Active Site Flexibility of Mycobacterium ... - ACS Publications

Aug 18, 2017 - However, the dynamic details of ICL which could give insights to the ICL–ligand interaction have yet to be solved. Therefore, a serie...
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The Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form Yie-Vern Lee, Sybing Choi, Habibah Wahab, and Yee Siew Choong J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00265 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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The Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form

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Yie-Vern Lee†, Sy Bing Choi┴, Habibah A. Wahab§, and Yee Siew Choong†,* †

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia ┴ Pharmaceutical Design and Simulation Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia *

Corresponding author E-mail: [email protected]. Tel.: +604 653 4837. Fax: +604 653 4803

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ABSTRACT

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Tuberculosis (TB) still remains as global threat due to the emerging of drug resistant strain.

15

Instead of focus on drug target of active stage TB, we are highlighting the isocitrate lyase (ICL)

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at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis

17

(MTB) to survive during dormant phase. In addition, the absent of ICL in human has made ICL a

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potential drug target for TB therapy. However, the dynamics details of ICL which could give

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insights to the ICL-ligand interaction is yet to be solved. Therefore, a series of ICL dimer

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dynamics study through molecular dynamics simulation was performed in this work. The ICL

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active site entrance gate closure is contributed by hydrogen bonding and electrostatic interactions

22

with the C-terminal. Analysis suggested that on the open-close behavior of ICL active site

23

entrance is depending on the type of ligand presence in the active side. We also observed the four

24

residues (Ser91, Asp108, Asp153 and Cys191) which could possibly be the nucleophile for

25

nucleophilic attack for the cleavage of isocitrate at the C2-C3 bond. We hope that the elucidation

26

of ICL dynamics can benefit in future works such as lead identification or antibody design

27

against ICL for TB therapeutic.

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INTRODUCTION

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Tuberculosis (TB) is one of the global threats that infected one-third of the world population.1

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Within 50 years of hard work from the global TB research society, TB has managed to be

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controlled and cured by chemotherapy.2 However, the emerging of drug resistant strain

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especially the newly emerged totally drug resistant strain (TDR) of Mycobacterium tuberculosis

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(MTB) in Iran,3 has alarmed the research to accelerate the pace of TB drug development before it

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reverts back into the incurable days.

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Isocitrate lyase (ICL) is one of the potential drug targets in MTB during dormant/latent

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stage.4, 5 It plays the key role to convert isocitrate into succinate and glyoxylate, facilitating MTB

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to enter glyoxylate bypass (bypass the Krebs Cycle) and to utilize lipid as sole carbon source

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during dormancy.6 This theory is further supported by studies showing the importance of ICL in

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hypoxic condition and how MTB failed to survive in murine model (latent TB model) without

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isocitrate lyase.7-10 In year 2000, ICL X-ray crystal structure of MTB was solved11 and enable the

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extensive study on ICL.

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In previous studies, MD simulations have been employed to study ICL in terms of its

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flexibility and stability for different point mutation and different potential inhibitors.12-15 In this

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work, MD simulations for three systems of ICL dimers have been performed: one system in apo-

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form and two systems in complex form. This study is initiated by two objectives: to study the

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open-close conformational changes of the active site with and without the presence of the

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substrate. However, the full cycle for the opening and closing of the active site is realized within

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30 ns MD simulation. But the simulations are still able to gain insights of some useful

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information. The flexible loops as well as substrate conformation were monitored and non-

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bonded interactions involved in the active site were analyzed.

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MATERIALS AND METHODS

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Molecular Dynamics (MD) Simulation. Three simulations were performed. The initial atomics

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coordination was obtained from X-ray crystal structure in Protein Data Bank. The three systems

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were named: Apo_ICL, Complex1_ICL and Complex2_ICL. Apo_ICL is ICL dimer in apo-form

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(PDB id: 1F6111), Complex1_ICL is ICL dimer with glyoxylate and succinate (PDB id: 1F8I11)

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and Complex2_ICL is ICL dimer (PDB id: 1F8I11) with isocitrate (PDB id: 1XG416).

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The following descriptions were the system setup details of these three systems: All system

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was incorporated with Mg2+ as ICL cofactor, neutralized with Na+ as counterions and solvated

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with 12 Å truncated octahedral water box (TIP3PBOX). The protein and ligand topology files

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were prepared using FF03 AMBER force field17 and generalized AMBER force field.18 The total

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number of atoms was 100919, 98092 and 98118 for Apo_ICL, Complex1_ICL and

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Complex2_ICL, respectively (Supplementary Information Table S1). All MD simulations were

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performed with AMBER 8 package.19

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Energy minimization was started with 500 steps of steepest descent and followed with

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1000 steps of conjugate gradients to remove unfavorable contacts using Sander module of

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AMBER 8. The solvent of the system was first heated to 100 K with NVT ensemble followed by

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heating of the whole system to 300 K with NPT ensemble. Throughout MD simulation, 0.2 fs

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time step, SHAKE algorithm, periodic boundary and 10 Å cut-off were applied. The

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equilibration phase was 2 ns and the production phase of the simulation was 28 ns.

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Principal Component Analysis (PCA). Prior to PCA analysis with PCAsuite20, essential

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dynamics of ICL was extracted in order to eliminate noises and redundancy. Production

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trajectory (2 to 30 ns) of each system was compressed with 90% quality using PCAzip module of

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PCAsuite. Within this step, naïve data of trajectories had arranged into matrixes, hence their

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covariance matrixes were calculated. Covariance matrix then subsequently diagonalized into

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eigenvectors with corresponding eigenvalues, whereby the noises and redundant data were

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reduced along with the diagonalization process. PCZdump module was used to retrieve the

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eigenvectors and eigenvalues that represent the vector and magnitude of movement of the

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systems and porcupine plot is used to view the retrieved motion.

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MM-PBSA/GBSA Free Energy Calculation. The free energy calculation was performed using

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AMBER 8. A total of 1000 snapshots were collected from the last 5 ns (with 5 ps interval) of

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MD simulation. The MM calculation with dielectricity constant of 1.0 was performed.

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Subsequently, PB calculation was done by pbsa program with the parameter: solute dielectric

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constant of 1, solvent dielectric constant of 80, lattice spacing of 2 Å, solvent probe radius of 1.4

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Å, iterations number of 1000 and cavity radii was set according to system prmtop file. Non-polar

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contribution towards desolvation was calculated by molsurf module.

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Hydrogen Bond Analysis and Radial Distribution Function Calculation. Hydrogen bond was

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analyzed using hbond module of PTRAJ, with cutoff 3 Å and 120o for distance and angle,

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respectively. Radial distribution function was performed with radial module in PTRAJ, with bin

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spacing of 0.5 and maximum histogram of 15 Å.

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RESULTS AND DISCUSSION

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ICL dimer MD simulation. ICL is reported to be functioned as tetramer but stable as dimer.11,21

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In addition, ICL monomer is lacking of α-12 and α-13 helix swap and causing the C-terminal of

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ICL to be is rather instable.12 The study reflected that the active site of ICL has open and close

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conformation in its apo- and holo-form. The active loop (residue 185-196) at the active site

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served as the “entrance gate" for the substrate or ligand entry. This “entrance gate” is shifted

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about 10-15 Å away to close the active site and the C-terminal (residue 411-428) of adjacent unit

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should overtake the original place of active loop and lay on top of it to secure the closure of

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active site (Figure 1). As the above-mentioned activities involved 2 units of ICL monomer, MD

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simulation for ICL was therefore performed in dimer format.

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All systems were well equilibrated with overall root mean square deviation (RMSD) below

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3.5 Å (Figure 2). In order to identify the regions that contributed to the fluctuation of ICL, the

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RMSD of active sites and terminal loops were also calculated (Supplementary Information

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Figure S1 and Table S1 shows the location and residue number of the active sites, terminal loops

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and loops near to active site). Figure 3 shows that active site RMSD was below the value of 2 Å.

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The dimer fluctuation was contributed mostly by the C-terminal loops at RMSD nearly 5 Å. This

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RMSD analysis is well correlated with reported work11 on the open and close conformation of

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ICL active site. The tightly close of C-terminal on top of the entrance gate is highly contributed

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by hydrogen bond and electrostatic interactions between the C-terminal and the entrance gate.

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The phenomena was also evidenced from mutagenesis work whereby the mutations of a few

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glutamate residues at C-terminal destroyed the hydrogen bonding and electrostatic interactions

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with neighboring residues, thus ICL loses its enzymatic activity.12

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Figure 1. Active site of Apo_ICL and Complex_ICL. Active loop (residue 185-196) in open and close conformation for respective system is in yellow representative, the C-terminal (residue 411-428) in cyan representative and the other loops around the active sites is in purple representation. Once the active loop closed the active site, C-terminal from adjacent unit will overtake the original place of active loop and lay on top of the active loop to secure the closure of the active site. Figure is generated using VMD 1.9.1.

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Figure 2. Overall root mean square deviation (RMSD) analysis as the function of time during MD simulation for Apo_ICL, Complex1_ICL and Complex2_ICL system.

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Open-close behavior of active site entrance gate could be affected by the substrate in the

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active site. Figure 3 also shows that the C-terminals were heavily fluctuated with the RMSD

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around 5 Å for Complex1_ICL and Complex2_ICL. However, the shifting of the C-terminals out

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from the active sites is not significant after 30 ns MD simulation. The active sites were remained

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very stable with RMSD less than 1.5 Å. This shows that the active loop is tightly close with C-

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terminal on top of it. We further studied the 147 active site residues (Supplementary Information

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Table S2) by PCA. Total eigenvalue of top 1 eigenvalue of each simulated system is tabulated in

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Table 1 (Supplementary Information Table S3 further detailed the top 3 eigenvalue from 2 to 30

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ns of MD simulation in each active site; Supplementary Information Table S4 further detailed the

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top eigenvalue of each active site sorted from highest to lowest eigenvalue). Apo_ICL has

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highest eigenvalue at the 16th ns (18.19) for active site 1 and 22nd ns (7.12) for active site 2. Their

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eigenvalue represented 10.77% and 5.65% out of total eigenvalue of 168.77 and 126.09 of

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respective active site. Comparatively, Complex1_ICL and Complex2_ICL have relatively lower

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eigenvalues, indicating that they were less dynamics compare to Apo_ICL. Highest eigenvalues

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for Complex1_ICL active site 1 and 2 is obtained from 28th ns (7.55) and 4th ns (4.16),

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representing 10.28% and 6.26% of total eigenvalue of 73.4 and 66.41 of respective active site.

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Lastly, highest eigenvalues for Complex2_ICL active site 1 and 2 is obtained from 12th ns (3.82)

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and 26th ns (5.00), representing 7.41% and 7.28% of total eigenvalue of 51.64 and 68.71 of

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respective active site. Figure 4 shows the porcupine plot of each active site for all simulated

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systems. The direction and thickness of the arrow indicates the vector and magnitude of the

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motion in the system.

152

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Figure 3. Active and terminal loops RMSD of all systems. Loop 1 and 2 represent the loop from monomer 1 and 2, respectively, in ICL dimer. Figure is generated using VMD 1.9.1. 10 ACS Paragon Plus Environment

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Table 1. Highest eigenvalue of each active site in all systems from 2 to 30 ns of MD simulation System Total eigenvector Percentage of highest eigenvector Highest Eigenvalue Time (ns) with observed highest eigenvector

Apo_ICL Active Active Site 1 site 2 168.77 126.09 10.77 5.65 18.19 16

7.12 22

Complex1_ICL Active Active Site 1 site 2 73.40 66.41 10.28 6.26 7.55 28

4.16 4

Complex2_ICL Active Active Site 1 site 2 51.64 68.71 7.41 7.28 3.82 12

5.00 26

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Figure 4. Porcupine plot of highest eigenvalue eigenvector of the active site in (a) Apo_ICL, (b) Complex1_ICL, and (c) Complex2_ICL. Red arrow summarized the vector and magnitude of motion. Figure is generated using PCA suite.

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Although the 30 ns MD simulation was not able to observe a complete open-close

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movement of the entrance gate, the PCA analysis manage to demonstrate the movement of the

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entrance gate along with its vector and magnitude. Both Apo_ICL and Complex1_ICL, the

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entrance gate is moving towards the active site. For Apo_ICL, the entrance gate is in “open”

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conformation. Its gate closing behavior is very significant compare to Complex1_ICL which is in

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“close” conformation. However, Complex2_ICL (also having its entrance gate in “close”

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conformation as in Complex1_ICL) exhibits opening behavior. Hence, it is postulated that

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different substrate presence inside the active site could affect the open-close behavior differences

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of the entrance.

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In addition, radial distribution function calculation showed that there could be less than

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2.5% and 25% of possibility for water to be found 3 Å away from the substrates for

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Complex1_ICL and Complex2_ICL system, respectively (Figure 5). Although the occurrence of

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water molecule within the distance in Complex1_ICL is 10 times lesser compare with that of

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Complex2_ICL, but the possibility of the water to exist the binding site is still relatively low.

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Therefore, unlikely water molecules can be found within 3 Å from the substrates to form any

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water mediated hydrogen bond in both complex systems. Hydrogen bond analysis was also

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performed on the active site residues and the substrates. In Complex1_ICL and Complex2_ICL

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system, no hydrogen bond was found to be directly contributed towards the substrates. Table 2

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summarizes the top 3 hydrogen bonding (Detail information is available at Supplementary

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Information Table S5). Most of them appeared to occupy > 80% throughout the simulation time.

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No hydrogen bond with occupancy > 80% of the simulation time was found in nearby loops

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around the active site. Therefore, it is unlikely that water molecules are involved in the open-

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closed behavior of ICL active site entrance gate.

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Figure 5. Radial distribution function plot for (a) Complex1_ICL and (b) Complex2_ICL.

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Table 2. The top three hydrogen bonding occupancy in all systems System Apo_ICL Monomer 1 Apo_ICL Monomer 2 Complex1_ICL Monomer 1 Complex1_ICL Monomer 2 Complex2_ICL Monomer 1 Complex2_ICL Monomer 2

Donor Val117 Ile282 Phe345 Ala645 Ala595 Ile568 Phe345 Ser116 Ile282 Lys770 Ile710 Ala605 Ser116 Phe345 Ile282 Phe778 Tyr516 Ala776

Acceptor Val121 Ala311 Cyc314 Trp701 Val642 Ala595 Cys314 Gln129 Ala311 Val494 Ala739 Val652 Val120 Cys314 Ala311 Asn782 Ala498 Ala780

Occupancy (%) 91.06 85.85 81.86 85.40 81.31 80.91 88.53 85.87 82.48 96.34 84.40 78.19 88.91 84.56 83.07 60.87 58.06 46.78

193 194 195

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ICL mechanism of action. From MM-PBSA calculation, total estimated binding free energy

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was in negative value for all monomers (indicated good binding of ligands) except

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Complex2_ICL chain B (Table 3). Binding free energy for each of the monomer was

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significantly contributed by electrostatics interaction. The polar solvation free energy of all was

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unfavorable thus suggesting that the substrate-ICL interactions favour in hydrophobic condition.

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The binding free energy of glyoxylate and succinate (in Complex1_ICL) is -338.23 kcal/mol,

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whereas the binding free energy for isocitrate (in Complex2_ICL) is -55 kcal/mol. Despite the

203

cleavage and formation of isocitrate is a reversible reaction, the above evident concluded that the

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binding of isocitrate in the active site is less favourable compare to glyoxylate and succinate.

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ICL catalyzes the cleavage and formation of isocitrate (Figure 6). From the ICL X-ray

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crystal structure11 and previous mechanism study by William et. al,22 the sequence of substrate

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binding should begin with glyoxylate and followed by succinate. The formation of isocitrate

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involved Claisen condensation process. However, not much study is available for isocitrate

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cleavage mechanism. It is suggested that a nucleophilic attack at the C2 position of isocitrate for

210

cleavage.11

211

mechanical/molecular mechanical (QM/MM) calculation on isocitrate lyase superfamily, 2.3-

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Dimethylmalate lyase suggested the cleavage of C2 and C3 bond is the result of nucleophilic

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attack by neighboring base.23 Besides, another kinetic calculation proved that Cys191 is a

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catalytic base that able to perform this nucleophilic attack.24 From the MD simulation of

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Complex2_ICL complex (isocitrate bounded ICL), the observation is indeed in line with the

216

mentioned nucleophilic attack mechanism. In addition, other than Cys191, we observed another

217

three residues namely Ser91, Asp108 and Asp153 (located within 5 Ǻ from isocitrate) could also

218

be potential nucleophiles.

This

hypothesis

is

further

supported

by

recent

study

on

quantum

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Figure 6. Reaction scheme of isocitrate lyase. Table 3. MMPB/SA calculation for Complex_ICL systems. Complex1_ICL has glyoxylate and succinate and Complex2-ICL has isocitrate in their active sites respectively. Energy term ∆EELE ∆EVDW ∆EMM ∆GPB ∆GGB ∆GSA ∆GMMPBSA ∆GMMGBSA

Complex1_ICL Monomer 1 -1232.47 ± 19.52 4.17 ± 5.77 -1228.30 ±17.28 894.06 ± 14.65 1050.33 ± 15.12 -3.98 ± 0.10 -338.23 ± 14.06 -181.96 ± 10.15

Energy (kcal/mol) Complex1_ICL Complex2_ICL Monomer 2 Monomer 1 -1202.28 ± 17.62 -853.63 ± 17.48 3.48 ± 5.18 -0.08 ± 6.20 -1198.79 ± 15.80 -853.71 ± 17.29 872.25 ± 10.64 801.85 ± 13.37 1007.12 ± 11.81 822.04 ± 13.80 -3.75 ± 0.10 -3.14 ± 0.11 -330.29 ± 13.24 -55.00 ± 13.95 -195.43 ± 9.96 -34.81 ± 10.22

Complex2_ICL Monomer 2 -588.35 ± 15.39 0.49 ± 7.19 -587.86 ± 15.86 762.33 ± 14.06 681.43 ± 11.10 -2.92 ± 0.07 171.56 ± 15.38 90.66 ± 11.83

226 227

228

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Future study. We once doubted if the catalysis activity of ICL is influenced by the dynamical effect of

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ICL. In fact, catalysis of enzyme including ICL should obey the transition state theory without accounting

231

dynamical effects.25-26 “Dynamical effects” is a rather confusion term used in the field to relate dynamics

232

of the enzyme like conformational change prior to the mechanism of catalysis. For instance,

233

conformational is first to be changed, thus bring the respective residues closer prior to the formation of

234

the transition state region for the catalysis to happen.25 This is in line with the evidence from previous

235

study11 and our results that ICL possess conformational change in order to catalyze isocitrate. However, a

236

number of reported studies disagree that the dynamics drive the catalysis.26-28 It is the reacting region

237

fulfills the transition state theory value that leads to the enzymatic catalysis.25 Therefore, understanding

238

the ICL catalysis mechanism based on the information from MD simulation alone could be

239

inadequate. The detail insights of the catalysis will need the information on the transition state of

240

the isocitrate. As isocitrate has four ionizable group for various protonation state, i.e. the ~OH

241

group, the study on protonation and pKa calculation to elucidate the catalytic event would be the

242

appropriate approach.29-31 Besides, by involving QM/MM calculations that have been developed

243

and ready to be used29,32-36 at the active site or catalytic steps will provide more detail on the

244

catalytic mechanism. Once the important residues that involved in catalytic steps were identified,

245

point mutation experiments regardless of in silico or in vitro, could contribute more valuable

246

information towards the ICL catalytic mechanism.12,15

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CONCLUSION

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A total of 30 ns MD simulation was performed on both open (Apo_ICL) and close conformation

249

(Complex1_ICL and Complex2_ICL) of ICL crystal structures. From the simulation, C-terminal

250

loop of ICL contributed majority of the dynamics as it involved in securing the closing of active

251

site. Our study showed that the dynamics of each monomer were found behaved in a different

252

manner. The active loops of each monomer of Apo_ICL, which expected to exhibit “gate-

253

opening” behavior, were found to fluctuate at different magnitude in essential dynamics

254

calculation (PCA). Whereas in close conformation of ICL, active loop of Complex1_ICL and

255

Complex2_ICL which expected to show “gate-closing” behavior, showed fluctuation with

256

opposite magnitude. This means that Complex1_ICL showed “gate-closing behavior but

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Complex2_ICL showed “gate-opening” behavior. All these strengthen the postulation that

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different substrate within the active site determines the gate-opening or closing behavior. This is

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further supported by evidence from free energy calculation, where the binding of

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glyoxylate/succinate (gate-closing) is stronger than isocitrate (gate-opening). Electrostatic

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interactions were found to be contributed the most on the binding affinity. In addition, the

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dynamics of the active site is not only favors for hydrophobic interaction but also reflected that it

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is unlikely to have water presences in the active site during the interaction. This is in line with

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the proposed nucleophilic attack mechanism of action of ICL where no water was involved.

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ACKNOWLEDGEMENTS

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This work is supported by Fundamental Research Grant Scheme (FRGS; 203/CIPPM/6711439)

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and the computational facilities of Higher Institution Centre of Excellence Grant (HICoE;

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311/CIPPM/44001005) from the Malaysian Ministry of Higher Education. Besides, we would

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like to express our sincere gratitude for Professor Hideo Matsuda for his support in Genome

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Information Engineering Laboratory, Department of Bioinformatic Engineering, Graduate

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School of Information Science and Technology, Osaka University, Japan. Y.-V. Lee would like

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to acknowledge Malaysia Ministry of Science, Technology and Innovation (MOSTI) for

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National Science Fellowship.

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ASSOCIATED CONTENT

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Supporting Information:

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The supporting information is available free of charge via the Internet at http://pubs.acs.org.

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The loops around M. tuberculosis ICL active site (Figure S1); detail atomic information of

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isocitrate lyase (ICL) dimer systems for molecular dynamics simulation (Table S1); the

280

active site residues, C-terminal residues, N-terminal residues and the loops around

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Mycobacterium tuberculosis ICL active site (Table S2); top 3 eigenvalues of all simulated

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systems (Table S4); the top eigenvalue of each active site in all the simulated system sorted

283

from highest to lowest eigenvalue; Hydrogen analysis for all simulated systems (Table S5).

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AUTHOR INFORMATION

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Corresponding Author

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* Email: [email protected]

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ORCID

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Yee Siew Choong: 0000-0001-5067-2073

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Notes

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The authors declare no competing financial interest.

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(17) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. A Point-Charge Force Field for Molecular Mechanics Simulations of Proteins Based on Condensed-Phase Quantum Mechanical Calculations. J. Comput. Chem. 2003, 24, 1999-2012. (18) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25, 1157-1174. (19) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D. A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.; Ross, W. S.; Kollman, P. A. AMBER 8. 2004. (20) Luque, F.; Orozco, M. PCA Suite: Software Package for Glossy Trajectory Compression using Principle Component Analysis Techniques. http://mmb.pcb.ub.es/software/pcasuite/ Molecular Recognition and Bioinformatics Group, University of Barcelona. (accessed 1-082015). (21) Giachetti, E.; Pinzauti, G.; Bonaccorsi, R.; Teresa Vincenzini, M.; Vanni, P. Isocitrate Lyase from Higher Plants. Phytochemistry 1987, 26, 2439-2446. (22) McFadden, B. A.; Williams, J. O.; Roche, T. E. Mechanism of Action of Isocitrate Lyase from Pseudomonas indigofera. Biochemistry 1971, 10, 1384-1390. (23) Jongkon, N.; Chotpatiwetchkul, W.; Gleeson, M. P. Probing the Catalytic Mechanism Involved in the Isocitrate Lyase Superfamily: Hybrid Quantum Mechanical/Molecular Mechanical Calculations on 2,3-Dimethylmalate Lyase. J. Phys. Chem. B. 2015, 119, 1147311484. (24) Moynihan, M. M.; Murkin, A. S. Cysteine is the General Base that Serves in Catalysis by Isocitrate Lyase and in Mechanism-based Inhibition by 3-Nitropropionate. Biochemistry 2013, 53, 178-187. (25) Tuñón, I.; Laage, D.; Hynes, J. T. Are There Dynamical Effects in Enzyme Catalysis? Some Thoughts Concerning the Enzymatic Chemical Step. Arch. Biochem. Biophys. 2015, 582, 42-55. (26) Warshel, A.; Bora, R. P. Perspective: Defining and Quantifying the Role of Dynamics in Enzyme Catalysis. J. Chem. Phys. 2016, 144, 180901. (27) Kamerlin, S. C. L.; Mavri, J.; Warshel, A. Examining the Case for the Effect of Barrier Compression on Tunneling, Vibrationally Enhanced Catalysis, Catalytic Entropy and Related Issues. FEBS Lett. 2010, 584, 2759-2766. (28) Glowacki, D. R.; Harvey, J. N.; Mulholland, A. J. Taking Ockham's Razor to Enzyme Dynamics and Catalysis. Nat. Chem. 2012, 4, 169-176. (29) Olsson, M. H. M.; Siegbahn, P. E. M.; Blomberg, M. R. A.; Warshel, A. Exploring Pathways and Barriers for Coupled ET/PT in Cytochrome C Oxidase: A General Framework for Examining Energetics and Mechanistic Alternatives. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 244-260. (30) Adamczyk, A. J.; Cao, J.; Kamerlin, S. C. L.; Warshel, A. Catalysis by Dihydrofolate Reductase and Other Enzymes Arises from Electrostatic Preorganization, Not Conformational Motions. Proc. Nat. Acad. Sci. 2011, 108, 14115-14120. (31) Borštnar, R.; Repič, M.; Kamerlin, S. C. L.; Vianello, R.; Mavri, J. Computational Study of the pKa Values of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B. J. Chem. Theory Comput. 2012, 8, 3864-3870.

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(32) Warshel, A.; Levitt, M. Theoretical Studies of Enzymic Reactions: Dielectric, Electrostatic and Steric Stabilization of the Carbonium Ion in the Reaction of Lysozyme. J. Mol. Biol. 1976, 103, 227-249. (33) Warshel, A. Computer Modelling of Chemical Reactions in Enzymes and Solutions; Wiley: New York, 1991. (34) Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, M. H. M. Electrostatic Basis for Enzyme Catalysis. Chem. Rev. 2006, 106, 3210-3235. (35) Frushicheva, M. P.; Cao, J.; Warshel, A. Challenges and Advances in Validating Enzyme Design Proposals: The Case of Kemp Eliminase Catalysis. Biochemistry 2011, 50, 3849-3858. (36) Poberznik, M.; Purg, M.; Repic, M.; Mavri, J.; Vianello, R. Empirical Valence Bond Simulations of the Hydride-Transfer Step in the Monoamine Oxidase A Catalyzed Metabolism of Noradrenaline. J. Phys. Chem. B 2016, 120, 11419-11427.

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FIGURE CAPTIONS Figure 1. Active site of Apo_ICL and Complex_ICL. Active loop (residue 185-196) in open and close conformation for respective system is in yellow representative, the C-terminal (residue 411-428) in cyan representative and the other loops around the active sites is in purple representation. Once the active loop closed the active site, C-terminal from adjacent unit will overtake the original place of active loop and lay on top of the active loop to secure the closure of the active site. Figure is generated using VMD 1.9.1. Figure 2. Overall root mean square deviation (RMSD) analysis as the function of time during MD simulation for Apo_ICL, Complex1_ICL and Complex2_ICL system. Figure 3. Active and terminal loops RMSD of all systems. Loop 1 and 2 represent the loop from monomer 1 and 2, respectively, in ICL dimer. Figure is generated using VMD 1.9.1. Figure 4. Porcupine plot of highest eigenvalue eigenvector of the active site in (a) Apo_ICL, (b) Complex1_ICL, and (c) Complex2_ICL. Red arrow summarized the vector and magnitude of motion. Figure is generated using PCA suite. Figure 5. Radial distribution function plot for (a) Complex1_ICL and (b) Complex2_ICL. Figure 6. Reaction scheme of isocitrate lyase.

TABLE CAPTIONS Table 1. Highest eigenvalue of each active site in all systems from 2 to 30 ns of MD simulation. Table 2. The top three hydrogen bonding occupancy in all systems. Table 3. MMPB/SA calculation for Complex_ICL systems. Complex1_ICL has glyoxylate and succinate and Complex2-ICL has isocitrate in their active sites respectively.

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Table of Contents The Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form Yie-Vern Lee†, Sy Bing Choi┴, Habibah A. Wahab§, and Yee Siew Choong†,*

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Figure 1. Active site of Apo_ICL and Complex_ICL. Active loop (residue 185-196) in open and close conformation for respective system is in yellow representative, the C-terminal (residue 411-428) in cyan representative and the other loops around the active sites is in purple representation. Once the active loop closed the active site, C-terminal from adjacent unit will overtake the original place of active loop and lay on top of the active loop to secure the closure of the active site. Figure is generated using VMD 1.9.1. 203x138mm (300 x 300 DPI)

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Figure 2. Overall root mean square deviation (RMSD) analysis as the function of time during MD simulation for Apo_ICL, Complex1_ICL and Complex2_ICL system. 203x249mm (300 x 300 DPI)

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Figure 3. Active and terminal loops RMSD of all systems. Loop 1 and 2 represent the loop from monomer 1 and 2, respectively, in ICL dimer. Figure is generated using VMD 1.9.1. 203x325mm (300 x 300 DPI)

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Figure 4. Porcupine plot of highest eigenvalue eigenvector of the active site in (a) Apo_ICL, (b) Complex1_ICL, and (c) Complex2_ICL. Red arrow summarized the vector and magnitude of motion. Figure is generated using PCA suite. 203x291mm (300 x 300 DPI)

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Figure 5. Radial distribution function plot for (a) Complex1_ICL and (b) Complex2_ICL. 269x357mm (300 x 300 DPI)

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Figure 6. Reaction scheme of isocitrate lyase. 152x37mm (300 x 300 DPI)

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