Báo cáo khoa học: Molecular characterization of secretory proteins Rv3619c and Rv3620c fromMycobacterium tuberculosisH37Rv docx

Molecular characterization of secretory proteins Rv3619c
and Rv3620c from Mycobacterium tuberculosis H37Rv
Anjum Mahmood
1
, Shubhra Srivastava
1
, Sarita Tripathi
1
, Mairaj Ahmed Ansari
2
, Mohammad
Owais
2
and Ashish Arora
1
1 Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow, India
2 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India
Keywords
binding constant;
Mycobacterium tuberculosis; Rv3619c;
thermal unfolding; vaccine
Correspondence
A. Arora, Molecular and Structural Biology
Division, Central Drug Research Institute,
Lucknow 226001, India
Fax: +91 522 223405
Tel: +91 522 2612411 ext: 4329
E-mail:
(Received 5 September 2010, revised 1
November 2010, accepted 9 November
2010)

doi:10.1111/j.1742-4658.2010.07958.x
Rv3619c and Rv3620c are the secretory, antigenic proteins of the ESAT-
6 ⁄ CFP-10 family of Mycobacterium tuberculosis H37Rv. In this article, we
show that Rv3619c interacts with Rv3620c to form a 1 : 1 heterodimeric
complex with a dissociation constant (K
d
) of 4.8 · 10
)7
M. The thermal
unfolding of the heterodimer was completely reversible, with a T
m
of
48 °C. The comparative thermodynamics and thermal unfolding analysis of
the Rv3619c–Rv3620c dimer, the ESAT-6–CFP-10 dimer and another
ESAT family heterodimer, Rv0287–Rv0288, revealed that the binding
strength and stability of Rv3619c–Rv3620c are relatively lower than those
of the other two pairs. Molecular modeling and docking studies predict the
structure of Rv3619c–Rv3620c to be similar to that of ESAT-6–CFP-10.
Spectroscopic studies revealed that, in an acidic environment, Rv3619c and
Rv3620c lose their secondary structure and interact weakly to form a com-
plex with a lower helical content, indicating that Rv3619c–Rv3620c is
destabilized at low pH. These results, combined with those of previous
studies, suggest that unfolding of the proteins is required for dissociation
of the complex and membrane binding. In the presence of membrane
mimetics, the a-helical contents of Rv3619c and Rv3620 increased by 42%
and 35%, respectively. In mice, the immune response against Rv3619c pro-
tein is characterized by increased levels of interferon-c, interleukin-12 and
IgG
2a
, indicating a dominant Th1 response, which is mandatory for protec-

tion against mycobacterial infection. This study therefore emphasizes the
potential of Rv3619c as a subunit vaccine candidate.
Structured digital abstract
l
MINT-8056093: Rv0288 (uniprotkb:P0A568) and Rv0287 (uniprotkb:O53692) bind (MI:0407)
by isothermal titration calorimetry (
MI:0065)
l
MINT-8055978: Rv3620c (uniprotkb:O07932) and Rv3619c (uniprotkb:P96364) bind
(
MI:0407)bycircular dichroism (MI:0016)
l
MINT-8055964: Rv3620c (uniprotkb:O07932) and Rv3619c (uniprotkb:P96364) bind
(
MI:0407)byisothermal titration calorimetry (MI:0065)
Abbreviations
ASA, accessible surface area; BCG, bacille Calmette–Gue
´
rin; DMPC, dimyristoylphosphatidylcholine; DPC, dodecylphosphocholine;
HRP, horseradish peroxidase; IFN, interferon; IL, interleukin; ITC, isothermal titration calorimetry; MRE, mean residual ellipticity;
MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; RD, region of deletion; SI, stimulation index; TFE, trifluoroethanol.
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 341
Introduction
Comparative genomic studies based on whole genome
DNA microarrays have led to the identification of 16
regions of deletion (RDs) in Mycobacterium bovis
bacille Calmette–Gue
´
rin (BCG), which is currently used
as a vaccine, with respect to Mycobacterium tuberculosis,

and five RDs with respect to M. bovis. The RD encom-
passing ORFs Rv3619c and Rv3620c is absent from all
vaccine strains of M. bovis. This region has been classi-
fied as RD9 by Behr et al. and as RD8 by Gordon et al.
[1,2]. It is a stretch of 5516 bp encompassing seven
ORFs (Rv3617 to Rv3623). Rv3619c and Rv3620c are
the ESAT-6 ⁄ CFP-10 family members. ORFs Rv3621c
and Rv3622c belong to the family encoding proteins
containing sequence motif Pro-Pro-Glu (PPE) and Pro-
Glu (PE), respectively. The region also contains an epox-
ide hydrolase encoded by Rv3617, which may be
involved in detoxification, catabolism and regulation of
signaling molecules [3]. Rv3618 and Rv3623 encode a
probable monooxygenase and lipoprotein, respectively.
Rv3619c and Rv3620c are secretory proteins of 94
and 98 amino acids, respectively, reported in culture
filtrates of M. tuberculosis [4–6]. In silico studies have
predicted their presence in Mycobacterium leprae,
Mycobacterium avium and Mycobacterium marinum [7].
They belong to the ESAT-6 family, which comprises
23 members. However, they share only 20% sequence
identity with ESAT-6 and CFP-10. Within the ESAT-6
family, Rv3619c and Rv3620c are constituted within
a subfamily comprising Rv1037c ⁄ Rv1038c, Rv1197 ⁄
Rv1198, Rv1792 ⁄ Rv1793 and Rv2346c ⁄ Rv2347c [8].
The members within this subfamily share > 90%
amino acid sequence identity with Rv3619c ⁄ Rv3620c.
Overlapping synthetic peptide studies have demon-
strated that the Rv3619c ⁄ Rv3620c subfamily consists
of potent T-cell antigens [9].

Detailed studies of ESAT-6 and CFP-10 have
revealed that they interact strongly to form a 1 : 1
heterodimeric stable complex with a four-helix bundle
in which each protein bears a central WXG motif
[10,11]. Similar results have been obtained with
Rv0287–Rv0288 [12]. However, deviations from the
basic prototype structure of the ESAT-6–CFP-10 com-
plex have also been reported. Recent crystallographic
studies have suggested that Rv3019c–Rv3020c (ESAT-6 ⁄
CFP-10 homologs) exists as a heterotetramer. Rv3020c
contains histidine in place of tryptophan in the WXG
motif, which induces the formation of a small helix
that joins the N-terminal and C-terminal domains [13].
The ESAT-6 ⁄ CFP-10 homologs in Staphylococcus
aureus (Sa
EsxA and SaEsxB) do not form a hetero-
dimer complex; rather, they homodimerize. The crystal
structure of the homodimer of SaEsxA has been deter-
mined. Furthermore, sequence analysis has predicted
that SaEsxB (CFP-10 homolog) will have a structure
similar to that of SaEsxA; on the basis of this, it has
been suggested that the two proteins may work inde-
pendently [14]. These variations among complexes indi-
cate that different homologs and paralogs of the ESX
family may have different structural properties that
may lead to further functional dissimilarities. There-
fore, a separate detailed analysis for each pair is
required, structurally as well as functionally.
One of the major functions associated with ESAT-6
is its cytolytic activity. Hsu et al. have shown the

cytolysis of host cells by ESAT-6 secreted by intracel-
lular mycobacteria [15]. We have previously shown
that ESAT-6 adopts significant helical structure in the
presence of dimyristoylphosphatidylcholine (DMPC)
vesicles and dodecylphosphocholine (DPC) micelles,
indicating membrane binding. Furthermore, only
ESAT-6, and not CFP-10 or the complex, was found
to interact with lipid membranes [16]. Moreover, Smith
et al. have shown that ESAT-6 induces pore formation
in M. marinum in a dose-dependent manner, enabling
the bacterium to escape from the vacuole to the host
cell cytosol [17]. The membrane-binding ability was
related to disruption of the complex by Jonge et al.
[18]. These authors demonstrated that, in the acidic
phagosomal environment, the 1 : 1 ESAT-6–CFP-10
complex dissociated to free ESAT-6 to exert its cyto-
lytic activity. However, no studies have been per-
formed on other ESAT-6 ⁄ CFP-10 paralogs to
determine whether other members have similar mem-
brane-destabilizing functions.
The ESAT-6 family is a potential source of T-cell
antigens, which can be exploited for the development
of suitable vaccines against mycobacteria. ESAT-6 and
CFP-10 are the most widely studied proteins of this
family. ESAT-6 and CFP-10 activate the Th1 response
which is marked by T-cell proliferation and interferon
(IFN)-c release [19,20]. Subunit-based or DNA-based
ESAT-6 vaccines have been prepared, and their protec-
tive efficacy has been evaluated. The first DNA-based
vaccine involving ESAT-6 was reported by Kamath

et al. [21]. Although this vaccine provided a significant
level of protection against mycobacteria, its potency
was found to be lower than that of BCG. Brandt et al.
prepared the first single protein subunit tuberculosis
vaccine, using ESAT-6 with dioctadecylammonium
bromide and monophosphoryl lipid A, that conferred
protection similar to that provided by BCG [22]. A
recombinant chimeric fusion protein of ESAT-6 and
Characterization of Rv3619c and Rv3620c A. Mahmood et al.
342 FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS
Ag85B was found to provide better protection than
individual proteins or a mixture of them, and is cur-
rently being evaluated as a promising vaccine candi-
date [23,24]. Besides ESAT-6, other members evaluated
for immune response were Rv0288, Rv3019c, and
Rv3017c [25]. Rv3019c demonstrated its potential to
be used as a heterologous prime booster in conjunction
with BCG.
In the present study, we characterized the forma-
tion of the complex between the ESAT-6 family
protein Rv3619c and its pairing partner Rv3620c,
using isothermal titration calorimetry (ITC) and CD
spectroscopy. We also examined the structural prop-
erties of the proteins by spectroscopy and molecular
modeling. Furthermore, we evaluated the immune
response of Rv3619c in free antigenic form in mice
by determining lymphocyte proliferation, cytokine
levels, and antigen-specific antibody levels. Our study
provides insights into the properties of these secre-
tory proteins.

Results and Discussion
Interaction of Rv3619c and Rv3620c
ITC experiments were performed to determine the
thermodynamic parameters governing formation of the
complex between Rv3619c and Rv3620c. The raw ITC
data and integrated areas under each peak versus
Rv3619c ⁄ Rv3620c molar ratio are shown in Fig. 1A.
The binding isotherm was fitted to a single-site binding
model for determination of thermodynamic parame-
ters. The parameters used in fitting were the stoichiom-
etry of association (n), the binding constant (K
b
), and
the change in enthalpy (DH
b
). The values of these
parameters obtained from the nonlinear least-squares
fit to the binding curve are as follows: n = 1.0,
K
b
= (2.05 · 10
6
) ± (3.24 · 10
5
) m
)1
and DH
b
=
)3.35 · 10

4
± 760.1 calÆmol
)1
. The saturation of heat
released at a molar ratio of 1.0 strongly suggests that
the proteins form a 1 : 1 heterodimeric complex. The
Fig. 1. Thermodynamic and spectroscopic studies on Rv3619c–Rv3620c. (A) ITC measurements of the interaction between Rv3619c and
Rv3620c in phosphate buffer at 25 °C; raw data of heat effect (lcalÆs
)1
) of 30 injections (10 lL each) of 0.1 mM Rv3619c into 1.43 mL of
0.01 m
M Rv3620c. The data points (j) were obtained by integration of heat signals plotted against the Rv3619c ⁄ Rv3620c molar ratio in the
reaction cell. The solid line represents a calculated curve using the best-fit parameters obtained by a nonlinear least square fit. The heat of
dilution was subtracted from the raw data of titration of Rv3619c with Rv3620c. (B) Far-UV CD spectra of Rv3619c, Rv3620c, and the 1 : 1
complex. CD spectra of 5 l
M Rv3619c (j), Rv3620c (•) and the 1 : 1 complex (m) in phosphate buffer (pH 6.5, 25 °C. (C) Normalized transi-
tion curves for temperature-induced transition of the complex monitored in the far-UV CD region at 222 nm. The thermal unfolding (j) and
thermal refolding (•) profiles of the complex were plotted as fraction of protein folded versus temperature in °C.
A. Mahmood et al. Characterization of Rv3619c and Rv3620c
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 343
dissociation constant of the complex ( K
d
=1⁄ K
b
) was
4.8 · 10
)7
m. The free energy change (DG) and entropy
change (DS) associated with complex formation were
)8.55 kcalÆmol

)1
and )83.7 calÆmol
)1
ÆK
)1
, respectively,
at 25 °C.
A comparative thermodynamic analysis of ESAT-6⁄
CFP-10 family members is shown in Table 1. Although
Rv0287–Rv0288 is well characterized [12], we performed
ITC experiments with Rv0287 and Rv0288 to generate
the thermodynamic data for their interaction. The ITC
data for ESAT-6–CFP-10 have already been obtained
[16]. Data analysis revealed that Rv0288–Rv0287 has
the strongest binding affinity (K
d
$ 10 nm), followed by
ESAT-6–CFP-10 (K
d
$ 50 nm). Rv3619c–Rv3620c
showed the weakest binding (K
d
$ 480 nm). The bind-
ing free energy (DG) of Rv3619c–Rv3620c revealed dif-
ferences of 2.26 kcalÆmol
)1
relative to Rv0287–Rv0288
and 1.4 kcalÆmol
)1
relative to ESAT-6–CFP-10. This

suggests that binding of Rv0287 and Rv0288 is energeti-
cally more favored. The difference in entropy values
indicates that the conformational freedom of side chains
in Rv3619c–Rv3620c is comparatively greater. The dif-
ferences in binding affinity among ESAT-6 ⁄ CFP-10 par-
alogs suggest that the binding equilibrium in loosely
bound complexes may be shifted to the reactant side,
resulting in the release of unbound proteins under cer-
tain specific conditions, such as those in the acidic phag-
osomal environment.
The conformational changes associated with com-
plex formation were estimated by recording far-UV
CD spectra. The CD spectra of Rv3619c, Rv3620c and
the 1 : 1 complex were recorded (Fig. 1B) at 25 °C,
and data were analyzed by the k2d server. The CD
spectra showed that the two proteins and their 1 : 1
complex adopt a predominantly a-helical conforma-
tion. The a-helical contents of Rv3619c, Rv3620c and
the 1 : 1 complex were approximately 33%, 34% and
70%, respectively. The secondary structure of
Rv3619c–Rv3620c was very similar to that of ESAT-
6–CFP-10 and Rv0288–Rv0287. However, unlike CFP-
10 and Rv0287, which are unstructured, Rv3620c had
a significant a-helical content, even in the uncomplexed
state. The lower values of DH and DS (Table 1)
observed for Rv3619c–Rv3620c than for ESAT-6–
CFP-10 and Rv0287–Rv0288 could result from the dif-
ference in the folding state of Rv3620c from that of
CFP-10 and Rv0287.
Thermal unfolding of proteins

The presence of any stable tertiary structure in
Rv3619c and Rv3620c was ruled out by thermal
unfolding experiments (data not shown). The two pro-
teins were denatured when the temperature was
increased from 25 °Cto80°C, following non-coopera-
tive unfolding, and the structure was not regained on
cooling, indicating that they lack any stable tertiary
structure. The complex, however, demonstrated signifi-
cant resistance to denaturation before melting. The
unfolding started at 38 °C, following a cooperative
pathway with a denaturation midpoint (T
m
)of48°C
(Fig. 1C). Rv3619c–Rv3620c showed lower thermal
stability than ESAT-6–CFP-10 (T
m
=54°C) and
Rv0287–Rv0288 (T
m
=70°C) [12]. This indicates that
Rv3619c–Rv3620c has a smaller intermolecular hydro-
phobic overlapping interface. Overall, the thermal
denaturation profile and ITC data suggest that
Rv3619c forms a loose complex with its genomic part-
ner Rv3620c, because of a smaller protein–protein
interaction surface area than that in ESAT-6–CFP-10
and Rv0287–Rv0288.
The thermal renaturation profile of the 1 : 1 com-
plex was recorded by cooling the sample from 80 °C
to 25 °C (Fig. 1C). On reversal of the temperature, the

complex completely regained its secondary structure,
retracing a similar path. This characteristic feature was
also observed for ESAT-6–CFP-10 [16]. The two pro-
teins complement each other to attain a folded struc-
ture. However, in the absence of the other partner,
they lose their structure irreversibly. This implies that
Rv3620c, like CFP-10, functions to keep Rv3619c in a
structured and soluble form under physiological
conditions.
Modeling and docking
The secondary structure was also analyzed by molecu-
lar modeling and docking experiments. On the basis of
Table 1. A comparative analysis of thermodynamic parameters of ESAT–CFP-10, Rv0287–Rv0288, and Rv3619c–Rv3620c. The stoichiome-
try of interaction and values of K
b
and DH were determined by ITC. DG and DS were calculated from the thermodynamic formula
DG = )RT ln K
b
= DH ) TDS.
ESX pairs NK
b
(M
)1
) K
d
(M) DH (kcalÆmol
)1
) DS (calÆmol
)1
ÆK

)1
) DG (kcalÆmol
)1
)
Rv3619c–Rv3620c 1.0 2.0 · 10
6
4.8 · 10
)7
)33.5 )83.7 )8.55
ESAT-6–CFP-10 1.0 2.0 · 10
7
5.0 · 10
)8
)40.3 )101 )9.95
Rv0287–Rv0288 1.0 9.2 · 10
7
1.0 · 10
)8
)40.8 )100 )10.81
Characterization of Rv3619c and Rv3620c A. Mahmood et al.
344 FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS
the solution structure of the 1 : 1 ESAT-6–CFP-10
complex, we generated molecular models of Rv3619c
and Rv3620c using i-tasser and swiss model, respec-
tively, and molecules were docked using the patch
dock server. The model Rv3619c–Rv3620c is shown in
Fig. 2. We determined the accessible surface area
(ASA) for the Rv3619c–Rv3620c model and the
ESAT-6–CFP-10 solution structure by using the dis-
covery studio 2.1 package. The ASA for docked

Rv3619c–Rv3620c was 12 190 A
2
, and the buried
surface area was 1211 A
2
. The ASA for the ESAT-6–
CFP-10 solution structure was 11 512 A
2
, and the
buried surface area was 1675 A
2
. The buried surface
area for docked Rv3619c–Rv3620c was smaller than
that for the ESAT-6–CFP-10 solution structure, which
is in complete agreement with our ITC data.
The model suggests that the two proteins interact
with 1 : 1 stoichiometry, lying antiparallel to each
other, with each one having two helixes separated by a
loop containing the WXG motif. The predicted struc-
ture resembles the four-helix bundle packing of ESAT-
6–CFP-10. The crystal structure of Rv3019c–Rv3020c
suggested that replacement of tryptophan by histidine
in the WXG motif induced the formation of a small
helix in Rv3020c, joining the N-terminal and C-termi-
nal helices, conferring a tetramer structure. However,
as the WXG motif in the Rv3620c is strictly con-
served (Trp45 and Gly47), it is highly unlikely that
Rv3619c–Rv3620c would form a tetrameric complex
like Rv3019c–Rv3020c. Despite the predicted structure
being similar to that of ESAT-6–CFP-10, a consider-

able difference at the C-terminus of Rv3620c was
noticed. The C-terminal end of CFP-10 is unstruc-
tured, whereas the modeling and docking results pre-
dicted that Rv3620c would possess a C-terminal helix.
The C-terminal end of SaEsxA from S. aureus also
contains a folded region [14].
In order to identify the residues forming the inter-
molecular contact surface of Rv3619c–Rv3620c, we
generated a contact map for docked Rv3619c and
Rv3620c, using the acclerys discovery studio 2.1
software package. The contact map analysis predicted
that Val10, Ile17, Ala21, Leu24, Ala26, Ala30, Ile31,
Ile32, Val35, Leu36, Ala38, Phe41, Cys50, Phe53,
Leu57, Phe61, Val63, Ile64, Ala68, Ala70, Val75,
Ala77, Ala78, Met82, Val89 and Ala94 of Rv3619c
and Met12, Met15, Ala16, Phe19, Val21, Ala23,
Val26, Ala30, Met33, Ala35, Ala37, Ile40, Ala43,
Met48, Ala49, Leu54, Met57, Met60, Phe64, Ile67,
Val68, Met70, Leu71, Val74, Leu78, Val79 and Ala82
of Rv3620c are likely to form hydrophobic contact
surfaces. We plotted the N-terminal and C-terminal
helices of Rv3619c and Rv3620c on a heptad repeat
helical wheel, on the basis of optimum sequence
alignment (Figs S1 and S2). Hydrophobic residues
suggested by contact map analysis occupied predomi-
nantly ‘a’ and’d’ positions, suggesting that they form
the core of helix bundle packing. This is in full agree-
ment with the models suggested for ESAT-6–CFP-10
and Rv0287–Rv0288. The complex was predicted to be
stabilized by formation of a single salt bridge between

Glu25 of Rv3619c and Arg31 of Rv3620c.
The residues highlighted in Fig. 2, in the Rv3619c
helix, represent the nonconserved, semiconserved or
substituted conserved residues at the corresponding
positions among the related members Rv1037c,
Rv1198, Rv1793 and Rv2346c. Substitution of residues
at these positions severely alters antigenic recognition
by the cell. Alderson et al. demonstrated that a T-cell
line specific for Rv1198 failed to recognize peptides
from Rv1793 and Rv3619c with amino acid substitu-
tions at the 22nd and 23rd positions [9].
Effect of pH on complex stability
We analyzed pH-induced conformational changes in
Rv3619c, Rv3620c and the 1 : 1 complex by recording
the far-UV CD spectra at different pH values. The
mean residual ellipticity (MRE) at 222 nm was plotted
against different pH values, as shown in Fig. 3A. On
G22 / A22
S23 / L23
S33 / R33
T37 / A37
S39 / G39
A
48 / V48
G52 / E52
I32 / V32
N
Rv3620c
C
Rv3620c

N
Rv3619c
C
Rv3619c
Fig. 2. In silico modeling and docking of Rv3619c and Rv3620c.
The two proteins form a 1 : 1 complex. Rv3619c is shown in blue
and Rv3620c is shown in gray. The nonconserved, semiconserved
and substituted conserved regions of paralogs of Rv3619c (Rv1037,
Rv1198, Rv1793, and Rv2346c) are highlighted in blue, orange, and
yellow.
A. Mahmood et al. Characterization of Rv3619c and Rv3620c
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 345
lowering of the pH from 6.5 to 4.5, Rv3619c and
Rv3620c showed considerable loss of conformation,
although Rv3619c resisted conformational change until
pH 5.5. However, on mixing of the two proteins at
pH 4.5, the resultant spectra, shown in Fig. 3B, dem-
onstrated significant loss of structure. This could be
attributable to weak interactions between two unfolded
or partially folded proteins. Furthermore, it indicates
that the structural unfolding of individual proteins
could be the event that results in dissociation of the
complex at acidic pH. Recently, Arbing et al. also
reported the pH-induced dissociation of Rv3019c–
Rv3020c (unpublished data) [13].
Effect of trifluoroethanol (TFE) and DPC micelles
on the conformation of Rv3619c and Rv3620c
Previous work suggested that ESAT-6 adopts a helical
structure, and is sequestered and induces pore formation
in the cell membrane. Furthermore, only ESAT-6, and

not CFP-10, was associated with cytolytic activity
[16,26]. To investigate the role of Rv3619c, Rv3620c and
the 1 : 1 complex in membrane binding, CD spectros-
copy experiments were performed in the presence of
TFE and DPC micelles, as shown in Fig . 4A. In 40%
TFE, Rv3619c, Rv3620c and the 1 : 1 complex adopted
a highly folded structure. In 20 mm DPC, Rv3619c and
Rv3620c demonstrated 42% and 35% increases, respec-
tively, in helical content. However, the 1 : 1 complex did
not show any significant change in conformation in
20 mm DPC. This implies that the two proteins bind to
the membrane individually, but not after they form a
complex, as also observed for ESAT-6 and CFP-10.
Furthermore, we recorded the intrinsic tryptophan fluo-
rescence of Rv3619c and Rv3620c in 20 mm DPC at
wavelengths ranging from 300 to 400 nm (Fig. 4B). The
k
max
values of Rv3619c and Rv3620c shifted to lower
wavelengths by 5 nm and 10 nm, respectively, and this
was accompanied by enhancements of fluorescence
intensity, suggesting the relocation of tryptophans in the
hydrophobic environment because of membrane bind-
ing. We also checked the binding of the two proteins to
small unilamellar vesicles of DMPC, using CD spectros-
copy. Our preliminary results suggested that Rv3619c,
but not Rv3620c, underwent a change in helicity in the
presence of DMPC small unilamellar vesicless. This does
not correlate with the change in CD spectra observed in
the presence of DPC micelles. However, interestingly, it

does correlate with the binding of the fluorescent dye 8-
anilinonapthalene-1-sulfonate, which was observed for
Rv3619c but not for Rv3620c (data not shown).
Previous membrane-binding studies with ESAT-6
suggested that the deeper integration of the protein
into phospholipids is related to its unfolding and struc-
tural transition [16]. We also found unfolding of
Rv3619c and Rv3620c at pH 4.5. In some cases,
phagosomes containing mycobacteria may advance to
the phagolysosomal stage, with concomitant lowering
of the pH from 5.1 to 4.8–4.5. It has been suggested
that, under such conditions, ESAT-6 could dissociate
from the complex and bind the lipid membranes [17].
Considering the two events together, it can be inferred
that unfolding of proteins and formation of a struc-
tural intermediate is associated with membrane bind-
ing. The acidic phagolysosome may provide an
environment that stimulates dissociation of the com-
plex and structural reorientation of proteins, allowing
them to assemble and penetrate the membrane deeply.
Taking into account that mycobacteria in infected cells
inhibit the fusion of lysosomes with phagosomes,
Rv3619c–Rv3620c would most likely exist as a com-
plex. However, under certain conditions when the
infected cells also contain phagolysosomes [27,28], the
1 : 1 complex may be disrupted, and Rv3619c and
Fig. 3. pH-induced conformational changes in Rv3619c, Rv3620c,
and the 1 : 1 complex. (A) MREs of Rv3619c (j), Rv3620c (d) and
the 1 : 1 complex (m) at 222 nm were plotted against different pH
values. (B) Far UV-CD spectra of the 1 : 1 complex at pH values of

6.5 (j), 5.5 (d), and 4.5 (m).
Characterization of Rv3619c and Rv3620c A. Mahmood et al.
346 FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS
Rv3620c may execute their functions independently
and bind to lipid membranes. Although no pH-based
structural studies have so far been performed with
ESAT-6, our study supports the hypothesis of Simeone
et al. [26] that the biological activity of ESAT-6
depends on the pH of phagosomal compartments.
Proteins of the ESAT-6 family are not only involved
in spreading virulence by exhibiting cytolytic activity;
they also confer protection against M. tuberculosis
through the antimicrobial Th1 host immune response.
ESAT-6, Rv0288 and Rv3019c are known to stimulate
T-cells to proliferate and protect against mycobacteria
[25,29,30]. Th1 activation results in IFN-c and inter-
leukin (IL)-12 release, whereas Th2 activation releases
IL-4. A balanced Th1 ⁄ Th2 response is important for
clearance of mycobacteria. To determine whether
Rv3619c also activates T-cells to release cytokines, we
examined its role in the immune response in mice.
Analysis of immune response of Rv3619c
We evaluated the immune response of Rv3619c by
injecting the free antigen in NaCl ⁄ P
i
in Balb ⁄ c mice,
with NaCl ⁄ P
i
as a control. Initially, the antigen-induced
lymphocyte proliferation activity of Rv3619c was

assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay. The proliferative
response of Rv3619c was demonstrated by stimulation
Fig. 4. Membrane binding of Rv3619c, Rv3620c, and the 1 : 1 complex. (A) Far-UV CD spectra of 5 lM Rv3619c, Rv3620c and the 1 : 1
complex in the presence of phosphate buffer (j), 40% TFE (•), and 20 m
M DPC (m) are shown. All of the spectra were recorded at 25 °C.
(B) Fluorescence emission spectra of Rv3619c and Rv3620c in phosphate buffer (j) and 20 m
M DPC (•).
A. Mahmood et al. Characterization of Rv3619c and Rv3620c
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 347
index (SI), as shown in Fig. 5A; it was determined to be
4.695 ± 0.24 pgÆmL
)1
postbooster, with a statistically
significant difference (P < 0.05) from the NaCl ⁄ P
i
group. The significant augmentation of antigen-specific
proliferation clearly demonstrates the presence of
immunologically active lymphocytes in immunized
mice. Statistically significant levels of IFN-c (832
± 30.61 pgÆmL
)1
; P < 0.001) and IL-12 (481 ± 5.46
pgÆmL
)1
; P < 0.05) were produced by spleenocytes of
mice immunized with Rv3619c (Fig. 5B); IL-4, a Th2
cytokine, was also detected in immunized groups, with a
significant difference, but the level of IFN-c was very
high when compared with that of IL-4. The humoral

response was determined by measuring the Rv3619c-
specific serum IgG level (Fig. 6A). Class switching
showed a predominant IgG
2a
response, as indicated by
a postbooster IgG
2a
⁄ IgG
1
ratio greater than 1 (Fig. 6B)
The high levels of IFN-c and IL-12 secretion, suggesting
a biased Th1-type response of Rv3619c, is consistent
with the IgG
2a
⁄ IgG
1
ratio. The study clearly demon-
strates that Rv3619c is a potent T-cell antigen that may
provide protection against mycobacterial infection if
used in combination with a suitable adjuvant or in a
mixture with other antigens.
Rv3619c paralogs, i.e. Rv1037c, Rv1198, Rv1793
and Rv2346c and their genomic partners, share greater
than 90% amino acid sequence identity. Structurally,
they may form similar complexes to those observed for
Rv3619c, Rv3620c, and other pairs, but the immune
response may vary, as they display unique epitopes.
The presence of similar structure and function but
different immune responses has been related to the
A

B
Fig. 5. Study of immune response to Rv3619c antigen. Analysis of
the Rv3619c-specific cytokine profile of 6–8-week-old female
Balb ⁄ c mice immunized with 20–25 lg of antigen. (A) Lymphocyte
proliferation response of Rv3619c expressed in terms of SI. Each
bar represents mean ± standard deviation. (B) IFN-c, IL-12 and IL-4
levels determined 2 weeks postimmunization and 2 weeks post-
booster. Three mice per group were used, and the data obtained
were statistically significant different, with ***P < 0.001,
**P < 0.01, or *P < 0.05, from those obtained with NaCl ⁄ P
i
.
A
B
Fig. 6. Estimation of humoral response to Rv3619c antigen. The
antibody response against Rv3619c in mice (three mice per group)
is shown. Serum was collected 2 weeks postimmunization and
2 weeks postbooster. The antibody level was estimated by record-
ing the absorbance at 490 nm. (A) Total IgG content. (B) IgG
2a
⁄ IgG
1
ratio, indicating the biased Th1 response.
Characterization of Rv3619c and Rv3620c A. Mahmood et al.
348 FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS
mycobacterial strategy for escaping the host immune
recognition system [30]. Studies with tuberculosis reac-
tor animals also revealed that immunodominant epi-
topes of the ESAT-6 family come from variable
regions more than homologous regions, suggesting that

mycobacteria may vary their antigenic load according
to requirements, leading to antigenic drift that enables
mycobacterial escape [31]. The presence of several
pairs of ESAT family proteins within the M. tuberculo-
sis genome suggests that they might be expressed under
different physiological conditions, and are able to sub-
stitute for each other functionally; this strategy enables
them to survive longer in host cells. However, at the
same time, sequence variations among the family mem-
bers provide a pool of antigens that can generate the
effector molecules that restrict mycobacterial growth.
Evaluation of the formation of complexes and their
protective efficacy will help in the development of suit-
able vaccine candidates.
Conclusion
The ESAT-6 ⁄ CFP-10 family has 23 members, consisting
of 11 pairs and one unpaired member. Although these
11 pairs are likely to form complexes in a manner simi-
lar to the formation of ESAT-6–CFP-10, they may exhi-
bit subtle variations in affinity, stability and immune
response, which in turn may further define their individ-
ual functional roles. A systematic study of various com-
plex-forming pairs would improve our understanding of
the evolutionary and functional relevance of the whole
ESAT family. Our study further consolidates the
hypothesis that the structural unfolding of individual
proteins under conditions of acidic pH may be the key
factor triggering the dissociation of complexes. How-
ever, we believe that this aspect still needs more elegant,
unambiguous, quantitative and time-resolved character-

ization. The sequence variation in the ESAT-6 ⁄ CFP-10
family not only determines the stability of the complex,
but also provides an antigenic pool in which a change in
a single amino acid can change the host immune
response. The efficacy of peptides and proteins incorpo-
rating these antigens must be evaluated so that those
conferring protection can be developed further. We are
currently working in this direction.
Experimental procedures
Materials
pET expression vectors were obtained from Novagen
(Darmstadt, Germany). Oligonucleotides for gene isolation
were from BIO Serve (Hyderabad, Andhra Pradesh, India).
Restriction endonucleases, T4 DNA ligase and DNA size
markers were from New England Biolabs (Beverly, MA,
USA). Taq polymerase and other reagents for PCR, the
plasmid miniprep kit and the gel extraction kit were from
Qiagen. The Ni
2+
–nitrilotriacetic acid superflow metal-
affinity chromatography matrix was from Qiagen. For
protein concentration, Centricon membrane were used
(molecular mass cut-off 3KDa: Millipore (India) Pvt. Ltd,
Bangalore, India). The rest of the chemical reagents were
from Sigma (New Delhi, India).
Cloning, expression and purification
Genomic DNA of M. tuberculosis H37Rv was prepared as
described by Kremer et al. [32]. The genes encoding
Rv3619c and Rv3620c were PCR-amplified with oligonu-
cleotide primers and pfu DNA polymerase, and cloned into

pET-NH6. This cloning strategy added an additional 30
residues at the N-terminus, including the six residues of the
His-tag. The vectors containing the genes encoding
Rv3619c and Rv3620c were then transformed into
BL21(kDE3) Escherichia coli cells, which were grown in LB
medium supplemented with ampicillin (100 lg Æ mL
)1
).
BL21(kDE3) cells containing the plasmids pET-NH6–
Rv3619c and pET-NH6–Rv3620c were grown in LB med-
ium supplemented with ampicillin (100 lgÆmL
)1
) and
induced at D
600 nm
= 1.0 with a final concentration of
0.5 mm isopropyl thio-b-d-galactoside. The Rv3619c culture
was grown for a further 12–14 h at 27 °C, and the Rv3620c
culture for 6 h at 37 °C. All proteins were purified over a
Ni
2+
–nitrilotriacetic acid matrix with a standard protocol
under denaturing conditions, according to the manufac-
turer’s instructions, except that NaCl and guanidine hydro-
chloride were excluded from the buffer. The column
fractions were checked for purity by SDS ⁄ PAGE (15%
gel). The proteins were refolded by dialysis, with a buffer
containing 25 mm NaH
2
PO

4
, 100 mm NaCl, and 1 mm
EDTA (pH 6.5). Refolded Rv3619c and Rv3620c were dia-
lyzed against buffer containing 20 mm NaH
2
PO
4
,50mm
NaCl, and 0.1% NaN
3
(pH 6.5). The pET-NH6–Rv3619c-
encoded and pET-NH6–Rv3620c-encoded proteins con-
tained 30 extra N-terminal residues with a His-tag.
ITC
The ITC experiments were performed at 25 °Cona
VP-ITC calorimeter from Microcal (Northampton, MA,
USA). The calorimeter was calibrated according to the user
manual of the instrument. The proteins were dialyzed
against buffer containing 20 mm NaH
2
PO
4
and 50 mm
NaCl (pH 6.5). Samples were degassed prior to titration at
20 °C. The ITC experiments were performed by adding
aliquots of Rv3619c to Rv3620c. The sample cell was filled
with 1.43 mL of 0.01 mm Rv3620c and titrated against
0.1 mm Rv3619c. Thirty injections of 10 lL each were
A. Mahmood et al. Characterization of Rv3619c and Rv3620c
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 349

made at intervals of 180 s. The ITC data were analyzed
with origin version 7. The amount of heat produced per
injection was calculated by integration of the area under
each peak with a baseline selected by origin.
CD spectroscopy
CD measurements were performed to determine the second-
ary structure of Rv3619c, Rv3620c, and the 1 : 1 complex.
The experiments were performed on a Jasco Spectropola-
rimeter model J-810. The instrument was calibrated with
(+)-10-camphorsulfonic acid. The protein spectra were
recorded with the protein samples in buffer containing
20 mm NaH
2
PO
4
and 50 mm NaCl (pH 6.5). The protein
concentration used was in the range 5–10 lm . Three scans
were averaged for each spectrum. Isothermal wavelength
scans were recorded in the range 250–200 nm, with a path-
length of 2 mm, a response time of 1 s, a scan speed of
20 nmÆmin
)1
, and a data pitch of 0.5. The CD results were
expressed as MRE, in degree cm
)2
Ædmol
)1
, calculated as
follows:
MRE ¼ðh  100  M

r
Þ=ðcdN
A
Þ
where h is the observed ellipiticity (°), c is the protein con-
centration (mgÆmL
)1
), d is the pathlength (cm), and N
A
is
the number of amino acids. Percentage secondary structure
was calculated with the online k
2D server (l.
de/
~
andrade/k2d/). For thermal denaturation studies, the
spectra were recorded in the temperature range 25–80 °Cat
a speed of 1 °CÆmin
)1
. For refolding, the temperature was
reversed at the same speed. The fraction of protein folded
corresponding to the MRE at 222 nm was calculated from
the equation
½h
obs
Àh
den
=½h
nat
Àh

den

where h
nat
and h
den
are the MREs at 222 nm when proteins
are in the native state at 25 °C, and in the denatured state
at 80 °C. h
obs
is the observed MRE.
To study the effect of membrane mimetic conditions on
conformation of proteins, far-UV CD spectra were acquired
in the presence of either 40% TFE or 20 mm DPC. DPC
stock (200 mm) was prepared in buffer containing 20 mm
NaH
2
PO
4
and 50 mm NaCl (pH 6.5), and centrifuged at
18 500 g to remove any suspended particles. DPC was added
to 5 lm protein to a final concentration of 20 mm. For TFE
experiments, 5 lm protein was added to 40% TFE. The spec-
tra were recorded in the wavelength range 250–200 nm and
analyzed on the k
2D server.
Protein modeling and docking
The protein sequences of Rv3619c and Rv3620c were taken
from the TB Structural Genomics Consortium (http://
www.doe-mbi.ucla.edu/TB/), and sequences were aligned

using a server ( Models of
Rv3619c and Rv3620c were generated using online servers
( and http://
swissmodel.expasy.org//SWISS-MODEL.html, respectively).
The modeled structures of Rv3619c and Rv3620c were
docked using patchdock ( />PatchDock), a geometry-based molecular docking algo-
rithm. The docked complex was analyzed with accelrys
discovery studio 2.0.
Fluorescence spectroscopy
Fluorescence spectra were acquired to record the intrinsic
tryptophan fluorescence changes of Rv3619c, Rv3620c and
the 1 : 1 complex in the presence of 40% TFE and 20 mm
DPC. Fluorescence spectra were acquired at 25 °Cona
Perkin-Elmer Life Sciences LS 50B spectroluminescence
meter, with a 5-mm-pathlength quartz cell. Protein (1 lm)
was mixed with either 40% TFE or 20 mm DPC. The maxi-
mum intrinsic fluorescence was monitored to record the
wavelength shift. The fluorescence emission spectra were
recorded in the range 300–400 nm, with an excitation wave-
length of 280 nm.
Animals and immunization
Female Balb ⁄ c mice (6–8 weeks old) were purchased from the
JALMA Institute for Leprosy and Other Microbial Diseases,
Agra, India. Mice were maintained in the animal facility, and
the techniques used for injection and bleeding of animals
were performed in strict accordance with the mandates
approved by the Animal Ethics Committee (CPCSEA, Gov-
ernment of India). Ten mice in two groups were taken for
study. One group was immunized with antigen in NaCl ⁄ P
i

,
and the other group (control) was injected with NaCl ⁄ P
i
only.
The immunization volume was 100 lL per animal, and the
antigen dose was 20–25 lg per injection. The mice were
immunized by subcutaneous injection in the lower abdominal
region. Two weeks postimmunization, a single booster with
same amount of antigen was given to each animal.
Lymphocyte proliferation
Cells were grown in RPMI-1640 with 10% fetal bovine
serum and 1% antimycotic solution for 48 h in a CO
2
incu-
bator at 37 °C, 5% CO
2
and appropriate humidity under
aseptic conditions, with 20 lg of antigen and 2 lgÆmL
)1
concanavalin A. Four to five hours before completion of
incubation, 25 lLof55mgÆmL
)1
of MTT stock solution
was added to each well in 96-well plates to attain a final
concentration of 1 mgÆmL
)1
. One well was kept blank; that
is, before addition of the MTT, 100 lL of lysis buffer was
added to the well. When dark crystals appeared, 50 l Lof
tissue culture-grade dimethylsulfoxide was added to each

well. Plates were then incubated for 2 h in a CO
2
incubator
Characterization of Rv3619c and Rv3620c A. Mahmood et al.
350 FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS
at 37 °C. The absorbance was then measured at 540 nm.
The data are presented here in the form of SI. An SI > 2
was considered to be significant:
SI = (D
540 nm
of activated cells – D
540 nm
of inactivated
cells) ⁄ D
540 nm
of inactivated cells.
Cytokine assay – determination of IFN-c, IL-4 and
IL-12 levels by sandwich ELISA
Cytokine levels were estimated with appropriate purified
and biotinylated antibody pairs according to the manufac-
turer’s protocols. Briefly, 50 lL of the purified capture
antibodies was adsorbed overnight on polystyrene microt-
iter plates at 4 °C in carbonate buffer (pH 9.5). Plates
were washed five times with buffer containing NaCl ⁄ P
i
with 0.05% tween twenty, and blocked with 5% skimmed
milk. After the usual steps washing steps, the 50-lL
supernatant of spleenocytes cultured for 48 h were used
for detection of cytokines. After the stipulated incubation
time, the plate was thoroughly washed and incubated with

biotinylated polyclonal goat anti-(mouse IFN-c) (detection
antibody). Then, plates were washed three times with buf-
fer containing NaCl ⁄ P
i
with 0.05% tween twenty, and
100 lL of streptavidin–horseradish peroxidase (HRP) was
added to each well; the plates were then incubated for
30 min at room temperature. Plates were again washed
three times with buffer containing NaCl ⁄ P
i
and 0.05%
tween twenty, and finally developed with tetramethylbenzi-
dine; the absorbance was then read at 450 nm. Recombi-
nant IFN-c standards were used for calculation of
cytokine concentrations in the samples tested. IL-12 and
IL-4 levels were estimated similarly.
Determination of total IgG and isotyping
The production of antigen-specific total IgG and isotype
antibodies was measured in the sera of the immunized mice
bled 2 weeks postimmunization and 2 weeks postchallenge,
as described previously [33]. Briefly, ninety-six-well microtit-
er plates were incubated overnight with 100 lL of antigen
(0.5 ngÆmL
)1
) in carbonate ⁄ bicarbonate buffer (0.05 m,
pH 9.6) at 4 °C. After washing and blocking steps, test and
control sera were serially diluted, and plates were then incu-
bated at 37 °C for 1 h. After several washings of the plate,
total IgG was determined by using HRP-tagged rabbit anti-
(mouse IgG); in isotyping, plate was further incubated with

100 lL of (1 : 5000 dilution of stock) goat anti-mouse
(IgG
1
) and goat anti-mouse (IgG
2a
). The plates were incu-
bated at 37 °C for 1 h. After the washing steps, 100 lLof
(1 : 5000 dilution of stock) HRP-conjugated rabbit anti-
goat antibody was added to each well, and the plate was
incubated at 37 °C for 1 h. The plate was washed again
before addition of 100 lL of substrate solution (6 mg of
o-phenylenediamine dihydrochloride in 12 mL of substrate
buffer with 5 lL of 30% H
2
O
2
), and was finally incubated
at 37 °C for 40 min. The reaction was terminated by the
addition of 50 lLof1m H
2
SO
4
. The absorbance was read
at 490 nm with a microtiter plate reader (Bio-Rad, Life
Science Research, Hercules, CA).
Statistical analysis
The data on cytokine expression, lymphocyte proliferation
and serum IgG levels were analyzed with Student’s t-test,
using sigma plot version 2010. The values were considered
to be significant with ***P < 0.001, **P < 0.01, or

*P < 0.05.
Acknowledgements
A. Mahmood is the recipient of research fellowships
from the Council of Scientific and Industrial Research
(CSIR), New Delhi, India. S. Srivastava is the recipient
of project assistantship from the CSIR network pro-
ject. The work was supported by grants from CSIR
network project NWP0038 and from the Department
of Biotechnology. M. A. Ansari is the recipient of a
fellowship from ICMR.
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Supporting information
The following supplementary material is available:
Fig. S1. Helical wheel projection of N-terminal and
C-terminal Rv3619c.
Fig. S2. Helical wheel projection of N-terminal and
C-terminal Rv3620c.
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
A. Mahmood et al. Characterization of Rv3619c and Rv3620c
FEBS Journal 278 (2011) 341–353 ª 2010 CDRI. Journal compilation ª 2010 FEBS 353