Article-2015-Insight into the structural flexibility and function of Mycobacterium tuberculosis isocitrate lyase

Biochimie 110 (2015) 73e80
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Biochimie
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Research paper
Insight into the structural flexibility and function of Mycobacterium
tuberculosis isocitrate lyase
Harish Shukla a, Vikash Kumar a, Amit Kumar Singh a, Neha Singh a, Md. Kashif a,
Mohammad Imran Siddiqi a, c, Manju Yasoda Krishnan b, c, Md. Sohail Akhtar a, c, *
a
b
c
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, PIN 226 031, India
Microbiology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, PIN 226 031, India
Academy of Scientific and Innovative Research, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, PIN 226 031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 August 2014
Accepted 26 December 2014
Available online 7 January 2015
Isocitrate lyase (ICL), is a key enzyme of the glyoxylate shunt crucial for the survival of Mycobacterium
tuberculosis (Mtb) in macrophages during persistent infection. MtbICL catalyses the first step of this
carbon anaplerosis cycle and is considered as a potential anti-tubercular drug target. The MtbICL is a
tetramer with 222 symmetry, and each subunit of the enzymeis composed of 14 a-helices and 14 bstrands. We studied the conformational flexibility of the enzyme to get a deeper insight into its stability
and function. Our studies show that the mutation of His180, close to the MtbICL signature sequence
(K193KCGH197) completely abolishes the oligomeric conformation and function of the enzyme. Molecular
dynamics studies suggest that the loss of interaction between His180 and Tyr89 most likely alters the
orientation of Tyr89 side chain, thereby causing the movement of helices a6, a12, a13 and a14 in the
vicinity and affecting the tetrameric assembly. We further show that the oligomerization of MtbICL is
primarily mediated by the inter subunit interactions, and strengthened by the helix swapping of a12
ea13 between adjacent subunits. Furthermore, the enzyme activity is influenced by the interactions
between the residues of lid region (P411NSSTTALTGSTEEGQFH428) and the loop region (T391KHQREV397).
Mutation of glutamates of the lid region to non homologous residues (E423A or E424A) or basic residues
(E423K or E424K) inactivates the enzyme, whereas the activity is not much compromised in case of
homologous mutations (E423D or E424D).
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Keywords:
Isocitrate lyase
Structure
Activity
Drug target
Kinetics
1. Introduction
Tuberculosis (TB) remains an expanding global health crisis
responsible for about 2 million deaths annually [1,2]. Nearly about
one-third of the world's population harbors latent Mycobacterium
tuberculosis (Mtb) and would have a positive skin test for the
infection [1e4]. There is no effective vaccine against the infection
and the prolonged therapy frequently leads to the emergence of
multi-drug resistance not only to the first line, but also to some of
the second-line drugs [1e5]. The extended therapy is needed to
tackle the non replicating dormant population of Mtb in patient
lesions which are refractory to killing by the currently used anti-TB
Abbreviation: Mtb, Mycobacterium tuberculosis; ICL, isocitrate lyase; MtbICL,
Mtuberculosis isocitrate lyase; SEC, size-exclusion chromatography.
* Corresponding author. Molecular and Structural Biology Division, CSIR-Central
Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, PIN 226 031,
India.
E-mail address: sohail@cdri.res.in (Md.S. Akhtar).
drugs, which are capable of killing only the actively dividing bacilli
[6]. Thus combating TB requires new therapeutic strategies which
can effectively target the dormant bacilli and act in synergy with
the available frontline drugs [1]. Hence, the hunt for novel, safer TB
drugs effective in treating drug resistant as well as persistent
infection is still on.
Many recent works are beginning to elucidate the metabolic
adaptation by Mtb during persistence, for instance, the dependence
on purine, amino acid and pantothenate biosynthesis, iron acquisition, glyoxylate shunt etc [7]. Beta-oxidation, gluconeogenesis
and glyoxylate shunt are important for the survival of Mtb inside
the phagosomes of macrophages, which are glucose deficient, but
fatty acid replete [8]. Isocitrate lyase (ICL), one of the key enzymes
of the glyoxylate shunt catalyses the conversion of isocitrate to
succinate and glyoxylate. The enzyme is essential for carbon anaplerosis in the TCA cycle during growth on C2 substrates such as
fatty acids [9,10]. The glyoxylate shunt is absent invertebrates, but
are widespread among prokaryotes, lower eukaryotes and plants
http://dx.doi.org/10.1016/j.biochi.2014.12.016
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0300-9084/© 2015 Elsevier B.V. and Socie
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H. Shukla et al. / Biochimie 110 (2015) 73e80
[11]. The expression of ICL is upregulated during infection of macrophages by Mycobacterium sp. and the disruption of ICL gene inhibits the persistence of Mtb in macrophage and in mice [12e14].
Hence ICL of Mtb (MtbICL) is considered as one of the potential and
attractive drug targets against persistent infection.
The crystal structures of MtbICL in apo form as well as in complex with inhibitors are solved [15]. MtbICL (428 amino acids) is a
tetramer with 222 symmetry (Fig. 1). Each subunit of MtbICL is
composed of 14 a-helices and 14 b-strands. The core of the structure is an unusual a/b-barrel composed of eight a-helices (a4ea11)
and eight b-strands (b2eb5, b8, b12eb14) forming the larger
domain of the enzyme. Helix a12 (residues 349e367) projects away
from the barrel and together with the two consequent helices a13
(residues 370e384) and a14 (residues 399e409), forms interactions exclusively with the neighboring subunits. A small bdomain consisting of a short five stranded b-sheet (b6, b7, b9, b10,
b11) lies atop of the a/b barrel and contains several of the active site
residues. The striking feature of this structure is the inter-subunit
helix-swapping of a12 and a13 between two non crystallographically related subunits responsible for the tetrameric structure. A
similar helix swapping has been proposed to enable the formation
of stable dimers in other proteins [16].
The mechanism of MtbICL function was elucidated based on
the crystal structure of apo MtbICL and MtbICLC191Sin complex
with glyoxylate and the non-reactive succinate analog 3nitropropionate [15]. The conformation of apo MtbICL differs
from its substrate analog bound forms, especially in regions that
control access to the active site [1,15]. The first region is an active
site loop (L185ASEKKCGHLGG196) that contains the ICL signature
sequence (K189KCGH193) and the second region consists of the last
18 residues (P411NSSTTALTGSTEEGQFH428) at the C-terminal end of
the adjacent subunit (lid). The active site loop is flexible in the
‘open’ conformation where as in the substrate bound form it
moves by 10e15 Ǻ and attains a closed conformation. The closed
conformation brings catalytic Cys191 of ICL signature sequence
next to the substrate and completely closes off the active site from
bulk solvent. The electrostatic interaction of Lys189 within the
negative patches triggers movements in the active site loop to
adopt the ‘closed’ conformation. Closure of the active site loop
blocks the accessibility to the catalytic residues and invokes a
movement of C-terminus lid (residues 411e428) of the adjacent
subunit completing the catalytic conformation. It is not clear what
invokes the movement of lid on top of the active site loop, locking
it into a catalytic conformation? It appears that the two conserved
glutamates in the lid region might play an important role during
enzyme catalytsis [17]. Furthermore, apart from the residues of
signature sequence (KKCGH), the function of ICL is also influenced
by the histidine residue (His184) adjacent to the ICL signature
sequence in Escherichia coli. The mutation of His184 to Lys, Arg,
Leu or Gln resulted in either inactive ICL or a minimally active
ICL [17].
Conformational analysis of MtbICL with respect to its stability
and function is important and might help in the identification of
suitable inhibitors. The biological phenomena depend on the molecular recognition and play a significant role in protein folding,
stability and ligand binding. Understanding of the relationship
between the structure of proteins, their stability, binding with
micro or macromolecules etc. are useful to understand how biological systems work. This would facilitate drug discovery, such as
in molecular mechanism and energetics of drug-macromolecules
interactions or in order to estimate binding constants of two molecules [18,19].
In the present study, we systematically looked into the structural elements involved in the oligomerization and deactivation of
MtbICL.
2. Experimental
2.1. Cloning and site directed mutagenesis
The cloning of full length MtbICL (MtbICL/MtbICLa12,a13,a14) has
been described previously and was a kind gift from Dr. Ranjeet
Kumar, Lucknow [20]. Truncated ICL without a14 (tMtbICLa12,a13),
tMtbICLa12 (without a13 and a14) and tMtbICL (without a12, a13
and a14) were amplified using suitable primers (Table 1). For all
the samples, PCR reactions were carried out in a total volume of
50 ml with Platinum Pfx DNA polymerase (Invitrogen). The
amplification condition was 94 C-3 min; 94 C-30 s, 53 C-1 min,
68 C-1 min (30 cycles); 68 C-10 min. These amplified gene
fragments were digested with NheI and HindIII and then ligated
into the pET-23a (þ) vector cut with the same enzymes. Competent E. coli DH5-a cells were transformed with the plasmid constructs and screened for positive clones. The mutant ICLs,
MtbICLH180A, MtbICLY89A, MtbICLE423D, MtbICLE424D, MtbICLE423D
E424D, MtbICLE423A, MtbICLE424A, MtbICLE423AE424A, MtbICLE423K,
MtbICLE424k, MtbICLE423KE424K, MtbICLE423L, MtbICLE424L and
MtbICLE423LE424L were generated using mutagenic primer pairs
(Table 1). The DNA sequencing of all the amplified genes
confirmed the homogeneity of the sequences.
Fig. 1. Diagrammatic representation of the structure of MtbICL e Crystal structure of monomeric, dimeric and tetrameric MtbICL (left to right). The helices a12 and a13 involved
in the oligomeization are marked as red. The structure was generated with the help of PISA server [28].
H. Shukla et al. / Biochimie 110 (2015) 73e80
75
2.2. Preparation of recombinant proteins
The expression of MtbICL was carried out as described previously [20]. For the expression of remaining proteins, the corresponding plasmids were introduced into C41(DE3) cells and
initially grown in LuriaeBertani media containing 100 mg/ml
ampicillin at 30 C until A600~ 0.6 and later induced at 20 C with
0.1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG). Induced
cultures were grown further for 8 h with rigorous shaking at the
same temperature. The cleared cell lysates were loaded onto a NiNTA column pre-equilibrated with 20 mM TriseCl, pH 8 and
0.3 M NaCl and washed with the same buffer followed by washing
with 20 mM and 40 mM imidazole buffer. The proteins were eluted
with a linear gradient of 40e400 mM imidazole buffer and the
desired peaks were dialyzed against 50 mM phosphate buffer, pH 8
containing 0.1 M NaCl. The purity and molecular weight determinations of the recombinant proteins were carried out by SDS
as well as with size exclusion chromatography (SEC) experiments
on a SuperdexTM 200,10/300 GL column (manufacture's exclusion
limit 600 kDa) on AKTA FPLC (GE Healthcare). The column was preequilibrated and run with 50 mM phosphate buffer pH 8.0 at 25 C
with a flow rate of 0.5 ml/min, with detection at 280 nm. The
column was calibrated with standard molecular weight markers.
Table 1
Different primers used in PCR for MtbICL, its truncations and mutants.
Serial
no.
Gene/truncations/
mutants name
Primers
1
tMtbICLa12,a13
2
tMtbICLa12
3
tMtbICL
4
MtbICLH180A
5
MtbICLY89A
6
MtbICLE423D
7
MtbICLE424D
8
MtbICLE423D,E424D
9
MtbICLE423A
10
MtbICLE424A
11
MtbICLE423A,E424A
12
MtbICLE423K
13
MtbICLEE424K
14
MtbICLE423K,E424K
15
MtbICLE423L
16
MtbICLE424L
17
MtbICLE423L,424L
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CCC AAG CTT GCC TAC TTC ACG CTG GTG
CTT GGT 30
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CAA AAG CTT CTG GTT CTG GGC GTA GCC
GTA GGC 30
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CCC AAG CTT CAG CGT GAT GAA CTG GAA
CTT GAA30
F 50 GGC GTT GCG GGT TCG GCT TGG GAG GAT
CAG TTG30
R 50 CAA CTG ATC CTC CCA AGC CGA ACC CGC
AAC GCC 30
F 50 GGC CTG AAG GCC ATC GCC CTG TCG GGC
TGG CA30
R 50 TGC CAG CCC GAC AGG GCG ATG GCC TTC
AGG CC30
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC GTC CTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CTC GTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC GTC GTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CGC CTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CTC CGC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CGC CGC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAGT 30
R 50 CTC AAG CTT GTG GAA CTG ACC TTT CTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CTC TTT
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC TTT TTT
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC TTG CTC
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC CTC TTG
GGT GGA3
F 50 CCC GCT AGC ATG TCT GTC GTC GGC ACC
CCG AAG T 30
R 50 CTC AAG CTT GTG GAA CTG ACC TTG TTG
GGT GGA3
2.3. Activity assay
The activity of recombinant proteins was measured at 30 C in
20 mM HEPES buffer, pH 7.5, by a modification of the continuous
method as described previously [21]. The reaction mixture
comprised of 20 mM Hepes, pH 7.5, containing 5 mM MgCl2, 4 mM
phenyl hydrazine hydrochloride, 30 mM threo-DL-isocitrate and
enzyme in a final volume of 1 ml. The reaction was initiated by the
addition of the enzyme (25 nM), and the isocitrate cleavage was
measured at 30 C by the change in absorbance at 324 nm associated with the formation of glyoxylate phenylhydrazone (extinction
coefficient at 324 nm, 17,000/M/cm).
2.4. Glutaraldehyde crosslinking
50 mg of desired protein was incubated with 5 ml of 1:25 dilution
(w/v) of 25% glutaraldehyde in a 100 ml reaction volume in 20 mM
HEPES buffer pH 7.5 for 30 min. The reaction was stopped by the
addition of 10 ml of 1 M TriseHCl, pH 8 in the reaction mixture and
the samples analyzed on 8% SDS-PAGE.
2.5. Spectroscopy measurements
Tryptophan fluorescence spectra were recorded with a Perkin
Elmer Life Sciences LS 50-B spectrofluorimeter in a 5 mm path
length quartz cell at 25 C. Excitation wavelength of 280 nm was
used and the spectra were recorded between 300 and 400 nm. The
protein concentration of 3 mM was used for the studies.
Circular dichroism measurements were made on JASCO J810
spectropolarimeter calibrated with ammonium (þ)-10-camphorsulfonate with a 2 mm path length cell at 25 C. The values
obtained were normalized by subtracting the baseline recorded for
the buffer under similar conditions.
2.6. Molecular dynamics simulation
Crystal structure of MtbICL (PDB-id: 1F61) [16] was retrieved
from protein data bank (www.rcsb.org). Mutant model of MtbICL
was obtained after replacing His180 with alanine in USCF
Chimera1.6 [22]. Molecular dynamics simulation of wild and
mutant MtbICL was carried out for 10 ns using Gromacs4.5 [23].
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H. Shukla et al. / Biochimie 110 (2015) 73e80
Protein molecule was solvated in cubic box with TIP3P water model
under periodic boundary condition. Whole system was neutralized
by adding Naþ ion. After energy minimization solvated system was
subjected to NVT and NPT equilibration. Final production simulation was performed under NPT condition for 10 ns. For trajectory
analysis VMD software [24] and xmgrace were used.
3. Results and discussion
3.1. Purification of full length, truncated and mutant MtbICL
The recombinant enzyme and mutants such as MtbICL/MtbICLa12,a13,a14, tMtbICLa12,a13, tMtbICLa12, tMtbICL, MtbICLH180A,
MtbICLY89A MtbICLE423D, MtbICLE424D, MtbICLE423D E424D, MtbICLE423A, MtbICLE424A, MtbICLE423AE424A, MtbICLE423K, MtbICLE424k,
MtbICLE423KE424K,
MtbICLE423L,
MtbICLE424L
and
MtbICLE423LE424L were purified by the methods described under
“Experimental”. The molecular mass of the purified proteins was
determined by SDS-PAGE and the oligomeric status by size exclusion chromatography. The secondary and tertiary structure of
mutants was determined by spectroscopic studies and were
observed to be similar to those of MtbICL (Supplemental Fig. 1).
3.2. H180A mutation compromises the subunit assembly of MtbICL
Among the four highly conserved regions of ICL derived from
different organisms, the first conserved region (residues 173e197)
contains an ICL signature sequence (K193KCGH197) (Fig. 2A) [25,17]
and a conserved histidine at position 180 (His180). The ICL signature sequence is important for substrate binding and catalysis
because any non-homologous mutation in the region severely affects the function of ICL in E. coli [25,26]. We mutated the corresponding histidine (His180) of MtbICL (MtbICLH180A) and checked
its effect on the oligomerization of the enzyme by SEC (Fig. 2B). The
elution volumes of MtbICL (12.4 ml) and MtbICLH180A (15.3 ml)
correspond to the molecular weights of ~192 kDa (tetramer) and
~48 kDa (monomer) and are suggestive of the dissociation of
tetrameric MtbICL. The subsequent glutaraldehyde crosslinking
experiment further suggested the complete dissociation of the
mutant MtbICLH180A to monomer (Fig. 2B, inset A). The dissociation
of MtbICLH180A from tetramer to monomer also leads to the complete loss in the enzymatic activity (Fig. 2C). We subsequently
looked for the salt strength at which the MtbICL could be dissociated without His180 mutation and observed the formation of
dimeric as well as monomeric MtbICL at about 0.75 M Na2SO4(Supplemental Fig. 2). The dimeric and monomeric MtbICL were
enzymatically inactive (data not shown). We subsequently
measured the enzymatic activity of MtbICL between 0.5 M2.0 M
NaCl to help differentiate, whether the observed inactivity of
MtbICL was due to high Na2SO4 concentration or because of the
change in the oligomeric conformation. The MtbICL had no
apparent change in the enzymatic activity up to 1.5 M NaCl
(Supplemental Fig. 3) however, tended to precipitate after 1.75 M
NaCl with the loss in the enzymatic activity. The oligomeric status
of MtbICL remained unchanged up to 2 M NaCl (data not shown).
3.3. H180A mutation affects subunit assembly through
destabilization of the a6 and C-terminal helices (a12, a13 and a14)
With molecular dynamics simulation (10 ns), we analyzed the
trajectories of MtbICL and its His180 mutant (MtbICLH180A). Both
the proteins showed large deviation (up to 4 Ǻ) from the starting
structure (Fig. 3A and Supplemental Fig. 4). The main reason behind
the high value of root mean square deviation (RMSD) can be
attributed to the highly flexible C-terminal region. It has been reported that the C-terminal region of MtbICL is more dynamic and
helps in oligomerization through helix swapping of a12 and a13
with the neighboring subunits [15]. We carried out MD study on the
single subunit of MtbICL, in which the C-terminal region is no
longer stabilized due to the loss of swapping. Despite this, any effect
of H180A mutation on monomeric structure could be analyzed. As
evident from RMSD trajectory, mutant MtbICL showed irregular
deviation with respect to wild MtbICL, indicating its structural
instability. The observed effects of H180A mutation on the structure
Fig. 2. H180A mutation breaks oligomerization in MtbICL e (A) The conserved region of Isocitrate Lyases containing the signature sequence (KKCGH) and histidine. (B) The SEC
profiles of MtbICL and MtbICLH180A at pH 8.0 and 25 C. The inset A shows the glutaraldehyde cross-linked samples where lanes 1e4 represent, crosslinked MtbICL, molecular
weight marker, uncrosslinked MtbICL and cross-linked MtbICLH180A respectively. The inset B shows the standard molecular weight marker used for the calibration of SEC column.
(C) Relative enzymatic activity of MtbICL and MtbICLH180A.
H. Shukla et al. / Biochimie 110 (2015) 73e80
77
Fig. 3. MtbICLH180A destabilizes the a6 and C-terminus helices e (A) Trajectory showing Ca-RMSD of wild and mutant MtbICL during 10 ns of dynamics simulation. (B)
Superimposed wild (Orange) and mutant (Sky blue) MtbICL after 10 ns of MD simulation. Here last frame coordinates were used. C-terminal domains of both proteins are
highlighted in the square, where major differences are visible. (C) Superimposed wild (Orange) and mutant (Sky blue) MtbICL after 10 ns of MD simulation (left). Close view of
differences in and near active site pocket where H180A mutation exists (right). (D) Trajectory showing per residue RMSF of wild and mutant ICL during 10 ns of dynamics
simulation.
of MtbICL are largely on the a6 and C-terminal helices a12, a13 and
a14 (Fig. 3B). In case of Aspergillus nidulans, His185 makes hydrogen
bond with hydroxyl group of Tyr95 side [27]. The corresponding
residues (His180 and Tyr89) are also conserved in Mtb and our
simulation studies support the above finding. We observed that
His180 and Tyr89 side chains are involved in pep stacking interaction and H-bonding (approximately 50% occupancy) in MtbICL
(Fig. 3C). In addition, HE2 hydrogen of His180 appeared to make Hbond with the backbone of Leu90 residue. We propose that the loss
of the above interactions between His180 and the hydroxyl group
of Tyr89, alters the orientation of Tyr89 side chain leading to the
movement of the helices in the vicinity. Like MtbICLH180A, the Tyr89
mutant (MtbICLY89A) also leads to the formation of inactive
monomer (Supplemental Fig. 5). Supplemental Fig. 6 shows the key
residues of the active site in the simulated structures of MtbICL.
Root mean square fluctuation (RMSF) plot (Fig. 3D) clearly shows
that the a6 and C-terminal helices of mutant MtbICL undergo more
fluctuation than those of wild MtbICL. Overall, our simulation results show that the H180A mutation alters the orientation of a6 and
C-terminal helices, which in turn may impede the oligomerization
process.
3.4. Inter helix domain swapping is not exclusive for the tetrameric
conformation of MtbICL
We have observed that the mutant MtbICLH180A show structural
instability and affects largely the helices a6, a12, a13 and a14. The
helix a12 (residues 349e367) together with the two helices a13
(residues 370e384) and a14 (residues 399e409) interacts with the
neighboring subunits. Helix a12 and a13 are mainly involved in
helix swapping, essential for tetrameric oligomerization [15]. For
studying the effect of helix-swapping on the molecular assembly of
MtbICL, we systematically truncated the a-helices and looked for
the effect on oligomerization and enzymatic activity. Fig. 4A summarizes the result of the size exclusion profile of MtbICL/MtbICLa12,a13,a14, tMtbICLa12,a13, tMtbICLa12 and tMtbICL showing
oligomeric stability. For full length as well as truncated MtbICLs a
similar peak at about 12.25 ± 0.4 ml was observed corresponding to
the tetramer (~192 ± 24 kDa). The oligomeric stability of full length
as well as the truncated MtbICLs was also confirmed by glutaraldehyde crosslinking (Fig. 4A inset). The crosslinked band of proteins observed at about 192 kDa corresponds to a tertameric
configuration. The truncation of MtbICL leads to a slight change in
the secondary structure (Supplemental Fig. 7).
The stability of the truncated enzyme was also studied by optical
spectroscopy in the presence of increasing concentrations of chaotropic agents. Fig. 4B and C summarize the GdnHCl and urea
induced changes in the structural properties of recombinant
MtbICLs. The observed Cm (concentration of denaturant where 50%
denaturation is observed) of MtbICL/MtbICLa12,a13,a14, tMtbICLa12,a13, tMtbICLa12 and tMtbICL are presented in Table 2 which
primarily suggest that the removal of a-helices affects the overall
stability of MtbICL. In case of the truncated MtbICLs, the presence of
small peaks in the void volume suggests their overall instability as
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H. Shukla et al. / Biochimie 110 (2015) 73e80
Fig. 4. The oligomeric conformation of MtbICL is not dependent on helix swapping e (A) The SEC profiles represent MtbICL/MtbICLa12,a13,a14, tMtbICLa12,a13, tMtbICLa12 and
tMtbICL at pH 8.0 and 25 C. The inset shows the glutaraldehyde cross-linked MtbICL and its truncations. Lanes 1e6 represents, molecular weight markers, uncrosslinked MtbICL,
cross-linked MtbICL, cross-linked tMtbICLa12,a13, cross-linked tMtbICLa12 and cross-linked tMtbICL respectively. (B) Plot of the fractional change in the wavelength maxima of
fluorescence of MtbICL ( ), tMtbICLa12,a13 (◊), tMtbICLa12 (D) and tMtbICL (7) with increasing concentration of urea. (C) The plot of the fractional change in the wavelength
maxima of fluorescence of MtbICL ( ), tMtbICLa12,a13 (◊), tMtbICLa12 (D) and tMtbICL (7) with increasing concentration of GdnHCl. (D) The dimer of MtbICL showing complementarity of surface involved in inter-subunit interaction and the interacting residues of a6 helix from both the monomeric subunits. Chain A and B are colored in orange and blue
respectively. Hydrogen bonds are displayed in black dotted lines. The non-polar hydrogens are not shown to avoid crowding. (E) The relative enzymatic activity of MtbICL,
tMtbICLa12,a13, tMtbICLa12 and tMtbICL.
▫
▫
compared to the full length MtbICL (Fig. 4A). A similar peak in the
void volume was also observed in the case of MtbICLH180A (Fig. 2B).
However, as mentioned above, these instabilities do not affect the
oligomeric configuration and hence the other interactions in
addition to helix swapping are also essential for stable MtbICL
oligomerization.
As our experimental study suggested that the truncation of
three C-terminal helices i.e., a12, a13 and a14 respectively did not
affect the oligomerization state, we looked for inter-subunit interactions essential for holding the subunits. We selected the dimer
structure of MtbICL and subsequently truncated it. The resulting
dimer of MtbICL had all structural elements except the a12, a13 and
a14 helices. Fig. 4D shows that the interface region of the dimer,
containing all the residues may be involved in the inter-subunit
interactions. Detailed analysis of the interaction pattern showed a
substantial number of H-bonds between two monomer units (for
H-bond details, please see the Supplemental Table). Important
residues, which were involved in H-bonds, are Asn75, Asp98,
Gly103, Thr105, Arg123, Asn126 and Arg130 from both subunits.
Also, we observed electrostatic interaction between Asp98 and
Arg123 from both subunits. Apart from the above interactions,
shape complementarity can be another factor that holds the subunits together. A recent study shows that besides electrostatic and
hydrophobic complementarity, shape complementarity is the third
key factor affecting proteineprotein interactions. As we see, the
contact surfaces of the subunits appear complementary. Involvement of inter-subunit interaction in oligomerization is further
substantiated by the results of MD simulation studies on the
monomeric mutant MtbICL (Supplemental Fig. 4).
The substrate binding to MtbICL leads to a large conformational
changes in the active site loop as well as the carboxy terminal end
residues (lid region) of the adjacent subunit. We studied the effect
Table 2
Comparison of Urea and Guanidine hydrochloride Cm values for full length MtbICL
and truncations.
Protein
Oligomeric state
Cm urea (M)
Cm GdnHCl (M)
MtbICL/MtbICLa12,a13,a14
tMtbICLa12,a13
tMtbICLa12
tMtbICL
Tetramer
Tetramer
Tetramer
Tetramer
3.8
2.8
2.4
1.5
1.9
1.4
1.24
0.75
H. Shukla et al. / Biochimie 110 (2015) 73e80
79
Fig. 5. Closure of lid is influenced by electrostatic interaction e (A) Close view of lid region and active site interface showing important residues of MtbICL. (B) Relative enzymatic
activity of MtbICL and the mutants where the glutamic acid of the lid region was replaced with homologous, non homologous, basic and hydrophobic residues.
of truncations on enzymatic activity and observed that the truncated MtbICLs were enzymatically inactive (Fig. 4E). The loss in
activity was mainly because of the absence of lid region and non
closure of the active site loop as necessary to attain the catalytic
conformation.
3.5. Lid closure to accomplish catalytic conformation is influenced
by electrostatic interactionThe amino acid sequences of the active site loop
(L185ASEKKCGHLGG196) and the C-terminal lid (P411NSSTTALTGSTEEGQFH428) suggest the possibility of electrostatic interaction
between the two. From the crystal structure of closed form of
MtbICL (PDB:1F8M, 1F8I) it is evident that the C-terminal lid residues participate in hydrogen bonding and electrostatic interactions
with the neighboring residues. The above interactions appear
crucial in terms of maintaining the appropriate conformation of lid
region for the closure of active site. In the closed form of MtbICL,
side chain of E423 forms H-bonds with main chain NH of Gln394
and Arg395 residues (Fig. 5A). E424 does not appear to form any Hbond with neighboring residues and the orientation of its side
chain slightly differs in the crystal structures of the closed form. To
check the role of E423 and E424, we mutated these residues to
homologous (aspartic acids), non homologous (alanine), basic
(lysine) and hydrophobic (leucine) amino acids and measured the
enzymatic activity of MtbICL and its single and double mutants
Table 3
Enzyme kinetic parameters of MtbICL and its mutants.
Protein
Km (mM)
MtbICL
MtbICLE423D
MtbICLE424D
MtbICLE423D,E424D
MtbICLE423A
MtbICLE424A
MtbICLE423A,E424A
MtbICLE423K
MtbICLE424K
MtbICLE423K,E424K
MtbICLE423L
MtbICLE424L
MtbICLE423L,E424L
1.142
1.423
1.861
2.031
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND: not determined.
±
±
±
±
0.065
0.097
0.097
0.157
(Fig. 5B). The homologous mutants (MtbICLE423D, MtbICLE424D and
MtbICLE423D E424D) were enzymatically active, albeit with reduced
activity and specificity as indicated by lower Kcat values (Table 3).
The other mutants (MtbICLE423A, MtbICLE424A, MtbICLE423AE424A,
MtbICLE423K, MtbICLE424k, MtbICLE423KE424K, MtbICLE423L, MtbICLE424L and MtbICLE423LE424L) were enzymatically inactive. The
mutations in E423 and E424 despite affecting the enzyme catalytic
properties, had no apparent effect on the oligomeric structure and
stability of MtbICL (Supplemental Fig. 1). From mutational analysis,
it is clear that E423 and E424 have critical roles in the function of
MtbICL. However, in the crystal structures, side chain of E424 is
sequestered and less likely to be involved in electrostatic interaction(s). Now the question is how E424 is important? The
crystal structure of the closed form of MtbICL was analyzed and it
was found that K392 and R395 lie in the close vicinity of E423 and
E424. However the side chains of these glutamates are not in the
optimal orientation to from electrostatic interactions. We tried
different rotamer combinations and a few combinations suggested
the possibility of H-bonding or electrostatic interaction with E424
also (Supplemental Fig. 8). B-factor analysis of the monomeric
subunit using UCSF chimera shows that the extreme C-terminal
residues (410e427) have high atomic B-factor and hence high
flexibility of this region. It appears that the extreme C-terminal
region can adopt a different orientation and with the course can
interact with K392. The observations suggest that the closure of lid
to accomplish the catalytic conformation is influenced not only by
the electrostatic interactions involving E423 but may also of E424.
4. Conclusion
Vmax (mM1$min1)
Kcat (s1)
14.67 ± 0.23
12.52 ± 0.25
12.7 ± 0.21
11.3 ± 0.28
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.78
8.34
8.46
7.53
ND
ND
ND
ND
ND
ND
ND
ND
ND
±
±
±
±
0.15
0.16
0.14
0.18
The mutation of His180 leads to the destabilization and formation of monomeric MtbICL. The RMSD and RMSF trajectories of
mutant MtbICL show irregular deviation with respect to wild
MtbICL, which renders major conformational changes in the Cterminal helices, i.e. a6, a12, a13 and a14. Truncation of a12ea14 in
MtbICL did not lead to the formation of monomer as proposed
before, suggesting that the oligomerization in MtbICL is further
strengthened by the inter-subunit interactions influenced mostly
by electrostatic interactions. The movement of lid containing the
carboxy terminus end residues (P411NSSTTALTGSTEEGQFH428) of
adjacent subunit over the catalytic loop (L185ASEKKCGHLGG196)
containing ICL signature sequences (K189KCGH193) is essential to
accomplish the enzymatic activity (15). The closure of lid seems to
80
H. Shukla et al. / Biochimie 110 (2015) 73e80
be influenced by electrostatic interactions in order to stabilize the
lid in a suitable conformation during closed form. Since the mutant
MtbICLs (glutamic acids to alanine)were inactive, we assume that
the movement of the lid was affected due to the absence of electrostatic interaction with neighboring K392 and R395 residues of
the lid.
The conformational freedom and structural dynamics associated
with MtbICL seems to be ideal for rational drug design, especially
based on structure-activity relationship. Apart from the screening
of inhibitors against the active site pocket of MtbICL, screening of
compounds docked against a region affecting the enzyme
function may also be useful. It is an urgent need to have a drug for
persistent infection not only of M. tuberculosis but also for other
microbial pathogens that shows persistent infection and contain
ICL eg. Pseudomonas21, Salmonella22, Yersinia23 and Leishmania24
etc. [15].
Conflict of interest
None.
Acknowledgments
This work was supported in part by CSIR-YSA (YSA0001) to MSA,
and CSIR-GENESIS (BSC0121) to MIS. HS, AKS, NS, MK is grateful to
CSIR and VK to DBT, New Delhi for financial assistance. We do not
have any financial conflict of interest. This is CDRI communication
number 8876.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biochi.2014.12.016.
References
[1] C.V. Smith, V. Sharma, J.C. Sacchettini, TB drug discovery: addressing issues of
persistence and resistance, Tuberculosis 84 (2004) 45e55.
[2] K.S.J. Duncan, Approaches to tuberculosis drug development, in: G.F. Hatfull,
W.R.J. Jacobs (Eds.), Molecular Genetics of Mycobacteria, ASM Press, Washington, DC, 2000, pp. 297e307.
[3] D.G. Russell, C.E. Barry 3rd, J.L. Flynn, Tuberculosis: what we don't know can,
and does, hurt us, Science 328 (2010) 852e856.
[4] A. Engstrom, N. Morcillo, B. Imperiale, S.E. Hoffner, P. Jureen, Detection of firstand second-line drug resistance in Mycobacterium tuberculosis clinical isolates
by pyrosequencing, J. Clin. Microbiol. 50 (2012) 2026e2033.
[5] D.G. Russell, Mycobacterium tuberculosis: here today, and here tomorrow, Nat.
Rev. Mol. Cell. Biol. 2 (2001) 569e577.
[6] J.E. Gomez, J.D. McKinney, M. tuberculosis persistence, latency, and drug
tolerance, Tuberculosis 84 (2004) 29e44.
[7] H.I. Boshoff, C.E. Barry 3rd, Tuberculosis e metabolism and respiration in the
absence of growth, Nat. Rev. Microbiol. 3 (2005) 70e80.
[8] D. Schnappinger, S. Ehrt, M.I. Voskuil, Y. Liu, J.A. Mangan, I.M. Monahan,
G. Dolganov, B. Efron, P.D. Butcher, C. Nathan, G.K. Schoolnik, Transcriptional
adaptation of Mycobacterium tuberculosis within macrophages: insights into
the phagosomal environment, J. Exp. Med. 198 (2003) 693e704.
[9] W. Sega, in: G.P. Kubica, L.G. Wayne (Eds.), The Mycobacteria: A Sourcebook,
Dekker, New York, 1984, pp. 547e573.
[10] P.R. Wheeler, C. Ratledge, Use of carbon sources for lipid biosynthesis in
Mycobacterium leprae: a comparison with other pathogenic mycobacteria,
J. Gen. Microbiol. 134 (1988) 2111e2121.
[11] P. Vanni, E. Giachetti, G. Pinzauti, B.A. McFadden, Comparative structure,
function and regulation of isocitrate lyase, an important assimilatory enzyme,
Comp. Biochem. Physiol. B 95 (1990) 431e458.
[12] K. Honer Zu Bentrup, A. Miczak, D.L. Swenson, D.G. Russell, Characterization of
activity and expression of isocitrate lyase in Mycobacterium avium and
Mycobacterium tuberculosis, J. Bacteriol. 181 (1999) 7161e7167.
[13] J.E. Graham, J.E. Clark-Curtiss, Identification of Mycobacterium tuberculosis
RNAs synthesized in response to phagocytosis by human macrophages by
selective capture of transcribed sequences (SCOTS), Proc. Natl. Acad. Sci. U S A
96 (1999) 11554e11559.
[14] J.D. McKinney, K. Honer zu Bentrup, E.J. Munoz-Elias, A. Miczak, B. Chen,
W.T. Chan, D. Swenson, J.C. Sacchettini, W.R. Jacobs Jr., D.G. Russell, Persistence of Mycobacterium tuberculosis in macrophages and mice requires the
glyoxylate shunt enzyme isocitrate lyase, Nature 406 (2000) 735e738.
[15] V. Sharma, S. Sharma, K. Hoener zu Bentrup, J.D. McKinney, D.G. Russell,
W.R. Jacobs Jr., J.C. Sacchettini, Structure of isocitrate lyase, a persistence
factor of Mycobacterium tuberculosis, Nat. Struct. Biol. 7 (2000) 663e668.
[16] M.J. Bennett, M.P. Schlunegge, D. Eisenberg, 3D domain swapping: a mechanism for oligomer assembly, Protein Sci. 4 (1995) 2455e2468.
[17] P. Diehl, B.A. McFadden, The importance of four histidine residues in isocitrate
lyase from Escherichia coli, J. Bacteriol. 176 (1994) 927e931.
[18] G. Bruylants, J. Wouters, C. Michaux, Differential scanning calorimetry in life
science: thermodynamics, stability, molecular recognition and application in
drug design, Curr. Med. Chem. 12 (2005) 2011e2020.
[19] F.R. Salemme, J. Spurlino, R. Bone, Serendipity meets precision: the integration
of structure-based drug design and combinatorial chemistry for efficient drug
discovery, Structure 5 (1997) 319e324.
[20] R. Kumar, V. Bhakuni, Mycobacterium tuberculosis isocitrate lyase (MtbICL):
role of divalent cations in modulation of functional and structural properties,
Proteins 72 (2008) 892e900.
[21] G.H. Dixon, H.L. Kornberg, Assay methods for the key enzyme of the glyoxalate cycle, Biochem. J. 7 (1959) 72e73.
[22] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng, T.E. Ferrin, UCSF chimera e a visualization system for exploratory
research and analysis, J. Comput. Chem. 25 (2004) 1605e1612.
[23] S. Pronk, S. Pall, R. Schulz, P. Larsson, P. Bjelkmar, R. Apostolov, M.R. Shirts,
J.C. Smith, P.M. Kasson, D. van der Spoel, B. Hess, E. Lindahl, GROMACS 4.5: a
high-throughput and highly parallel open source molecular simulation toolkit, Bioinformatics 29 (2013) 845e854.
[24] W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics, J. Mol.
Graph. 14 (1996) 27e38.
[25] P. Diehl, B.A. McFadden, Site-directed mutagenesis of lysine 193 in Escherichia
coli isocitrate lyase by use of unique restriction enzyme site elimination,
J. Bacteriol. 175 (1993) 2263e2270.
[26] A. Rehman, B.A. McFadden, Lysine 194 is functional in isocitrate lyase from
Escherichia coli, Curr. Microbiol. 35 (1997) 14e17.
[27] K. Britton, S. Langridge, P.J. Baker, K. Weeradechapon, S.E. Sedelnikova, J.R. De
Lucas, D.W. Rice, Turner, The crystal structure and active site location of
isocitrate lyase from the fungus Aspergillus nidulans, Structure 8 (2000)
349e362.
[28] E. Krissinel, K. Henrick, Inference of macromolecular assemblies from crystalline state, J. Mol. Biol. 372 (2007) 774e797.