCEase_TCH, was observed with the largest
fluctuations than the other two systems. The binding of proximal TCH is responsible for the
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Molecular Diversity
fluctuation of the residues in the large α/β domain whereas the binding of remote TCH is
responsible for the fluctuation of the residues in the small helical domain. Hence the required
flexibility of residues of the surface loops is more in double ligated system. The RMSF of
catalytic residues was calculated, though hCEase_TCH has shown more flexibility in its
surface loops region the catalytic site residues were well stabilized during the MD simulation.
The potential energies of the 4 systems were initially fluctuated and stabilized at the value of
-1.2 106 kJ/mol during the simulation time. This result suggested that the ligated systems are
energetically more stable like the apoform, which is determined crystallographically. The Rg
values were calculated for all the four systems to assure their compactness. The ligated
systems have shown better values than the non-ligated system. The average Rg values of
hCEase_Apo, hCEase_TCH, hCEase_Prx and hCEase_Rmt systems are 2.291, 2.282, 2.279
and 2.264, respectively. The results of stability analyses revealed that all systems were well
stabilized during the simulation time.
2.2. Intermolecular hydrogen bonds
Similarly, TCH-rmt ligand has shown more number of H-bonds when compared to Rrmt during the simulation time. The TCH-rmt and Rmt-rmt ligands have shown the average
H-bond interactions of 4.4 and 3.5 H-bonds, respectively (Fig. S6A). The binding mode of
TCH-rmt (Fig. S6B) was very stable due to the strong H-bonds, maximum of 11, with the
important residues. While remote alone is present (Rmt-rmt) the binding mode (Fig. S6C)
was not stable due to the less H-bond, maximum of 6, interactions. From this result it is very
clear that the H-bond interactions are very stable and strong in the double ligated system. A
synergistic effect is observed between proximal and remote TCH in the double ligated system
where the remote TCH assists the binding of proximal TCH similarly the proximal TCH
assists the binding of remote TCH. Therefore TCH-prx and TCH-rmt ligands have shown
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Molecular Diversity
more stable polar and non-polar interactions with hCEase when they are present together.
This signifies the importance of the presence of both the bile salts. The hydrophilic and
hydrophobic interactions were analyzed using DS, Ligplot, Molegro Virtual Viewer, and
Pose View programs.
2.3. Changes in the other loops
Apart from 120-loop there are some other important loops such as 70-loop (residues 65-75),
270-loop (residues 270-285), 420-loop (residues 423-433), and 220-loop (residues 222-229)
are present around the active site and these loops are called surface loops. The TCH binding
influenced the conformational changes of these loops. Like 120-loop the movements of other
surface loops were also measured for hCEase_TCH, hCEase_Prx and hCEase_Rmt with
respect to hCEase_Apo. The considerable movement was observed in the 70- and 420-loops
of all the systems (Fig. S9). The 270-loop is another important loop present near the small
domain, the conformational flexibility of this loop plays a crucial role in the accommodation
of different substrates. The hydrophobic pocket formed by the 270-loop and small domain
can be able to accommodate the fatty acid part of the substrate. This site was proposed as a
possible pathway for the product to exit from the catalytic site 1. In the double ligated systems
the 270-loop moved 5.2 Å away from the active site similarly in hCEase_Prx system this loop
moved 3.0 Å away from the active site whereas in hCEase_Rmt this loop moved 5.8 Å
towards the active site. From the observation, it is clear that in the double ligated system the
prominent open conformation and in hCEase_Prx the significant open conformation of 270loop were observed. In hCEase_Rmt the closed conformation of 270-loop was observed
which is not suitable for a substrate to bind (Fig. S10). The loop conformational changes
clearly explain that if both the TCH (proximal and remote) are present the surface loops have
shown favorable conformational flexibility. The appreciable conformational flexibility of the
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Molecular Diversity
loops was observed while prx alone binding at its site but this flexibility was very less in the
rmt alone system. This means both the bile salts are mandatory for the flexibility of surface
loops but particularly prx involved more. Thereby binding of proximal TCH not only shows
great influence on the 120-loop but also on the 270-loop. Hence binding of both the TCH
stabilized the protein and adjusted all the surface loops thereby made the active site big
enough to accommodate the substrate molecules.
2.4. Changes in the catalytic site
The distance analysis was carried out to observe the changes happened at the catalytic site of
the protein during MD simulations. The distance between two key residues Gly107 and
His435 from the oxyanion hole and the catalytic triad, respectively, was calculated. These
residues present in the direction extremely opposite to each other in the catalytic site. Thus
the calculation of distance between them was done to ascertain the changes happened due to
the presence of TCH. Interestingly, in the double ligated system the distance was less with an
average value of 0.527 nm. In case of single ligated systems, hCEase_Prx and hCEase_Rmt,
the distance was large with an average value of 0.660 and 0.611 nm respectively similar to
the value of hCEase_Apo which has shown an average value of 0.569 nm (Fig. S11). The
distance analysis revealed that the required distance between the key residues for an effective
hydrolysis process in the catalytic site were maintained well in the double ligated system.
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Molecular Diversity
Text S3
3. Importance of 7α-hydroxyl group in TCH
3.1. Overall system stability
The backbone RMSD calculated for both the systems (Fig. S13) were well converged
throughout the simulation with an average of 0.203 and 0.227 nm (Fig. S13A). The potential
energies and the Rg of the systems were measured and it was observed that both the systems
were equilibrated well and stable throughout the simulation. The Cα RMSF revealed that the
absence of 7α-OH group greatly influence the flexibility of the protein (Fig. S13B). When 7αOH is present the flexibility of the surface loops was high but in absence of 7α-OH the
fluctuation reduced considerably. The distance between the key residues Gly107 and His435
was measured to check the size of the catalytic site (Fig. S14). Interestingly, the increase in
the distance was observed from 0.527 to 0.681 nm when 7α-OH is not present thereby leads
to the disruption in the catalytic process. The basic analysis results revealed that the
flexibility of the surface loops required for the activation of the enzyme was reduced and it
also disturbed the catalytic machinery due to the absence of 7α-OH.
3.2. Analysis of intermolecular hydrogen bonds
Similarly the 7α-OH of TCH-rmt has formed strong H-bond with Leu224. It also aids TCH to
have interaction with Trp522, Lys231, Ile353, Thr354, and Tyr526. In case of TCH_XOHrmt which did not show interaction with Leu224, Lys231, and Trp522 has shown strong
interaction with Tyr526, Thr354, and Ile353 (Fig. S16). Throughout the MD simulation,
strong and stable H-bonds were maintained by TCH-rmt, maximum of 11 and an average of
4.4, whereas TCH_XOH-rmt has shown weak interactions, maximum of 7 and an average of
3.6. From this H-bond interaction analysis it has become very clear that missing a 7α-OH
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Molecular Diversity
group exerted a huge difference in the protein-ligand binding. This result provided the
molecular explanation for the fact that the key interaction of 7α-OH group is very important
for the activation of hCEase. The lack of stable H-bond interactions was mainly due to the
absence of 7α-OH group. Though the TCH_XOH forms minimum number of H-bonds the
necessary interactions required for the activation of enzyme was missing. As a result the
active site became collapsed and not suitable for a substrate or inhibitor to bind.
3.3. Interaction energetics
The interaction energies of the important residues in the TCH and TCH_XOH system were
compared. The results showed the favorable electrostatic interaction energy with the
important residues such as Ala117, Asn118, Asn121, Asn122, Tyr123, Leu124, Gln440,
Lys445, Thr449, Pro450, Thr451, Gly452, and Tyr453. In hCEase_TCH, the proximal site
residues such as Phe119, Asn121, Thr449, Pro450 and Thr451 has shown strong electrostatic
interaction of -6.348, -6.916, -4-869, -4.247 and -8.881 kcal mol-1 with the ligand whereas the
same residues have shown weak electrostatic interaction 1.597, -1.770, -0.300, -0.521 and 2.054 kcal mol-1 with the TCH_XOH (Table S3). Similarly, favorable interaction was
observed for TCH in the remote site (Table S4). The total interaction energy for all residues
of TCH and TCH_XOH in proximal and remote sites are -60.067, -53.210, and -16.123,
72.822 kcal mol-1 respectively.
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Molecular Diversity
Table S1 Non-bonded interaction energies in kcal/mol between proximal bile salts and
protein of hCEase_TCH, hCEase_CHA, hCEase_GCH systems
Residue
TCH
CHA
GCH
Coulomb
Vdw
Coulomb
Vdw
Coulomb
Vdw
Ala117
3.406
-2.312
3.693
3.788
-3.346
-0.118
Asn118
-1.134
-1.599
-1.249
-1.361
-0.680
-0.168
Phe119
-6.348
-2.921
-1.408
32.862
-2.076
-4.131
Leu120
-2.125
-0.105
-1.965
-0.275
1.644
43.199
Asn121
-6.916
-1.337
-3.779
-0.152
-10.282
-0.577
Asn122
0.162
-7.588
1.566
-2.894
-1.218
-1.224
Tyr123
3.404
-3.719
-2.589
-0.700
1.993
-0.258
Leu124
-0.893
1.273
-0.827
201.868
0.510
-1.224
Tyr125
2.411
-0.240
-0.730
-0.065
-0.018
-0.091
Gln440
0.141
-0.305
0.158
-2.094
4.921
-1.526
Lys445
11.877
-1.951
-0.851
-1.773
-10.749
-2.565
Ala448
2.469
-0.371
0.076
-0.179
-6.051
-0.256
Thr449
-4.869
-5.387
2.647
41.523
-1.464
-3.932
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Molecular Diversity
Pro450
-4.247
-0.562
-1.195
-0.207
-2.422
-1.209
Thr451
-8.881
9.597
-1.794
-2.944
-6.618
-5.803
Gly452
1.309
-0.475
-0.092
1.027
-2.803
-0.365
Tyr453
0.056
-2.639
-4.110
-4.638
1.933
-0.786
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Molecular Diversity
Table S2 Non-bonded interaction energies in kcal/mol between remote bile salts and protein
of hCEase_TCH, hCEase_CHA, hCEase_GCH systems
Residue
TCH
CHA
GCH
Coulomb
Vdw
Coulomb
Vdw
Coulomb
Vdw
Leu224
3.392
-2.523
-0.626
-2.055
-3.040
-2.708
Ser225
4.507
-0.328
2.666
-0.736
-5.624
-0.465
Phe351
-2.906
-2.131
-0.208
-3.333
2.923
-2.265
Thr352
-1.813
-1.417
2.965
-1.116
1.175
-1.774
Ile353
-2.015
-3.010
1.794
-2.151
-1.796
-1.236
Thr354
-3.070
16.543
-9.399
5.189
-2.886
-0.497
Pro396
-0.073
-2.452
1.545
-3.133
-3.118
-1.932
Ile399
-0.569
-2.414
1.282
1244.360
2.028
-4.125
Trp522
-0.710
-2.691
0.955
-4.742
1.054
-7.408
Thr523
-4.406
1.289
1.658
-0.776
10.953
-2.423
Tyr526
-6.170
5.508
-4.079
-3.641
-0.480
-5.091
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Molecular Diversity
Table S3 Non-bonded Interaction Energy values between proximal hCEase_XOH and
hCEase_TCH
Residue
TCH
TCH-XOH
Coulomb
Vdw
Coulomb
Vdw
Ala117
3.406
-2.312
-3.068
-0.466
Asn118
-1.134
-1.599
-1.729
-2.258
Phe119
-6.348
-2.921
1.597
14.147
Asn121
-6.916
-1.337
-1.770
-0.190
Leu120
-2.125
-0.105
-1.144
-0.315
Asn122
0.162
-7.588
-7.658
-3.786
Tyr123
3.404
-3.719
-1.990
-0.673
Leu124
-0.893
1.273
-5.815
-1.511
Tyr125
2.411
-0.240
4.304
-0.309
Gln440
0.141
-0.305
-0.664
0.221
Lys445
11.877
-1.951
-7.749
-1.584
Ala448
2.469
-0.371
1.596
-0.077
Thr449
-4.869
-5.387
-0.300
-2.474
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Molecular Diversity
Pro450
-4.247
-0.562
-0.521
-0.187
Thr451
-8.881
9.597
-2.054
7.242
Gly452
1.309
-0.475
-0.534
-1.286
Tyr453
0.056
-2.639
-0.198
-2.296
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Molecular Diversity
Table S4 Non-bonded interaction energy values between remote hCEase_XOH and
hCEase_TCH
Residue
TCH
TCH-XOH
Coulomb
Vdw
Coulomb
Vdw
Leu224
3.392
-2.523
-6.170
10.589
Ser225
4.507
-0.328
6.549
-0.298
Phe351
-2.906
-2.131
1.262
6.127
Thr352
-1.813
-1.417
-1.824
-1.644
Ile353
-2.015
-3.010
-4.437
-2.969
Thr354
-3.070
16.543
-6.016
2.494
Pro396
-0.073
-2.452
-0.177
-2.216
Ile399
-0.569
-2.414
1.946
122.338
Trp522
-0.710
-2.691
2.749
-4.284
Thr523
-4.406
1.289
0.508
-1.753
Tyr526
-6.170
5.508
-6.089
-2.950
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Molecular Diversity
Fig. S1 Non-ligated and different ligated systems used in MD simulations. The bile salt
binding sites are shown in black circles
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Molecular Diversity
Fig. S2 Basic analysis plots. A) The backbone RMSD graph was plotted to check the overall
stability of hCEase_TCH, hCEase_CHA and hCEase_GCH systems. The Cα-RMSF graph
for B) all protein residues C) catalytic site residues. The more fluctuating and catalytic
residues are highlighted in the RMSF plot
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Molecular Diversity
Fig. S3 Structural changes in the surface loop regions. The conformational changes of
surface loops were observed and compared between hCEase_TCH and (A) hCEase_CHA (B)
and hCEase_GCH systems. The distance between the loops were measured in Armstrong and
represented in black dotted lines. The thick arrows pointing the distance of the two major 120
and 270 loops
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Molecular Diversity
Fig. S4 Distances between the catalytic residues during MD simulation. The distances
between two important catalytic site residues, Gly107 and His435, located at the active site
were measured in hCEase_TCH, hCEase_CHA, and hCEase_GCH systems throughout the
MD simulations
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Molecular Diversity
Fig. S5 Stability analyses of four systems. The A) backbone RMSD and B) Cα-RMSF plot
of hCEase_Apo, hCEase_TCH, hCEase_Prx and hCEase_Rmt systems
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Molecular Diversity
Fig. S6 H-bond interactions and binding modes of TCH at the remote site. (A) The
number of H-bond interactions between protein and remote ligands of hCEase_TCH and
hCEase_Rmt systems throughout the MD simulations was measured. B) and C) The binding
modes of TCH at the active site of TCH-rmt and Rmt-rmt systems respectively. The key
interacting residues were shown in white and green color sticks whereas the ligands were
shown in deep teal color sticks. The H-bond interactions are shown in black dashed lines
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Molecular Diversity
Fig. S7 Structure overlay of hCEase and bCEase. Comparison of MD simulated hCEase
(gray color) with the crystal structure of bCEase (green color). The TCH was shown in stick
form and the surface loops around the active site are labeled
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Molecular Diversity
Fig. S8 Comparison of 120-loop movement. Distances between the residue Leu120 present
in the 120-loop of hCEase_Apo and hCEase_TCH, hCEase_Prx, hCEase_Rmt, respectively,
was measured and provided in Armstrong scale after 5 ns MD simulations. The distance was
shown in black dashed lines
Fig. S9 Conformational changes of surface loops. The conformational changes of surface
loops were observed between hCEase_Apo and A) hCEase_TCH, B) hCEase_Prx, and (C)
hCEase_Rmt systems. The distance were measured and represented in black dashed lines
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Molecular Diversity
Fig. S10 Conformational changes of 270-loop. Conformational changes of 270-loop were
observed between A) hCEase_TCH and hCEase_Prx and B) hCEase_TCH and hCEase_Rmt
systems. The important residue is shown in stick form
Fig. S11 Distances between the key residues in the catalytic site. The distances between
two important catalytic site residues, Gly107 and His435, located at the active site were
measured in hCEase_Apo, hCEase_TCH, hCEase_Prx, and hCEase_Rmt systems throughout
the MD simulations
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Molecular Diversity
Fig. S12 The 2D structures of TCH and TCH_XOH. The position of 7α-OH group is
highlighted in green color for TCH and red color for TCH_XOH
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Molecular Diversity
Fig. S13 Stability analyses plot. The A) RMSD and B) RMSF analyses plots for,
hCEase_TCH and hCEase_XOH systems, respectively. The most fluctuating residues were
labeled in the RMSF plot
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Molecular Diversity
Fig. S14 Distances between Gly107 and His435 plot. The distances between two important
catalytic site residues, Gly107 and His435, at the active site of hCEase_TCH (grey) and
hCEase_XOH (pink) were measured throughout the MD simulations. The average distances
for
both
the
systems
are
graph.
28
given
in
the
Molecular Diversity
Fig. S15 H-bond interaction analysis. The H-bond interactions between protein and ligand
in the A) proximal and B) remote sites were measured during 5 ns MD simulation
29
Molecular Diversity
Fig. S16 Binding modes of TCH and TCH_XOH at the remote site. The binding modes
and H-bond interactions between protein and ligand A) TCH and B) TCH_XOH are shown in
deep teal color and lime color stick forms. The missing 7α-OH group region is highlighted in
red circles. The H-bond interactions are shown in black dashed lines
30
Molecular Diversity
Fig. S17 Conformational changes of the surface loops. Comparison of conformational
changes of the surface loops between hCEase_TCH and hCEase_XOH systems
31