Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Chemical Information and Modeling is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

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

1 2

Journal of Chemical Information and Modeling

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

3 4 5 6 7 8 9 10 11 12

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

1 ACS Paragon Plus Environment


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

Page 2 of 32

13

ABSTRACT

14

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)

16

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

18

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

19

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

20

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

21

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.

28


Page 3 of 32

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


29

INTRODUCTION

30

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

31

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

32

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

33

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

34

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

35

reverts back into the incurable days.

36

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

37

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

38

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

39

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

40

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

41

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

42

extensive study on ICL.

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

bonded interactions involved in the active site were analyzed.



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

Page 4 of 32

52

MATERIALS AND METHODS

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

performed with AMBER 8 package.19

65

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

66

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

67

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

68

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

69

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

70

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

71 72

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

73

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

74

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


Page 5 of 32

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


75

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

76

covariance matrixes were calculated. Covariance matrix then subsequently diagonalized into

77

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

78

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

79

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

80

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

81 82

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

83

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

84

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

85

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

86

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

87

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

88

contribution towards desolvation was calculated by molsurf module.

89 90

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

91

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

92

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

93

spacing of 0.5 and maximum histogram of 15 Å.



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

Page 6 of 32

94

RESULTS AND DISCUSSION

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

simulation for ICL was therefore performed in dimer format.

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116 6 ACS Paragon Plus Environment

Page 7 of 32

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

117 118 119 120 121 122 123 124 125


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.



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

126 127 128 129 130

Page 8 of 32

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.


Page 9 of 32

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


131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

motion in the system.

152



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

153 154 155 156

Page 10 of 32

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

Page 11 of 32

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

157 158


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

159



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

160 161 162 163 164

Page 12 of 32

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.


Page 13 of 32

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


165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

of the entrance.

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

closed behavior of ICL active site entrance gate.



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

188 189 190 191

Page 14 of 32

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


Page 15 of 32

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

192


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



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

Page 16 of 32

196

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

197

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

198

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

199

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

200

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

201

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

202

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

204

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

205

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

206

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

207

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

208

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

209

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-

212

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

213

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

214

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

215

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


Page 17 of 32

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

219 220 221 222 223 224 225


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



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

Page 18 of 32

229

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

230

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


Page 19 of 32

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


247

CONCLUSION

248

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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



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

Page 20 of 32

265

ACKNOWLEDGEMENTS

266

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

267

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

268

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

269

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

270

Information Engineering Laboratory, Department of Bioinformatic Engineering, Graduate

271

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

272

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

273

National Science Fellowship.

274

275

ASSOCIATED CONTENT

276

Supporting Information:

277

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

278

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

279

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

281

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

282

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).

284 285


Page 21 of 32

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


286

AUTHOR INFORMATION

287

Corresponding Author

288

* Email: [email protected]

289

ORCID

290

Yee Siew Choong: 0000-0001-5067-2073

291

Notes

292

The authors declare no competing financial interest.



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

293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338

Page 22 of 32

REFERENCES (1) World Health Organization (W.H.O). Global tuberculosis report 2016. http://www.who.int/tb/publications/global_report/en/ (accessed 1-7-2017). (2) Nuermberger, E. L.; Spigelman, M. K.; Yew, W. W. Current Development and Future Prospects in Chemotherapy of Tuberculosis. Respirology 2010, 15, 764-778. (3) Velayati, A. A.; Masjedi, M. R.; Farnia, P.; Tabarsi, P.; Ghanavi, J.; ZiaZarifi, A. H.; Hoffner, S. E. Emergence of New Forms of Totally Drug-Resistant Tuberculosis Bacilli. Chest. 2009, 136, 420-425. (4) Murphy, D. J.; Brown, J. R. Computational Biology in Anti-Tuberculosis Drug Discovery. Infect. Disord. Drug Targets 2009, 9, 319-326. (5) Lee, Y.-V.; Wahab, H. A.; Choong, Y. S. Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary. BioMed Res. Int. 2015, 2015, 20. (6) Dunn, M. F.; Ramirez-Trujillo, J. A.; Hernandez-Lucas, I. Major Roles of Isocitrate Lyase and Malate Synthase in Bacterial and Fungal Pathogenesis. Microbiology 2009, 155, 3166-3175. (7) Muñoz-Elías, E. J.; McKinney, J. D. M. tuberculosis Isocitrate Lyases 1 and 2 are Jointly Required for In Vivo Growth and Virulence. Nat. Med. 2005, 638 - 644. (8) Gould, T., A.; Langemheen, H. v. d.; J. Muñoz-Elías, E.; McKinney, J., D. ; Sacchettini, J., C. Dual Role of Isocitrate Lyase 1 in the Glyoxylate and Methylcitrate Cycles in Mycobacterium tuberculosis. Mol. Microbiol. 2006, 61, 940-947. (9) Gengenbacher, M.; Rao, S. P. S.; Pethe, K.; Dick, T. Nutrient-Starved, Non-Replicating Mycobacterium tuberculosis Requires Respiration, ATP Synthase and Isocitrate Lyase for Maintenance of ATP Homeostasis and Viability. Microbiology 2010, 156, 81-87. (10) Betts, J. C.; Lukey, P. T.; Robb, L. C.; McAdam, R. A.; Duncan, K. Evaluation of a Nutrient Starvation Model of Mycobacterium tuberculosis Persistence by Gene and Protein Expression Profiling. Mol. Microbiol. 2002, 43, 717-731. (11) Sharma, V.; Sharma, S.; Bentrup, K. H. z.; McKinney, J. D.; Russell, D. G.; Jacobs Jr., W. R.; Sacchettin, J. C. Structure of Isocitrate Lyase, a Persistence Factor of Mycobacterium tuberculosis. Nat. Struct. Mol. Biol. 2000, 7, 663 - 668. (12) Shukla, H.; Kumar, V.; Singh, A. K.; Rastogi, S.; Khan, S. R.; Siddiqi, M. I.; Krishnan, M. Y.; Akhtar, M. S. Isocitrate Lyase of Mycobacterium tuberculosis is Inhibited by Quercetin Through Binding at N-terminus. Int. J. Biol. Macromol. 2015, 78, 137-141. (13) Shukla, H.; Kumar, V.; Singh, A. K.; Singh, N.; Kashif, M.; Siddiqi, M. I.; Yasoda Krishnan, M.; Sohail Akhtar, M. Insight into the Structural Flexibility and Function of Mycobacterium tuberculosis Isocitrate Lyase. Biochimie 2015, 110, 73-80. (14) Shukla, R.; Shukla, H.; Sonkar, A.; Pandey, T.; Tripathi, T. Structure-Based Screening and Molecular Dynamics Simulations Offer Novel Natural Compounds as Potential Inhibitors of Mycobacterium tuberculosis Isocitrate Lyase. J. Biomol. Struct. Dyn. 2017, 1-106. (15) Shukla, H.; Shukla, R.; Sonkar, A.; Tripathi, T. Alterations in Conformational Topology and Interaction Dynamics Caused by L418A Mutation Leads to Activity Loss of Mycobacterium tuberculosis Isocitrate Lyase. Biochem. Biophys. Res. Commun. 2017. (16) Liu, S.; Lu, Z.; Han, Y.; Melamud, E.; Dunaway-Mariano, D.; Herzberg, O. Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution. Biochemistry 2005, 44, 2949-2962. 22 ACS Paragon Plus Environment

Page 23 of 32

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

339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383


(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.



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

384 385 386 387 388 389 390 391 392 393 394 395 396 397

Page 24 of 32

(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.


Page 25 of 32

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

398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433


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.



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

434 435

Page 26 of 32

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†,*

436 437


Page 27 of 32

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


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)

ACS Paragon Plus Environment


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

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)


Page 28 of 32

Page 29 of 32

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


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)



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

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)


Page 30 of 32

Page 31 of 32

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


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



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

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


Page 32 of 32

Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in

Recommend Documents