We show that, simi-lar to WhiB3 and WhiB4, other freshly purified WhiB proteins also coordinate a [2Fe–2S] cluster which respond differently to the oxidizing environment.. tuberculosis en
Trang 1seven WhiB proteins of Mycobacterium tuberculosis
H37Rv
Md Suhail Alam, Saurabh K Garg* and Pushpa Agrawal
Institute of Microbial Technology, CSIR, Chandigarh, India
Mycobacterium tuberculosis has a remarkable ability to
survive under hostile conditions it encounters during
infection [1] Despite extensive research directed
towards understanding the physiology of M
tuberculo-sis and its molecular pathogenesis [1–3], many
funda-mental questions about the mechanisms of survival
during early infection and persistence remain poorly
understood Among several intriguing questions, are:
(a) what are the bacterial determinants necessary for
early infection, (b) how does the bacterium counteract
or evade its host’s defenses to survive the vigorous host-immune response, (c) what regulates the transition from initial growth to persistence and back to active growth, (d) are the bacteria present in a non-replicating ‘spore-like’ state or do they replicate at all during latency, and (e) how does the bacterium adapt to survive under the anaerobic and nutritionally altered environment within the granuloma? The answers to these questions are likely to provide insight into the mechanisms by which
M tuberculosis establishes infection and persists within
Keywords
iron–sulfur cluster; Mycobacterium
tuberculosis; protein disulfide reductase;
redox system; WhiB
Correspondence
P Agrawal, Institute of Microbial
Technology, Sector-39A, Chandigarh
160 036, India
Fax: +91 172 269 0585
Tel: +91 172 263 6680 ⁄ 263 6681; Ext 3264
E-mail: pushpa@imtech.res.in
*Present address
Department of Environmental and
Biomolec-ular Systems, Oregon Health and Science
University, Beaverton, OR, USA
(Received 16 September 2008, revised 22
October 2008, accepted 23 October 2008)
doi:10.1111/j.1742-4658.2008.06755.x
The whiB-like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins UV– Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe–2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea The [2Fe–2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments The [2Fe–2S] cluster of each WhiB protein responded differ-ently to the oxidative effect of air and oxidized glutathione In all cases, disassembly of the [2Fe–2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disul-fide of insulin, a model substrate However, the reduction efficiency varied significantly Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M tuberculosis
Abbreviations
ANS, 8-anilinonapthalene-1-sulfonate; GSH, reduced glutathione; GSSG, oxidized glutathione; IAA, iodoacetamide; ThT, thioflavin T;
Trx, thioredoxin.
Trang 2the host and the means to eliminate latent infection, a
phase of the disease that poses the most significant
obstacle to the eradication of tuberculosis To survive
and establish successful infection, M tuberculosis
appears to have acquired a strong network of genes to
sense and respond to stress conditions; the properties of
many of these are poorly understood
A family of genes, whiB, has received attention
because of their involvement in cell division (whiB2),
fatty acid metabolism and pathogenesis (whiB3),
antibi-otic resistance (whiB7) and in sensing a variety of stress
conditions [4–9] Seven genes, whiB1⁄ Rv3219, whiB2 ⁄
Rv3260c, whiB3⁄ Rv3416, whiB4⁄ Rv3681c, whiB5⁄
Rv0022c, whiB6⁄ Rv3862c and whiB7 ⁄ Rv3197A, have
been identified in M tuberculosis [10,11] as orthologs of
the whiB gene of Streptomyces coelicolor A3(2), which
has been shown to be involved in sporulation [12]
Although, WhiB proteins are annotated as putative
transcription factors [12], to date it has not been
shown directly that these proteins work as
transcrip-tion factors We have previously reported that WhiB1⁄
Rv3219 [13], WhiB3⁄ Rv3416 [14] and WhiB4 ⁄ Rv3681c
[15] are protein disulfide reductases WhiB4 has been
postulated to act as a sensor of oxidative stress, wherein
the inactive holo protein (containing a [4Fe–4S]
clus-ter) transformed into an active apo protein (without
an iron–sulfur cluster) in oxidizing environments and
gained protein disulfide reductase activity [15]
How-ever, to date the biochemical features of WhiB2, WhiB5,
WhiB6 and WhiB7 from M tuberculosis have not been
reported The observations that different whiB
muta-tions impart distinct phenotypes and respond differently
to stress conditions indicate importance of each member
separately in mycobacterial physiology The available
information on WhiB proteins demands careful
investi-gation of the biochemical and biophysical properties of
each
Mycobacterial WhiB proteins have 22–67% identity
with WhiB protein of S coelicolor A3(2) Sequence
analysis of M tuberculosis WhiB proteins shows the
presence of four conserved cysteines arranged as ‘C-X
19-36-C-X-X-C-X5-7-C’ [16] Notably, two cysteines are
present in a conserved CXXC motif, except in WhiB5⁄
Rv0022c where it is CXXXC (CLRRC) Proteins with
the CXXC motif have been implicated in diverse
func-tions, for example, protein disulfide oxidoreductase
activity [17], redox sensing [18] and the coordination of
metal cofactors [19] The functional importance of the
conserved cysteine residues in iron–sulfur cluster
coor-dination and protein disulfide reductase has been
dem-onstrated in WhiB4 [15] Recently, cysteines of WhiB3
have also been shown to act as a ligand for the O2- and
NO-responsive [4Fe–4S] cluster [9]
The presence of four conserved cysteines and a CXXC motif in WhiB proteins from M tuberculosis raises several questions: are all WhiB proteins coordi-nated with an iron–sulfur cluster? If yes, then what are their basic properties? Are the iron–sulfur clusters equally oxidation labile? Does removal of the iron– sulfur cluster lead to disulfide bond formation? Are the structural features of mycobacterial WhiB proteins similar? Do all WhiB proteins behave like protein disulfide reductase? The objective of this study is to answer several of the questions raised above
This is the first study to report the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M tuberculosis and also compare the prop-erties of all seven WhiB proteins We show that, simi-lar to WhiB3 and WhiB4, other freshly purified WhiB proteins also coordinate a [2Fe–2S] cluster which respond differently to the oxidizing environment Except WhiB2, apo WhiB5, WhiB6 and WhiB7 also reduce insulin in vitro, but the efficiency of the reduc-tion varies An extensive biophysical study suggested that the WhiB proteins of M tuberculosis are struc-turally different The functional relevance of their divergent molecular properties is discussed
Results
All seven whiB genes of M tuberculosis encode iron–sulfur proteins
Previous work on WhiB3 [9] and WhiB4 [15] identified the presence of cysteine-bound iron–sulfur cluster in these proteins We speculated that all seven WhiB pro-teins may also coordinate an iron–sulfur cluster There-fore, we overexpressed the recombinant WhiB proteins (with an N-terminal S-tag and C-terminal 6· His tag)
in Escherichia coli BL21 (DE3) Overexpression at
37C for 3 h led to the formation of light brown inclu-sion bodies However, induction at 16C for 20 h resulted in the expression of 10–20% of each WhiB protein in the soluble form On SDS⁄ PAGE, the mass
of Ni2+-NTA-purified WhiB proteins corresponded to their theoretically calculated molecular mass (predicted molecular mass + 5 kDa tags) (Fig S1) Proteins purified from a soluble fraction or after denaturation or
by in-column refolding were 98% pure and were brownish (Figs S1 and S2)
The presence of four conserved cysteines and the brownish appearance of purified WhiB1, WhiB2, WhiB5, WhiB6 and WhiB7 indicated the presence of
an iron–sulfur cluster To identify and confirm the presence of the iron–sulfur cluster, the absorption spectra of the purified proteins were recorded in the
Trang 3range 200–700 nm In addition to a peak at 280 nm,
two additional peaks at 333–340 and 420–
424 nm, along with two broad shoulders at 460 and
560–580 nm were observed (Fig 1) The peaks were
characteristic of an [2Fe–2S] cluster [20], therefore, it
was assumed that freshly purified WhiB1, WhiB2,
WhiB5, WhiB6 and WhiB7 also coordinated the [2Fe–
2S] cluster The absorption spectra of different WhiB
proteins were largely indistinguishable, however, in
WhiB6 and WhiB7, the shoulder at 460 nm was
more prominent than in others This subtle change in
the peak pattern may be because of their differential
electronic environment The nature and type of amino
acids and their side-chain orientations around iron–
sulfur cluster coordination sites are the likely cause of
minor variations in the electronic properties, which
were reflected in their absorption spectra
The brownish appearance of the protein purified in
the presence of 8 m urea indicated that the iron–sulfur
cluster of WhiB proteins had survived treatment by a
denaturant, a feature very similar to WhiB3 [14] and
WhiB4 [15] Unlike proteins purified from the soluble
fraction, which had a spectral feature typical of the
[2Fe–2S] cluster, proteins in 8 m urea showed a single
peak at 400–415 nm (Fig S3) The differential peak
features may be due to the solvent-induced
confor-mational change, which is possibly because of changes
in the chemical environment around the iron–sulfur
cluster, the partial destruction of the cluster or its
con-version to other forms In order to investigate the
probable reason(s) for the observed difference, the
pro-teins were processed for in-column refolding The
absorption spectra of the in-column refolded proteins
were similar to those of their native counterparts
(Fig S3) Interestingly, iron–sulfur cluster-specific peak
intensities were similar in both conditions These data
suggest that the coordination of iron–sulfur clusters to
the WhiB proteins was unaffected by 8 m urea and the
differences in peak patterns were due to the presence
of urea In order to acquire firm evidence for this
observation, the total iron content of proteins purified
under different conditions was measured
The total iron content of the native and in-column
refolded protein varied between 0.14 and 0.20 atoms
per monomer (Table 1) The sub-stoichiometric iron
content of iron–sulfur proteins is generally due to the
impaired incorporation of the cluster into the protein
during overexpression in E coli and⁄ or loss during
purification when conditions are not strictly anaerobic
[21] We attempted to reconstitute the iron–sulfur
clus-ter in WhiB proteins in vitro using FeCl3 and Na2S,
but did not succeed Therefore, incorporated
l-cys-teine as a sulfur source in the reconstitution assay
IscS⁄ Rv3025c, a cysteine desulfurase [9] of M tuber-culosis was cloned, expressed in E coli and purified by metal-affinity chromatography (data not shown) The WhiB proteins were incubated in the reaction mixture along with FeCl3, IscS and 35S-cysteine We observed
an IscS-dependent mobilization of sulfur from l-cyste-ine to the iron–sulfur cluster of WhiB proteins (Fig 2A) In the control reactions, where IscS was excluded or the iron concentration was limited (10-fold less), we did not observe any signal (Fig 2A) Further characterization of the iron–sulfur cluster of the recon-stituted samples could not be carried out because none
of the samples gave an EPR signal at 120K using liquid nitrogen (data not shown) It is possible that a further decrease in temperature (using liquid helium) would be required in order to detect the EPR signal Nevertheless, the absorption spectra of the reconsti-tuted proteins showed a single peak at 420 nm indi-cating the presence of a [4Fe–4S] cluster (Fig 2B) The presence of a similar cluster has been reported in WhiB3 and WhiB4
The iron content of proteins purified from the solu-ble fraction, from inclusion bodies, under denaturing conditions and after refolding was similar (Table 1) The data clearly suggested that the protein fold responsible for holding the iron–sulfur cluster was resistant to the denaturing effect of 8 m urea The abil-ity of the iron–sulfur cluster to survive the effects of protein denaturants is a feature of high potential iron– sulfur proteins [22] It is possible that WhiB proteins also fall into the same category However, detailed analysis would be required to establish this
Iron–sulfur clusters of M tuberculosis WhiB proteins are redox sensitive
Previously, we reported that the iron–sulfur cluster of WhiB4 disintegrates under an oxidizing environment, but not under reducing conditions [15] The rate of dis-integration was directly correlated with the duration and strength of the oxidizing environment Similarly,
in this study, the intensity of the brown color and the iron–sulfur cluster-specific peaks decreased gradually
as the time of exposure to air increased (Fig S4) The results suggest that the iron–sulfur clusters were sus-ceptible to oxidative degradation, although the rate of degradation varied significantly WhiB1 lost 65% of its iron–sulfur clusters in the initial 6 h, whereas in WhiB6 and WhiB7 the loss was 8–10% After 48 h
of air exposure, losses were as follows: 80% in WhiB1, 75% in WhiB2 and WhiB5, 65% in WhiB3, 60% in WhiB4, and 35–40% in WhiB6 and WhiB7 (Fig 3A) It was evident that the iron–sulfur
Trang 4300 350 400 450 500 550 600 650 700
0.000
0.015
0.030
0.045
0.060
0.075
0.090
WhiB1, 50 µ M WhiB1, 50 µ M , alkylated
WhiB2, 50 µ M WhiB2, 50 µ M , alkylated
WhiB3, 50 µ M
WhiB4, 50 µ M , alkylated
WhiB5, 50 µ M
M WhiB6, 50 µ M , alkylated
WhiB7, 50 µ M WhiB7, 50 µ M , alkylated
λ (nm)
300 350 400 450 500 550 600 650 700
0.000
0.015
0.030
0.045
0.060
0.075
0.090
300 350 400 450 500 550 600 650 700
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
300 350 400 450 500 550 600 650 700 0.00
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08
300 350 400 450 500 550 600 650 700 0.00
0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18
300 350 400 450 500 550 600 650 700 0.00
0.05 0.10 0.15 0.20 0.25 0.30 0.35
300 350 400 450 500 550 600 650 700 0.00
0.03 0.06 0.09 0.12 0.15 0.18
Fig 1 UV–Vis absorption spectra of WhiB proteins The absorption spectra of purified proteins (50 l M, thick line) show the presence of a [2Fe–2S] cluster in WhiB proteins Numbers (in nm) indicate the peak at the specified wavelength Alkylation was carried out by incubating the purified proteins (50 l M) with 20 m M IAA for 1 h at 25 C in the dark and the spectra were recorded (thin line) after the baseline correc-tion The spectra for WhiB3 and WhiB4 are taken from Alam & Agrawal [14] and Alam et al [15], respectievely.
Trang 5clusters were most stable against air oxidation in
WhiB6 and WhiB7, and most labile in WhiB1
To study the sensitivity towards oxidized glutathione
(GSSG), reduced glutathione (GSH) and dithiothreitol,
proteins were incubated with 10 mm of each agent and
the absorbance at 424 nm (A424) was recorded at
dif-ferent time intervals up to 42 h All WhiB proteins
showed differential sensitivity towards oxidation by
GSSG, and similar to air oxidation, the iron–sulfur
clusters of WhiB6 and WhiB7 were comparatively
more stable (Fig 3B) A reducing environment (in the
presence of GSH or dithiothreitol) significantly
low-ered the rate of disintegration of the iron–sulfur cluster
in each of the WhiB proteins (Fig 3C,D) Therefore,
disassembly of the iron–sulfur cluster under oxidizing
conditions and its stability under reducing conditions
suggested that the iron–sulfur clusters of M
tuberculo-sisWhiB proteins are redox sensitive We assume that
the iron–sulfur clusters of different WhiB proteins
would respond differently to the oxidative stress
encountered by M tuberculosis in vivo
Iron–sulfur clusters of WhiB proteins are
differentially exposed to the external environment
The differential sensitivity of the iron–sulfur cluster
towards different oxidizing agents could be attributed
to their relative surface accessibility We hypothesized that the iron–sulfur cluster of WhiB6 and WhiB7 may
be sequestered in the interior of the holo protein, thereby shielding it from oxidative degradation In
Table 1 Total iron content in WhiB proteins Proteins under
differ-ent conditions were purified as described in Experimdiffer-ental
proce-dures Data for each protein sample are expressed as means ± SD
(three independent protein preparations).
Samples
Atoms of iron per monomer
+ + – + + +
WhiB
+ – + + + +
WhiB1 WhiB2 WhiB3 WhiB4 WhiB5 WhiB6 WhiB7
Auto radiogram
Ponceau S stained
300 350 400 450 500 550 600 0.0
0.1 0.2 0.3 0.4 0.5
IscS (2 µ M ) WhiB (30 µ M )
WhiB (30 µ M ) + IscS (2 µ M )
λ (nm)
A
B
Fig 2 In vitro assembly of the iron–sulfur cluster in WhiB proteins (A) The upper panel is an autoradiogram showing IscS-dependent incorporation of radioactive sulfur in the iron–sulfur cluster of all seven WhiB proteins The lower panel is a representative ponceaue S-stained blot showing the status of protein spotting Reconstitu-tion was carried out in a 500-lL reacReconstitu-tion volume as described in Experimental procedures The concentration of FeCl3was either a 10-fold molar excess (10·) or 10-fold lower (0.1·) than that of WhiB proteins After the reaction, unwanted and free components were dialyzed The poly(vinylidene difluoride) membrane was first treated with methanol for 5 s and washed thoroughly The membrane was equilibrated with a buffer containing 50 m M Tris ⁄ HCl, pH 9.0,
150 m M NaCl and 10 m M dithiothreitol for 5 min In total, 30 lL (10 lL at one time) of the indicated samples were spotted, air dried and developed using Phosphorimager (Bio-Rad, Hercules, CA, USA) (B) Absorption spectra of a representative in vitro reconsti-tuted WhiB protein All seven WhiB proteins showed similar features.
Trang 6order to study the surface accessibility of the iron–
sulfur cluster of WhiB proteins, freshly purified proteins
were incubated with 20 mm EDTA and the degree of
iron chelation was monitored by recording the A424 at
different intervals up to 20 h Immediate chelation of
iron was not observed in any WhiB protein However,
as time increased, the degree of iron chelation
increased and the extent of chelation varied (Table 2)
Almost 30% of the iron was chelated within 2 h in
WhiB1 and WhiB2, whereas in WhiB6 and WhiB7 it
was negligible over the same period Even after 20 h of
incubation, iron chelation was 15–20% (minimum)
in WhiB6 and WhiB7, whereas it was 60% (maxi-mum) in WhiB1 and WhiB2; in the other proteins it varied between 20% and 60% (Table 2) From the data, it appears that the surface accessibility of the iron–sulfur cluster is significantly different in different WhiB proteins
Cysteine residues form two intramolecular disulfide bonds after removal of the iron–sulfur cluster
It has been shown in WhiB4 that the cysteine-thiols, which are ligands of the iron–sulfur cluster, undergo oxidation and form two intramolecular disulfide bonds after disassembly of the iron–sulfur cluster [15] The presence of two intramolecular disulfide bonds has also been demonstrated in apo WhiB1 [13] and apo WhiB3 [14] Proteins containing intramolecular disulfide bond(s) often show retarded mobility on SDS⁄ PAGE under reducing conditions [15,23] Both apo WhiB2 and apo WhiB5 showed significant retarded mobility
on SDS⁄ PAGE under reducing conditions, indicating the presence of intramolecular disulfide bond(s) (Fig 4) Alkylation of cysteine by iodoacetamide (IAA) coupled with MS was used to determine the sta-tus of cysteines after removal of the iron–sulfur cluster Reaction of IAA with a cysteine-thiol causes an increase in molecular mass of 57 Da, therefore, the
0
15
30
45
60
75
90
105
120
Effect of air 0 h
6 h
12 h
24 h
48 h
WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2
WhiB1
A42
0 15 0 30 3 45 0 60 6 75 0 90 9 105 0
2 h
6 h
20 h
30 h
42 h
WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2 WhiB1
A420
0
15
0
30
3
45
0
60
6
75
0
90
9
105
0
1 0 2
Effect of GSH (10 m M ) 0 h
2 h
6 h
20 h
30 h
42 h
WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2
WhiB1
15 0
0 30 3 45 0 60 6 75 0 90 9 105 0
1 0 2
Effect of dithiothreitol (10 m M ) 0 h
2 h
6 h
20 h
30 h
42 h
WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2 WhiB1
Fig 3 Bar diagram showing the kinetics of [2Fe–2S] cluster loss upon treatment with various oxidizing and reducing agents Effect of (A) air, (B) GSSG, (C) GSH and (D) dithiothreitol Freshly purified protein (50 l M) was incubated with 10 m M of the indicated agents at 25 C The loss of the [2Fe–2S] cluster was measured by recording A420at different time intervals The reading at t = 0 was set to 100% and the change in A 424 (residual) is expressed relative to the first reading A suitable baseline correction was made before recording each spectrum Results are an average of three independent protein preparations Values for WhiB4 were taken from Alam et al [15].
Table 2 Stability of the iron–sulfur cluster from various WhiB
proteins against EDTA The initial reading was set to 100% and
the change in A420(residual) is expressed relative to the reading
at t = 0 Data are expressed as means ± SD (three independent
protein preparations).
Proteins
Change in A420(%)
Trang 7total increase in the mass after reduction of the
disul-fide bond reflects the total number of cysteines present
in the thiol and disulfide forms In the oxidized state,
both WhiB2 and WhiB5 showed a major peak
corre-sponding to the theoretical molecular mass of the
recombinant protein However, the reduced proteins
had increased molecular masses, representing
alkyl-ation of four cysteine residues in each case (Fig 5)
Although, WhiB5 and WhiB6 did not show any
mobil-ity differences under reducing conditions, a similar
increase in mass was found after reduction (Fig 5)
The difference in mass between the oxidized and
reduced forms suggested the presence of four
cysteine-thiols in the reduced apo WhiB proteins Because none
of the cysteines was present in a thiol form in the
oxi-dized protein (except for one in WhiB6 which has five
cysteines), it was concluded that the apo form of all
WhiB proteins contained two intramolecular disulfide
bonds
All apo WhiB proteins, except WhiB2, reduce the insulin disulfide
Previously, we reported that apo WhiB1 [13] WhiB3 [14] and WhiB4 [15] are protein disulfide reductases The enzymatic activity of WhiB4 was shown to be gov-erned by the CXXC motif [15] Because the CXXC motif is present in all WhiB family members of
M tuberculosis, except WhiB5⁄ Rv0022c (CXXXC), we tested the protein disulfide reductase activity of WhiB2, WhiB5, WhiB6 and WhiB7 by insulin disulfide reduc-tion assay This is a standard assay to asses the disulfide reductase activity of any protein in which reduction of the insulin disulfide by dithiothreitol in the presence of
a test protein is monitored [24] Reductase activity was calculated by dividing the maximal slope of the curve (DA650Æmin)1) by the onset time of precipitation (time when A650reached 0.05) [25] Except WhiB2, all WhiB proteins catalyzed the reduction of insulin disulfide (Table 3, Fig S5) However, for WhiB2, the possibility
of the presence of a natural in vivo substrate protein(s) still remains Because M tuberculosis RshA also has
a C86XXC89 motif, and purified recombinant RshA (lab preparation) was therefore used as a control, but it did not catalyze insulin reduction
Formation of a reversible intramolecular disulfide bond between the cysteines of the CXXC motif is essential for protein disulfide reductase activity [16,26] Therefore, we assume that in WhiB proteins, one disul-fide bond is formed between the two cysteines of the CXXC motif (CXXXC in the case of WhiB5) and the other between the remaining two cysteines The assumption is supported by our earlier data, in which
a similar arrangement of intramolecular disulfide bonds in WhiB4 was established [15] In WhiB6, one
of the intramolecular disulfide bonds appeared to have formed between Cys53 and Cys56 but the involvement
of cysteines for the second bond is little hard to pre-dict, as it contains five cysteines (Cys12, Cys34, Cys53, Cys56 and Cys62)
Divergence in the secondary structure composition of WhiB proteins of M tuberculosis The multiple sequence alignment of WhiB proteins of
M tuberculosis showed 49–66% sequence homology and 31–50% identity with respect to each other (Table S1) However, because of the variation in amino acid composition, it is possible that structural variations may be an important determinant of their functional properties in vivo Therefore, the structural organization
of each M tuberculosis WhiB protein was studied using biophysical tools The secondary structure of each
WhiB5 26.9
20.0
36.5
WhiB6 18.4
14.4
25.0
WhiB7 18.4
14.4
25.0
26.9
20.0
kDa
Fig 4 Mobility shift of apo WhiB2, WhiB5, WhiB6 and WhiB7 on
15% SDS ⁄ PAGE Oxidized protein (5 lg) was incubated in the
absence or presence of different reducing agents, as indicated, for
1 h at 25 C in 50 m M Tris ⁄ HCl, pH 8.0, 200 m M NaCl After
reduc-tion, free thiols were alkylated with 20 m M IAA for 1 h at 25 C in
the dark Finally the samples were resolved by 15% SDS ⁄ PAGE
and proteins were visualized by Coomassie Brilliant Blue staining.
Trang 8WhiB protein was analyzed by CD spectroscopy The far-UV CD spectra of WhiB proteins were dissimilar because their molar ellipticities varied significantly (Fig 6) The spectra showed a-helix, b-strand and random coil features However, the proportion of each feature varied among WhiB proteins, as evident from the difference in negative molar ellipticity at specific wavelengths, i.e 208 and 222 nm (a helix signature),
218 nm (b strand signature), 202–204 nm (random coil signature) In WhiB5 and WhiB6, the structure was dominated by a helices and b strands and the propor-tion of these structural elements was higher in WhiB6 WhiB1, WhiB2 and WhiB4 showed relatively increased molar ellipticity at 202–204 nm, indicating the presence
of a significant proportion of random coils (Fig 6) The
Fig 5 MALDI-TOF spectroscopic analysis of oxidized and reduced form of apo WhiB proteins ‘Oxd’ represents the ‘oxidized and alkylated’ protein, whereas ‘Red’ represents ‘reduced and alkylated’ protein.
Table 3 Protein disulfide reductase activity of apo WhiB proteins.
The data for each protein sample (3 l M ) are expressed as
means ± SD (three independent protein preparations).
Samples
Reductase activity (· 10)3DA650nmÆmin)2)
a Taken from Alam & Agrawal [14] b Taken from Alam et al [15].
Trang 9–14 000 –12 000 –10 000 –8000 –6000 –4000 –2000 0
2000
WhiB2, Oxd WhiB2, Red
–6000
–5000
–4000
–3000
–2000
–1000
0
1000
2000
WhiB5, Oxd WhiB5, Red
λ (nm)
λ (nm)
λ (nm)
λ (nm)
λ (nm)
λ (nm)
–6000 –5000 –4000 –3000 –2000 –1000 0 1000 2000 3000
4000
WhiB6, Oxd WhiB6, Red
–5000
–4000
–3000
–2000
–1000
0
1000
WhiB3, Oxd WhiB3, Red
–10 000 –8000 –6000 –4000 –2000
WhiB4, Red
–14 000
–12 000
–10 000
–8000
–6000
–4000
–2000
0
2000
2 ·
–1 )
[θMRW
2 ·
2 ·
2 · –1 )
λ (nm)
–4000 –3500 –3000 –2500 –2000 –1500 –1000 –500 0
WhiB7, Red
[θMRW
2 ·
[θMRW
2 ·
2 ·
–1 )
WhiB1, Oxd WhiB1, Red
Fig 6 Secondary structure analyses of apo WhiB proteins Far-UV
CD spectra of apo proteins (0.2 mgÆmL)1) were recorded at 25 C.
Reduction was carried out by incubating the protein in buffer C
supplemented with 1 m M dithiothreitol for 1 h at 25 C The far-UV
CD spectrum of WhiB1 (0.2 mgÆmL)1) was recorded as described
in Garg et al [13], whereas the data of WhiB3 and WhiB4 were
taken from Alam & Agrawal [14] and Alam et al [15] respectively.
Trang 10proportion of all three secondary structural elements in
WhiB3 and WhiB7 appeared similar Disulfide bond
formation did not affect the secondary structure, except
for WhiB5 and WhiB6, and the effect was more
pronounced in WhiB6 (Fig 6)
It was observed that the secondary structure of
WhiB1 [13], WhiB3 [14] and WhiB4 [15] resists thermal
denaturation Thus, we asked would other WhiB
pro-teins also show similar features? Surprisingly, the
sec-ondary structures of WhiB5 and WhiB6 started to
melt at 50 and 77C respectively (Fig 7) At 70 C,
WhiB5 lost almost all its secondary structure, whereas
in WhiB6 some secondary structure was maintained
even at 95C Neither WhiB2 nor WhiB7 showed
thermal denaturation Together, the data suggest that considerable structural differences exist among the WhiB proteins of M tuberculosis, with WhiB5 and WhiB6 appearing to be the most structurally divergent family members
CD spectroscopy is considered more sensitive for the recognition of a helices and less reliable for b strands, thus the data obtained from CD spectroscopy are an approximate assessment To estimate the level of b-structures in different WhiB proteins, a thioflavin T (ThT)-binding assay was performed ThT shows strong fluorescence in the presence of crossed b-sheet struc-tures [27,28] The binding of ThT to each apo WhiB protein was measured by fluorescence spectroscopy
–8000
–6000
–4000
–2000
0
Temp (°C)
2 ·dmol
WhiB1, Oxd WhiB1, Red
–2400
–1600
–800
0
WhiB3, Oxd WhiB3, Red
2 ·dmol
Temp (°C)
–2000 –1600 –1200 –800 –400
0 WhiB7, Oxd WhiB7, Red
2 ·dmol –1 )
Temp (°C)
–6000 –5000 –4000 –3000 –2000 –1000 0
WhiB4, Oxd WhiB4, Red
2 ·dmol –1 )
Temp (°C)
–4000
–3000
–2000
–1000
0
WhiB5, Oxd WhiB5, Red
2 ·dmol
–1 )
Temp (°C)
–5000 –4000 –3000 –2000 –1000
0
WhiB6, Oxd WhiB6, Red
2 ·dmol –1 )
Temp (°C)
WhiB2, Oxd WhiB2, Red
–6000 –5000 –4000 –3000 –2000 –1000 0
2 ·dmol –1 )
Temp (°C)
Fig 7 Thermal denaturation kinetics of oxidized and reduceed apo WhiB proteins Far-UV CD spectra of each protein showed pronounced ellipticity at 222 nm therefore, the thermal stability of the secondary structure was tested by recording the change in ellipticity at 222 nm with increasing temperature from 25 to 90 C The thermal denaturation kinetics of WhiB1 (0.2 mgÆmL)1) was studied as described in Garg
et al [13], whereas the data of WhiB3 and WhiB4 were taken from Alam & Agrawal [14] and Alam et al [15] respectively.