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Studies on structural and functional divergence among seven 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. 76 FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS the 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-X 5-7 -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 O 2 - 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 37 °C for 3 h led to the formation of light brown inclu- sion bodies. However, induction at 16 °C for 20 h resulted in the expression of  10–20% of each WhiB protein in the soluble form. On SDS ⁄ PAGE, the mass of Ni 2+ -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 Md. S. Alam et al. Molecular properties of M. tuberculosis WhiB proteins FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 77 range 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 FeCl 3 and Na 2 S, 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 FeCl 3 , IscS and 35 S-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 Molecular properties of M. tuberculosis WhiB proteins Md. S. Alam et al. 78 FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 300 350 400 450 500 550 600 650 700 0.000 0.015 0.030 0.045 0.060 0.075 0.090 ~560–580 nm 420 nm 340 nm WhiB1, 50 µM WhiB1, 50 µM, alkylated WhiB2, 50 µM WhiB2, 50 µM, alkylated WhiB3, 50 µM WhiB3, 50 µM, alkylated WhiB4, 50 µM WhiB4, 50 µM, alkylated WhiB5, 50 µM WhiB5, 50 µM, alkylated WhiB6, 50 µM WhiB6, 50 µM, alkylated WhiB7, 50 µM WhiB7, 50 µM, alkylated Absorbance λ (nm) λ (nm) λ (nm) λ (nm) λ (nm) λ (nm) 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 ~560–580 nm 424 nm 340 nm Absorbance 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 ~560–580 nm 422 nm Absorbance 337 nm 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 ~560– 580 nm 420 nm 337 nm Absorbance 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 ~560–580 nm 424 nm 333 nm Absorbance 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 Absorbance ~560–580 nm ~460 nm 420 nm 340 nm 300 350 400 450 500 550 600 650 700 0.00 0.03 0.06 0.09 0.12 0.15 0.18 ~560–580 nm ~460 nm 424 nm 340 nm Absorbance Fig. 1. UV–Vis absorption spectra of WhiB proteins. The absorption spectra of purified proteins (50 lM, 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 mM 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. Md. S. Alam et al. Molecular properties of M. tuberculosis WhiB proteins FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 79 clusters 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 (A 424 ) 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- sis WhiB 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 Experimental proce- dures. Data for each protein sample are expressed as means ± SD (three independent protein preparations). Samples Atoms of iron per monomer WhiB1 (native) 0.131 ± 0.014 WhiB1 (in 8 M urea) 0.141 ± 0.052 WhiB1 (refolded) 0.138 ± 0.028 WhiB1 (alkylated) 0.008 ± 0.005 WhiB2 (native) 0.145 ± 0.022 WhiB2 (in 8 M urea) 0.142 ± 0.020 WhiB2 (refolded) 0.142 ± 0.035 WhiB2 (alkylated) 0.006 ± 0.005 WhiB5 (native) 0.185 ± 0.028 WhiB5 (in 8 M urea) 0.186 ± 0.036 WhiB5 (refolded) 0.188 ± 0.045 WhiB5 (alkylated) 0.010 ± 0.007 WhiB6 (native) 0.212 ± 0.065 WhiB6 (in 8 M urea) 0.198 ± 0.050 WhiB6 (refolded) 0.208 ± 0.072 WhiB6 (alkylated) 0.007 ± 0.003 WhiB7 (native) 0.182 ± 0.035 WhiB7 (in 8 M urea) 0.189 ± 0.066 WhiB7 (refolded) 0.175 ± 0.020 WhiB7 (alkylated) 0.007 ± 0.005 + + – + + + 10X 10X 10X 10X 10X 0.1X WhiB 35 S-Cys FeCl 3 IscS – + + + + + + – + + + + 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 µM) WhiB (30 µ M) WhiB (30 µM) + IscS (2 µM) ~420 nm Absorbance 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 reaction volume as described in Experimental procedures. The concentration of FeCl 3 was 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 mM 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. Molecular properties of M. tuberculosis WhiB proteins Md. S. Alam et al. 80 FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS order 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 A 424 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 7575 90 105 120 Effect of air 0 h 6 h 12 h 24 h 48 h WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2 WhiB1 % Change in A 420 0 15 0303 45 0606 7575 0909 105 021021 Effect of GSSG (10 mM) 0 h 2 h 6 h 20 h 30 h 42 h WhiB7 WhiB6 WhiB5 WhiB4 WhiB3WhiB2 WhiB1 % Change in A 420 0 15 0303 45 0606 75 75 0909 105 021021 Effect of GSH (10 mM) 0 h 2 h 6 h 20 h 30 h 42 h WhiB7 WhiB6WhiB5 WhiB4 WhiB3 WhiB2 WhiB1 % Change in A 420 15 0 0303 45 0606 7575 0909 105 021021 Effect of dithiothreitol (10 mM) 0 h 2 h 6 h 20 h 30 h 42 h WhiB7 WhiB6 WhiB5 WhiB4 WhiB3 WhiB2 WhiB1 % Change in A 420 AB CD 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 mM of the indicated agents at 25 °C. The loss of the [2Fe–2S] cluster was measured by recording A 420 at 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 A 420 (residual) is expressed relative to the reading at t = 0. Data are expressed as means ± SD (three independent protein preparations). Proteins Change in A 420 (%) 0h 2h 6h 20h WhiB1 100 62 ± 4 53 ± 4 44 ± 3 WhiB2 100 72 ± 5 44 ± 3 38 ± 6 WhiB3 100 82 ± 3 70 ± 5 58 ± 3 WhiB4 100 92 ± 2 59 ± 5 49 ± 6 WhiB5 100 80 ± 2 65 ± 3 58 ± 3 WhiB6 100 96 ± 3 89 ± 2 84 ± 2 WhiB7 100 98 ± 2 96 ± 4 80 ± 4 Md. S. Alam et al. Molecular properties of M. tuberculosis WhiB proteins FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 81 total 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 (DA 650 Æmin )1 ) by the onset time of precipitation (time when A 650 reached 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 aC 86 XXC 89 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 Marker Oxidized 1 m M dithiothreitol 2% β -ME 26.9 20.0 36.5 WhiB2 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 °Cin50m M Tris ⁄ HCl, pH 8.0, 200 mM NaCl. After reduc- tion, free thiols were alkylated with 20 m M IAA for 1 h at 25 °Cin the dark. Finally the samples were resolved by 15% SDS ⁄ PAGE and proteins were visualized by Coomassie Brilliant Blue staining. Molecular properties of M. tuberculosis WhiB proteins Md. S. Alam et al. 82 FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS WhiB 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 )3 DA 650 nmÆmin )2 ) WhiB1 4.78 ± 0.25 WhiB2 0.56 ± 0.16 WhiB3 a 4.19 ± 0.62 WhiB4 b 42.2 ± 0.86 WhiB5 10.96 ± 0.55 WhiB6 2.79 ± 0.22 WhiB7 5.44 ± 0.82 RshA 0.58 ± 0.10 Buffer control 0.62 ± 0.15 a Taken from Alam & Agrawal [14]. b Taken from Alam et al. [15]. Md. S. Alam et al. Molecular properties of M. tuberculosis WhiB proteins FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 83 –14 000 –12 000 –10 000 –8000 –6000 –4000 –2000 0 2000 WhiB2, Oxd WhiB2, Red 190 200 210 220 230 240 250 –6000 –5000 –4000 –3000 –2000 –1000 0 1000 2000 WhiB5, Oxd WhiB5, Red λ (nm) 190 200 210 220 230 240 250 λ (nm) 190 200 210 220 230 240 250 λ (nm) 190 200 210 220 230 240 250 λ (nm) 190 200 210 220 230 240 250 λ (nm) λ (nm) 190 200 210180 220 230 240 250 –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 0 WhiB4, Oxd WhiB4, Red –14 000 –12 000 –10 000 –8000 –6000 –4000 –2000 0 2000 [ θ] MRW (deg·cm 2 · dmol –1 ) [ θ] MRW (deg·cm 2 · dmol –1 ) [ θ] MRW (deg·cm 2 · dmol –1 ) [ θ] MRW (deg·cm 2 · dmol –1 ) 190 200 210 220 230 240 250 λ (nm) –4000 –3500 –3000 –2500 –2000 –1500 –1000 –500 0 500 WhiB7, Oxd WhiB7, Red [θ] MRW (deg·cm 2 · dmol –1 ) [ θ] MRW (deg·cm 2 · dmol –1 ) [ θ] MRW (deg·cm 2 · dmol –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. Molecular properties of M. tuberculosis WhiB proteins Md. S. Alam et al. 84 FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS proportion 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 77 °C respectively (Fig. 7). At 70 °C, WhiB5 lost almost all its secondary structure, whereas in WhiB6 some secondary structure was maintained even at 95 °C. 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. 20 30 40 50 60 70 80 90 100 –8000 –6000 –4000 –2000 0 Temp (°C) 222 (deg·cm 2 ·dmol –1 ) WhiB1, Oxd WhiB1, Red –2400 –1600 –800 0 WhiB3, Oxd WhiB3, Red 222 (deg·cm 2 ·dmol –1 ) 20 30 40 50 60 70 80 90 100 Temp (°C) –2000 –1600 –1200 –800 –400 0 WhiB7, Oxd WhiB7, Red 222 (deg·cm 2 ·dmol –1 ) 20 30 40 50 60 70 80 90 100 Temp (°C) –6000 –5000 –4000 –3000 –2000 –1000 0 WhiB4, Oxd WhiB4, Red 222 (deg·cm 2 ·dmol –1 ) 20 30 40 50 60 70 80 90 100 Temp (°C) –4000 –3000 –2000 –1000 0 WhiB5, Oxd WhiB5, Red 222 (deg·cm 2 ·dmol –1 ) 20 30 40 50 60 70 80 90 100 Temp (°C) –5000 –4000 –3000 –2000 –1000 0 WhiB6, Oxd WhiB6, Red 222 (deg·cm 2 ·dmol –1 ) 30 40 50 60 70 80 90 100 Temp (°C) WhiB2, Oxd WhiB2, Red –6000 –5000 –4000 –3000 –2000 –1000 0 222 (deg·cm 2 ·dmol –1 ) 20 30 40 50 60 70 80 90 100 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. Md. S. Alam et al. Molecular properties of M. tuberculosis WhiB proteins FEBS Journal 276 (2009) 76–93 ª 2008 The Authors Journal compilation ª 2008 FEBS 85 [...]... Fig S1 Purification of WhiB proteins Fig S2 Brownish color of WhiB proteins purified under different conditions Fig S3 UV–Vis spectra of WhiB proteins purified under denaturing or in-column refolding conditions Fig S4 Effect of air oxidation on the stability of [2Fe–2S] cluster of various WhiB proteins Fig S5 Insulin disulfide reduction assay of apoWhiB5, WhiB6 and WhiB7 Fig S6 Comparison of the amino acid... recombinant WhiB3 [9] and WhiB4 [15] of M tuberculosis, purified under normal conditions, coordinate a [2Fe–2S] cluster However, based upon the in vitro reconstitution assay, both were found to be [4Fe–4S] cluster coordinating proteins Iron–sulfur clusters are one of the most ancient and versatile cofactors of several important class of proteins and have been implicated in variety of functions [29,30] Iron and. .. fluorescence after reduction of the disulfide bonds, whereas WhiB1 , WhiB2 , WhiB5 and WhiB7 showed a low but reproducible increase in ANS fluorescence after reduction of the disulfide bonds, indicating a minor conformational change followed by the exposure of certain buried hydrophobic patches CD and fluorescence data clearly suggested structural variations among seven WhiB proteins of M tuberculosis Furthermore,... polyclonal antibodies raised against WhiB1 did not cross-react with WhiB4 and vice versa (data not shown) This may be because of variations in the antigenic epitopes of WhiB1 and WhiB4 , which is likely to be due to conformational differences between the two proteins The relative cross-reactivity of polyclonal antibodies was used to probe the conformational variations at the tertiary level among WhiB proteins. .. efficient network of specific gene products which assures survival and multiplication under unfavorable nutritional, pH and redox conditions [1] Among several mechanisms for resistance to intracellular killing, the scavenging of free radicals and the reactivation of degenerated proteins during infection by a family of proteins named ‘thioredoxins’ is one of the elegant mechanisms of defense and self-sustenance... the conformational difference among various WhiB proteins is the possible cause of this heterogeneity Because WhiB proteins share significant sequence homology minor cross-reactivity may be due to the presence of antibodies which are reacting to the linear epitopes or to conserved conformational epitopes The data showed that there are significant structural differences among WhiB proteins of M tuberculosis. .. Molecular properties of M tuberculosis WhiB proteins Reactivity (%) Md S Alam et al 87 Molecular properties of M tuberculosis WhiB proteins Md S Alam et al ever, in vivo, the possibility of the presence of [4Fe–4S] in WhiB proteins remains very high Protein ligands for the canonical clusters are typically sulfide ions of cysteines The involvement of four conserved cysteines in the coordination of the [4Fe–4S]... by fluorimetry The ANS fluorescence of various WhiB proteins was significantly different (Table 4) The fluorescence intensity of WhiB3 was maximal and threefold higher than that of WhiB6 (minimum) Based on ANS fluorescence, the surface hydrophobicity of various WhiB proteins decreases in the following order: WhiB3 > WhiB1 > WhiB4 > WhiB2 > WhiB5 > WhiB7 > WhiB6 (Table 4) WhiB4 showed a significant increase... for spectral studies and activity assays The molar extinction coefficient of various WhiB proteins was determined using vector-nti software The values were Molecular properties of M tuberculosis WhiB proteins as follows: 17 550 m)1Æcm)1 for WhiB1 , 13 140 m)1Æcm)1 for WhiB2 , 18 830 m)1Æcm)1 for WhiB3 and WhiB4 , 16 980 m)1Æcm)1 for WhiB5 , 27 200 m)1Æcm)1 for WhiB6 and 17 550 m)1Æcm)1 for WhiB7 Throughout,... major contributors in WhiB5 and WhiB6 , but the proportion is relatively low and similar in other WhiB proteins It should be noted that WhiB5 aligned only with WhiB3 and WhiB4 , whereas the WhiB6 sequence aligned only with WhiB4 (NCBI; http://www.ncbi.nlm.nih.gov/ blast/bl2seq/wblast2.cgi) (Table S1), suggesting that at the amino acid sequence level both WhiB5 and WhiB6 differ from the others, and the . Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37Rv Md. Suhail Alam, Saurabh K. Garg* and. 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 ,