Mycobacterium tuberculosis H37Rv ESAT-6–CFP-10 complex formation confers thermodynamic and biochemical stability Akshaya K. Meher 1 , Naresh Chandra Bal 1 , Kandala V. R. Chary 2 and Ashish Arora 1 1 Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India 2 Department of Chemical Science, Tata Institute of Fundamental Research, Mumbai, India Comparative genomic studies based on whole genome DNA microarray have led to the identification of 16 regions of deletion (RDs) in Mycobacterium bovis BCG, which is currently used as a vaccine, with respect to Mycobacterium tuberculosis and five RDs with respect to M. bovis. RD1 is absent from all strains of BCG and Mycobacterium microti, whereas it is present in all virulent strains of M. tuberculosis and M. bovis [1]. The RD1 region in M. tuberculosis is 9455 bp long, and encompasses nine ORFs (Rv3871–Rv3879c). Keywords association constant; ESAT-6–CFP-10 complex; limited proteolysis; lipid–protein interactions; thermal unfolding Correspondence A. Arora, Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India Fax: +91 522 223405 Tel: +91 522 261 2411 18 ext. 4329 E-mail: ashishcdri@yahoo.com (Received 4 January 2006, revised 30 January 2006, accepted 6 February 2006) doi:10.1111/j.1742-4658.2006.05166.x The 6-kDa early secretory antigenic target (ESAT-6) and culture filtrate protein-10 (CFP-10), expressed from the region of deletion-1 (RD1) of Mycobacterium tuberculosis H37Rv, are known to play a key role in viru- lence. In this study, we explored the thermodynamic and biochemical chan- ges associated with the formation of the 1 : 1 heterodimeric complex between ESAT-6 and CFP-10. Using isothermal titration calorimetry (ITC), we precisely determined the association constant and free energy change for formation of the complex to be 2 · 10 7 m )1 and )9.95 kcalÆ mol )1 , respectively. Strikingly, the thermal unfolding of the ESAT-6–CFP- 10 heterodimeric complex was completely reversible, with a T m of 53.4 °C and DH of 69 kcalÆmol )1 . Mixing of ESAT-6 and CFP-10 at any tempera- ture below the T m of the complex led to induction of helical conformation, suggesting molecular recognition between specific segments of unfolded ESAT-6 and CFP-10. Enhanced biochemical stability of the complex was indicated by protection of ESAT-6 and an N-terminal fragment of CFP-10 from proteolysis with trypsin. However, the flexible C-terminal of CFP-10 in the complex, which has been shown to be responsible for binding to macrophages and monocytes, was cleaved by trypsin. In the presence of phospholipid membranes, ESAT-6, but not CFP-10 and the complex, showed an increase in a-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced ther- modynamic and biochemical stability, and loss of binding to phospholipid membranes. These features of complex formation probably determine the physiological role of ESAT-6, CFP-10 and ⁄ or the complex in vivo. The ITC and thermal unfolding approach described in this study can readily be applied to characterization of the 11 other pairs of ESAT-6 family pro- teins and for screening ESAT-6 and CFP-10 mutants. Abbreviations ANS, 8-anilinonapthalene-1-sulfonate; CFP-10, 10-kDa culture filtrate protein; DPC, dodecylphosphocholine; DSC, differential scanning calorimetry; ESAT-6, 6-kDa early secretory antigenic target; ESAT-6–CFP-10 complex, 1 : 1 complex of ESAT-6 and CFP-10; HSQC, heteronuclear single quantum correlation; ITC, isothermal titration calorimetry; Myr 2 PtdCho, dimyristoyl-DL-a-phosphatidylcholine; Ni ⁄ NTA, nickel ⁄ nitrilotriacetic acid; RD1, region of deletion 1; trCFP-10, truncated 10-kDa culture filtrate protein. FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS 1445 Rv3874 or esxB and Rv3875 or esxA encode the proteins CFP-10 (10-kDa culture filtrate protein) and ESAT-6 (6-kDa early secretory antigenic target), respectively, which play a key role in virulence [2]. Both ESAT-6 and CFP-10 generate a specific Th-1 host immune response and have a strong diagnostic potential for both the virulent form and latent form of M. tuberculosis [3]. Several studies have shown that RD1 and its flanking regions comprising ORFs Rv3864–Rv3870 and Rv3880c–Rv3883c code for a spe- cialized secretion system Esx-1, which is responsible for secretion of ESAT-6 and CFP-10 [4,5]. Recently it has been shown that the secretion of ESAT-6 and CFP-10 is also dependent on Esx-1-associated protein EspA [6]. The genes encoding ESAT-6 and CFP-10 are organ- ized as an operon and are cotranscribed [7]. On the basis of tryptophan fluorescence, CD and 1D 1 H-NMR spectra, Renshaw et al. [8] have shown that ESAT-6 is a molten globule whereas CFP-10 is unstructured in the native form. Together, ESAT-6 and CFP-10 form a tight 1 : 1 complex. Recently, the NMR solution structure of the ESAT-6 and CFP-10 complex has been determined by Renshaw et al. [9] (PDB ID, 1WA8]. In the complex, both the proteins adopt helix–turn–helix hairpin conformation and are orientated antiparallel to each other. The contact sur- face between ESAT-6 and CFP-10 is primarily hydro- phobic, and van der Waals interactions between ESAT-6 and CFP-10 run all along the length of the helices of both proteins. The surface features of the complex, however, do not indicate its involvement with any specific function; rather DNA binding, enzyme activity and pore formation in lipid membranes can be excluded on the basis of the structure. Fluorescence microscopy studies have shown that the flexible C-ter- minal of CFP-10 in the complex is responsible for spe- cific binding to macrophages and monocytes, on the basis of which a role in receptor-mediated signaling has been attributed to the complex [9]. Whether CFP- 10 alone can bind to macrophages and monocytes in a specific manner was, however, not explored. The ESAT-6 family contains proteins consisting of nearly 100 residues. M. tuberculosis H37Rv has 22 members of this family, all of which are in tandem pairs arranged in clusters [10]. The ESAT-6 family of protein pairs expressed from Rv0287 and Rv0288 as well as Rv3019c and Rv3020c are secreted proteins and form 1 : 1 heterodimeric complexes. Moreover these protein pairs, because of their close sequence similarity, may also form nongenome Rv0287– Rv3020c and Rv0288–Rv3019c complexes. The ESAT- 6 and CFP-10 interaction is quite specific, and these proteins do not form nongenome complexes with either Rv0287 ⁄ Rv0288 or Rv3019c ⁄ Rv3020c pairs. Mutational analysis of ESAT-6 has been carried out recently to identify the key residues involved in com- plex formation with CFP-10, secretion, T-cell response and virulence of M. tuberculosis H37Rv [11]. Several residues essential for complex formation have been identified. Mutation of these key residues results in disruption of complex formation and attenuation of virulence. The results of mutational analysis have been explained in terms of a coiled-coil model for the ESAT-6–CFP-10 complex, with heptad repeats ‘abc- defg’ harboring positions at sites ‘a’ and ‘d’ for hydro- phobic residues. Hsu et al. [12] have demonstrated that either the deletion of RD1 or disruption of the Rv3874-Rv3875 (cfp-10-esat-6) operon of RD1 results in loss of cyto- toxicity towards both pneumocytes and macrophages. The behavior of these mutants is similar to that of BCG and in contrast with the well-established cytotox- icity of M. tuberculosis H37Rv to macrophages. Along similar lines, Guinn et al. [13] have reported that H37Rv RD1 mutants with disruption of either of the genes Rv3870, Rv3871, Rv3874 (cfp-10), Rv3875 (esat- 6) or Rv3876 grew minimally and produced no cell lysis in human macrophage-like THP-1 cell lines. In the studies of both Hsu et al. and Guinn et al. it was found that the H37Rv RD1 mutants grew inside the host cells but were unable to cause cytolysis. It was further demonstrated by Hsu et al. that ESAT-6, either alone or in combination with CFP-10, but not CFP-10 alone, could cause disruption and eventual lysis of black lipid membranes prepared from diphytanoyl- phosphatidylcholine. On the basis of this, Hsu et al. [12] proposed that ESAT-6 may mediate lethal ion fluxes through plasma membranes of the host, leading to cytolysis. In proteomic studies, ESAT-6 has been found in the cell membrane fraction of M. tuberculosis H37Rv [14]. However, Guinn et al. reported that addi- tion of purified ESAT-6, either alone or in combina- tion with CFP-10, did not show any toxic effect on THP-1 cells. Therefore, the nature of the interaction of ESAT-6, CFP-10 or the complex with phospholipid membranes is currently not very clear. A detailed characterization of biochemical and ther- modynamic changes associated with complex forma- tion is necessary to fully understand the biological role of ESAT-6, CFP-10 and the complex. In addition, the nature of the interaction of ESAT-6, CFP-10 and the complex with phospholipid membranes needs to be understood clearly. The results of our detailed bio- physical studies show that, compared with ESAT-6 or CFP-10, the complex has enhanced thermodynamic Stability of ESAT-6–CFP-10 complex A. K. Meher et al. 1446 FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS and biochemical stability. ESAT-6, but not CFP-10 or the complex, undergoes conformational change on binding to the phospholipid membranes. We also stud- ied complex formation with CFP-10 and interaction with phospholipid membranes for four mutants of ESAT-6. We suggest biophysical characterization of complex formation as a general approach that can be used for all 11 pairs of ESAT-6 family proteins in M. tuberculosis H37Rv, and furthermore for screening the entire set of ESAT-6 and CFP-10 mutants. Results Thermodynamic parameters governing ESAT-6 and CFP-10 complex formation Isothermal titration calorimetry (ITC) experiments were carried out to accurately measure the association constant for ESAT-6 and CFP-10 complex formation. The raw ITC data, generated by titration of 1.3 mL 0.42 mm ESAT-6 during the 50 injections of 4 lL 0.042 mm CFP-10 are shown in Fig. 1A, and the integ- rated areas under each peak versus molar ratio of ESAT-6 to CFP-10 are plotted in Fig. 1B. The binding isotherm of ESAT-6 with CFP-10 is characterized by strong heat release, which is indicated by a slope approaching infinity. The heat released decreases as ESAT-6 becomes saturated. In the last 23 injections of the titration, only heat of dilution is observed. The binding isotherm in Fig. 1B was fitted to a single-site binding model for determination of thermodynamic parameters. The solid line indicates best fit to the plot. The parameters used in fitting were the stoichiometry of association ( n), the binding constant (K B ) and the change in enthalpy (DH B ). The values of these parame- ters obtained from the nonlinear least-squares fit to the binding curve are: n ¼ 1.0, DH B ¼ )40.3 kcalÆ mol )1 , and K B ¼ 2 · 10 7 m )1 . The ITC binding iso- therm can be characterized by a unitless value c [15], which is given by c ¼ K B [M]n, where [M] is the con- centration of the macromolecule ESAT-6. For an accu- rate determination of the binding constant, a ‘c’ value between 1 and 1000 is recommended. In the case of ESAT-6 and CFP-10, the value of ‘c’ is 840, which is indicative of a tightly bound complex. The free energy change (DG) associated with complex formation is given by: DG ¼ –RTlnK B , where R is the gas constant and T is the temperature in Kelvin. At 25 °C, DG for complex formation is )9.95 kcalÆmol )1 . The entropy change associated with complex formation is deter- mined from the equation: DG ¼ DH ) TDS.At25°C, DS is )101 calÆmol )1 ÆK )1 . Both the entropy change and enthalpy change associated with complex forma- tion are characteristically high. However, typical enthalpy–entropy compensation results in a moderate value of DG of )9.95 kcalÆmol )1 . The free energy change for complex formation between ESAT-6 and CFP-10 is comparable to the DG associated with simi- larly sized protein–protein interactions, e.g. DG of )9.6 ± 0.5 kcalÆmol )1 was observed for interaction between turkey ovomucoid third domain with a-chy- motrypsin and DG of )11.3 ± 0.7 kcalÆ mol )1 was observed for interaction between T-cell factor 4 and b-catenin [16,17]. Thermal unfolding of the ESAT-6–CFP-10 complex is completely reversible Differential scanning calorimetry (DSC) studies were carried out to assess the thermal stability of the ESAT-6–CFP-10 complex and to accurately measure the enthalpy and heat capacity changes involved in the unfolding. A DSC thermogram of the thermal unfold- ing of the complex at a concentration of 0.105 mm in phosphate buffer and a scan rate of 60 °CÆh )1 , from 20 to 80 °C is shown by the solid line curve in Fig. 2. After the first heating scan, the sample was cooled from 80 to 20 °C and then a second heating scan was A B Fig. 1. Typical calorimetric isothermal titration measurements of the interaction of CFP-10 with ESAT-6 in phosphate buffer at 25 °C. (A) Raw data of heat effect (in lcalÆs )1 )of654-lL injections of 0.42 m M CFP-10 into 1.3 mL 0.042 mM ESAT-6 performed at 4-s intervals. (B) The data points (d) were obtained by integration of heat signals plotted against the molar ratio of ESAT-6 to CFP-10 in the reaction cell. The solid line represents a calculated curve using the best-fit parameters obtained by a nonlinear least squares fit. The heat of dilution was subtracted from the raw data of titra- tion of CFP-10 with ESAT-6. A. K. Meher et al. Stability of ESAT-6–CFP-10 complex FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS 1447 recorded, which is shown by the dotted line curve in Fig. 2. The peak shaped thermograms indicate co-op- erativity during unfolding [18]. The thermal unfolding transition is characterized by an enthalpy change (DH) of 69 kcalÆmol )1 , T m of 53.4 °C, and T 1 ⁄ 2 of 9.01 °C. However, no change in heat capacity (DC p ) was observed for the thermal unfolding transition. DSC scans recorded at scan rates of 20, 40, 60 and 90 °CÆh )1 showed only a small shift in the T m from 54 to 53.4 °C and a small decrease in transition enthalpy from 74 to 69 kcalÆmol )1 . As the first and second heat- ing scans completely overlap at every scan rate, it strikingly indicates that the thermal unfolding of the complex is completely reversible. The secondary and tertiary structural changes asso- ciated with thermal unfolding of the complex were followed by steady-state CD and 2D 15 N- 1 H heteronu- clear single quantum correlation (HSQC) NMR experi- ments, respectively. Far-UV CD spectra of CFP-10, ESAT-6 and ESAT-6–CFP-10 complex were similar to those reported previously by Renshaw et al. [8]. As CFP-10 is almost completely unstructured, the thermal unfolding and refolding experiments were performed only for ESAT-6 and the complex. Steady-state CD scans were recorded on a sample first at increasin g temperatures in the range 25–75 °C and then in decreasing order from 75 to 25 °C, at 5 °C intervals. The thermal unfolding and refolding profiles of ESAT-6 and the complex are shown in Fig. 3A. The midpoints of thermal unfolding transitions (T m )of Fig. 2. Thermal reversibility of 1 : 1 ESAT-6–CFP-10 complex monit- ored by DSC. DSC thermogram of 0.51 mL 0.105 m M ESAT-6–CFP- 10 from 20 °Cto80°C, at a scan rate of 60 °C per h. The raw data were baseline-corrected for buffer. The plots show excess heat capacity as a function of temperature in °C. The complex was hea- ted to 80 °C for the first thermogram shown by the solid line. The sample was then cooled down to 20 °C. The second thermo- gram recorded by reheating the same sample is shown by a dashed line. Fig. 3. Thermal reversibility of ESAT-6 and the 1 : 1 ESAT-6–CFP- 10 complex monitored by CD. (A) Normalized transition curves for temperature-induced transition of ESAT-6 and the complex monit- ored in the far-UV CD region at 222 nm. Thermal unfolding (h) and thermal refolding (s) profile of ESAT-6 and thermal unfolding (n) and thermal refolding (e) profile of the complex were plotted as fraction of protein folded versus temperature in °C. (B) Far-UV CD spectrum of ESAT-6 (h) was recorded in phosphate buffer, pH 6.5 at 25 °C. The sample was heated to 70 °C and cooled down to 25 °C, and the far-UV CD spectrum was recorded again (s). (C) CD spectrum of the 1 : 1 complex at 25 °C was recorded before therm- al unfolding (h) and after thermal refolding (s) as described for ESAT-6. Stability of ESAT-6–CFP-10 complex A. K. Meher et al. 1448 FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS ESAT-6 and the complex are at 33 °C and 53 °C, respectively. For the complex, the T m determined from CD (53 °C) matches well with that determined by DSC (53.4 °C). CD spectra recorded before and after unfolding, at 25 °C, for ESAT-6 and the complex are shown in Fig. 3B,C, respectively. Similar to the unfold- ing and refolding profiles mentioned above, entire CD spectra before and after unfolding overlapped at every temperature, suggesting that the molecular steps lead- ing to thermal unfolding are retraced on refolding for both ESAT-6 and the complex. The 2D 15 N- 1 H-HSQC spectrum serves as a finger- print of the overall structure of a protein. The HSQC spectrum recorded with 15 N-labeled CFP-10 at 30 °C is shown in Fig. 4A. The spectrum is characterized by sharp but narrowly dispersed peaks along the 1 H N dimension (within 7–8.5 p.p.m), which is consistent with CFP-10 being unstructured in its native form. The 2D 15 N- 1 H-HSQC spectrum of 15 N-labeled ESAT- 6 is shown in the Fig. 4B. The broad peaks and peak dispersion pattern in the HSQC spectrum are consis- tent with the previously reported molten globular state of free ESAT-6. The HSQC spectrum of the complex formed between 15 N-labeled CFP-10 and unlabeled ESAT-6 is shown in Fig. 4D, and that of the complex formed between 15 N-labeled ESAT-6 with unlabeled CFP-10 is shown in the Fig. 4E. Figure 4C shows the 2D 15 N- 1 H-HSQC spectrum of the complex in which both the proteins are 13 C, 15 N-labeled. The sum of the HSQC spectra of individually labeled proteins in com- plex, i.e. the sum of spectra in Fig. 4D,E, is shown in the Fig. 4F. The spectrum in Fig. 4F overlaps very well with the spectrum of the complex shown in Fig. 4C. To find any change in tertiary structure of the AB C DE F Fig. 4. Conformational change observed individually in ESAT-6 and CFP-10 on complex formation. (A) and (D) show 15 N- 1 H-HSQC spectra of 15 N-labeled CFP-10 in the free state and in complex with unlabeled ESAT-6, respectively. (B) and (E) show 15 N- 1 H-HSQC spectra of 15 N-label- ed ESAT-6 in the free state and in complex with unlabeled ESAT-6, respectively. (C) 15 N- 1 H-HSQC spectrum of 1 : 1 [ 13 C, 15 N]ESAT-6– [ 13 C, 15 N]CFP-10 complex. (F) Spectrum produced by addition of the spectra in (D) and (E). All spectra were recorded in NMR buffer (see Experimental procedures) containing 5% (v ⁄ v) D 2 Oat30°C on a 600-MHz NMR spectrometer. A. K. Meher et al. Stability of ESAT-6–CFP-10 complex FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS 1449 complex during the unfolding and refolding process, 15 N- 1 H-HSQC spectra on 1 mm complex in phosphate buffer were first recorded at 30, 40, 50, 55, 60 and 65 °C, in increasing order (Fig. 5A,C,E,G,I,K, respect- ively), after which HSQC spectra on the same sample were recorded at 60, 55, 50, 40 and 30 °C (Fig. 5J,H,F,D,B, respectively), in decreasing order. The tertiary structure is retained up until 60 °C. Strik- ingly, the peaks in the HSQC spectrum at any partic- ular temperature before and after unfolding almost completely overlap, and are representative of the HSQC spectrum of the complex, but not the HSQC spectra of the individual proteins ESAT-6 and CFP- 10. This indicates that the tertiary structure of the complex is also completely regained after thermal unfolding. Molecular recognition between ESAT-6 and CFP-10 exists even when the two proteins are in unstructured form As the secondary structure of ESAT-6 is highly dependent on the temperature, we investigated whether any residual secondary structure of ESAT-6 is neces- sary for complex formation with CFP-10. CD scans were recorded for samples in which ESAT-6 and CFP- 10 were mixed at 25, 30, 35, 40, 45, 50 and 55 °C, and compared with CD scans of the complex formed between the two proteins at 25 °C and heated to equivalent temperatures. Fig. 6 shows thermograms generated by plotting mean residue ellipticity at 222 nm as a function of temperature for ESAT-6, CFP-10, the 1 : 1 complex of ESAT-6–CFP-10, and equimolar CFP-10 and ESAT-6 mixed at different temperatures. As can be seen, there was an increase in helical content equivalent to that of the complex when ESAT-6 and CFP-10 were mixed together at tempera- tures up to 55 °C, indicating formation of helices locally by interactions between specific segments of CFP-10 and ESAT-6. These results indicate that the secondary structure of ESAT-6 is not necessary for the AB CD EF GH I K J Fig. 5. Thermal reversibility of 1 : 1 ESAT-6–CFP-10 complex monit- ored by NMR spectroscopy. 1 m M [ 15 N]ESAT-6–[ 15 N]CFP-10 com- plex in NMR buffer, pH 6.5, with 5% (v ⁄ v) D 2 O was used to monitor thermal reversibility of the complex. 15 N- 1 H-HSQC spectra were recorded on a 500-MHz NMR spectrometer at 30 °C(A), 40 °C(C),50°C(E),55°C(G),60°C (I) and 65 °C (K), in increasing order, after which 15 N- 1 H-HSQC spectra on the same sample were recorded at 60 (J), 55 (H), 50 (F), 40 (D) and 30 °C (B), in decreas- ing order. Fig. 6. Temperature dependence of the interaction of ESAT-6 and CFP-10. Isothermal CD spectra were recorded at 5 °C temperature interval from 25 to 55 °C. A plot is shown of mean residue elliptici- ty values at 222 nm as a function of temperature, recorded for ESAT-6 (h), CFP-10 (e), and 1 : 1 ESAT-6–CFP-10 complex formed by mixing equimolar proteins at 25 °C(n), and equimolar ESAT-6 and CFP-10 mixed together at 25, 30, 35, 40, 45, 50 and 55 °C(d). Stability of ESAT-6–CFP-10 complex A. K. Meher et al. 1450 FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS complex formation, and specific molecular recognition between the interacting segments of ESAT-6 and CFP-10 exists even when the two proteins are in unstructured form. CFP-10 reduces its susceptibility to trypsin digestion on forming a complex with ESAT-6 To investigate the biochemical stability of the proteins, limited proteolysis with trypsin was performed at 4 °C, for ESAT-6, CFP-10 and the 1 : 1 ESAT-6–CFP-10 complex, and the digested products thus obtained were analyzed by SDS ⁄ PAGE (15% gel). The Coomassie- stained SDS ⁄ polyacrylamide gels are shown in Fig. 7A. On trypsinolysis, CFP-10 showed multiple bands on SDS ⁄ PAGE after 1 min of digestion at 4 °C, and was completely digested to oligopeptides in 20 min. ESAT-6 was stable for 60 min at 4 °C. Fur- ther degradation of ESAT-6 yielded two bands corres- ponding to molecular masses of 14 kDa and 3 kDa. The 14-kDa band may be an aggregate of trypsin- degraded products of ESAT-6. In contrast with ESAT- 6 and CFP-10, the complex displayed a characteristic pattern on trypsinolysis. On treatment of the complex with trypsin at 4 °C, one additional band appeared after 1 min incubation. The largest and smallest of these bands corresponded to CFP-10 and ESAT-6, respectively. A third band labeled trCFP-10 (for trun- cated CFP-10), in between CFP-10 and ESAT-6, with molecular mass % 2 kDa lower than CFP-10 was observed, which apparently results from truncation of CFP-10 by cleavage at a particular site by trypsin. On continued incubation, the intensity of the band corres- ponding to CFP-10 decreased, whereas that of trCFP- 10 increased with time, and no change in the intensity of the band corresponding to ESAT-6 was observed. After 2 h of trypsin treatment, the band corresponding to intact CFP-10 had disappeared completely, whereas the bands corresponding to trCFP-10 and ESAT-6 were still present. An essentially similar pattern of bands was observed for the complex after 3 h of tryp- sinolysis except that a weak band with an apparent mass of 6 kDa was observed, which resulted from fur- ther degradation of trCFP-10. Both ESAT-6 and CFP- 10 have C-terminal hexa-histidine tags. Western blots with antibody to histidine are shown in Fig. 7B. trCFP-10 was not detected, indicating that it results from cleavage of the C-terminus of CFP-10. Overall, these results indicate that complex formation leads to interdependent protection of an N-terminal fragment of CFP-10 and ESAT-6 from trypsinolysis. ESAT-6 possesses solvent-exposed hydrophobic clusters To assess the solvent-exposed hydrophobic surface of the proteins, we studied the change in fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) on A B Fig. 7. Limited proteolysis with trypsin of ESAT-6, CFP-10 and 1 : 1 ESAT-6–CFP-10 complex. (A) SDS ⁄ PAGE of aliquots removed at differ- ent time points for reaction of 40 l M ESAT-6, or CFP-10, or 1 : 1 ESAT-6–CFP-10 complex with 1 lg trypsin at 4 °C. Lanes 1, 4, 7, 10, 13, 16, and 19, CFP-10; lanes 2, 5, 8, 11, 14, 17, and 20, ESAT-6; lanes 3, 6, 9, 12, 15, 18, and 21 ESAT-6–CFP-10 correspond to aliquots withdrawn after 0, 1, 5, 20, 60, 120 and 180 min of trypsinolysis. LMW is low-molecular-mass protein marker. (B) Western blot developed with antibody to histidine. The lanes of the blot correspond to the lanes of SDS ⁄ PAGE, except for LMW. A. K. Meher et al. Stability of ESAT-6–CFP-10 complex FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS 1451 binding to ESAT-6, CFP-10 and ESAT-6–CFP-10. Figure 8 shows extrinsic fluorescence spectra of ANS in the presence of ESAT-6, CFP-10 and the complex, at 25 °C. The fluorescence intensities have been nor- malized with respect to the maximum fluorescence intensity of ANS bound to ESAT-6. As expected from its molten globule state, ESAT-6 showed high ANS binding. No change in fluorescence intensity of ANS was observed in the presence of CFP-10, indicating that ANS did not bind to CFP-10, as expected from the unstructured form of CFP-10. A decrease of 65 ± 5% in ANS fluorescence intensity was obtained on ESAT-6–CFP-10 complex formation. Myr 2 PtdCho vesicles stabilize the secondary structure of ESAT-6 above its melting temperature To investigate the binding of ESAT-6, CFP-10 and the complex to lipid membranes, 6 lm protein samples were incubated with dimyristoyl-dl-a-phosphatidylcho- line (Myr 2 PtdCho) vesicles in phosphate buffer, and the change in conformation was monitored by CD spectroscopy. CD spectra of CFP-10, ESAT-6–CFP-10 and ESAT-6 in the absence and presence of Myr 2 Ptd- Cho vesicles are shown in Fig. 9A,B,C. At 25 °C, the Fig. 9. Far-UV CD spectra of ESAT-6, CFP-10 and the 1 : 1 ESAT-6–CFP-10 complex in the presence of Myr 2 PtdCho vesicles. CD spectra of 6 l M CFP-10, ESAT-6–CFP-10 and ESAT-6 without Myr 2 PtdCho vesicles in phosphate buffer, pH 6.5, at 25 °C(h) and 37 °C(s) and with Myr 2 PtdCho vesicles in phosphate buffer, pH 6.5, at 25 °C(n)and37°C(,) are shown. The spectra obtained at 25 °C after cooling the pro- tein samples containing Myr 2 PtdCho vesicles from 37 °C, are shown with symbols (e). Fig. 8. Binding of ANS to ESAT-6, CFP-10 and the 1 : 1 ESAT-6– CFP-10 complex. The fluorescence emission spectra of 100 l M ANS in the presence of 10 lM ESAT-6 (s), CFP-10 (h) and ESAT- 6–CFP-10 complex (m) in phosphate buffer, pH 6.5, at 25 °C. Stability of ESAT-6–CFP-10 complex A. K. Meher et al. 1452 FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS CD spectra of CFP-10 and the complex did not show any significant change, whereas ESAT-6 showed a minor increase in helicity (from 49% to 52%) in the presence of Myr 2 PtdCho vesicles. When the tempera- ture of the sample was increased to 37 °C, CFP-10 and the complex still showed no change. However, ESAT-6 retained an a-helical content of 32% in contrast with 19% in the absence of Myr 2 PtdCho vesicles at 37 °C. On cooling the same ESAT-6 ⁄ Myr 2 PtdCho vesicle sample from 37 °Cto25°C, the a-helical content increased further to 63%, which is significantly higher than the helicity obtained on mixing ESAT-6 and Myr 2 PtdCho vesicles at 25 °C. Interaction of ESAT-6 mutants with CFP-10 and phospholipid membranes We have used a novel approach to select residues for mutations from the 26 residues of ESAT-6 that are at the interface between ESAT-6 and CFP-10 in the com- plex, as reported by Renshaw et al. [9]. Our approach was based on detection of NOEs from the backbone amide protons of ESAT-6 to the side chain protons of CFP-10. Residues of ESAT-6, the amide protons of which showed strongest NOEs with the side chain protons of CFP-10 in the labeled complex, were selected for mutation. For detecting NOEs, we prepared the complex from 13 C, 15 N-labeled CFP-10 and 2 H, 13 C, 15 N- labeled ESAT-6. A set of 3D triple-resonance experi- ments HNCO, HNCA, and HN(CA)CB were recorded to validate our sample. Strips from HNCA and HN(CA)CB spectra demonstrating the sequential assignments of residues Leu39 to Trp43 are shown in Fig. 10A,B, respectively. These assignments are similar to those reported by Renshaw et al. [9]. An 15 N-edited NOESY-HSQC spectrum was recorded for the complex for detecting the NOEs. NOEs from backbone amide protons of ESAT-6 and side chain protons of CFP-10 A B C Fig. 10. Sequential assignments and inter- protein NOEs for a segment of ESAT-6 interacting with CFP-10. (A) and (B) Strips showing the sequential assignments from 3D HNCA and HN(CA)CB spectra, respect- ively, recorded from 1 m M 1 : 1 complex of 2 H, 13 C, 15 N-labeled ESAT-6 and 13 C, 15 N-labe- led CFP-10 in NMR buffer with 5% (v ⁄ v) D 2 Oat30°C on a 600-MHz NMR spectro- meter. The strips are taken at the indicated 15 N chemical shifts that were assigned to residues 39–43 of ESAT-6. They are cen- tered about the corresponding amide proton chemical shifts. The top of the sequence- specific assignments is indicated by one- letter amino-acid code and by sequence number. The one directional arrows in these figures indicate a sequential walk through 2D 13 C a - 1 H N and 13 C b - 1 H N planes taken in the position of the corresponding 1 H N , 15 N, 13 C a and 1 H N , 15 N, 13 C b resonances in 3D HNCA and HN(CA)CB spectra, respectively. (C) Strips from 1 H, 15 N-NOESY- HSQC spectrum recorded with s mix of 150 ms. In these strips, NOEs are shown between downfield amide protons and upfield aliphatic protons. The amide protons correspond to the sequentially assigned seg- ment 39–43 of ESAT-6. The backbone amide protons of this segment show NOEs with protons at 0.808 p.p.m. from a side chain of CFP-10. A. K. Meher et al. Stability of ESAT-6–CFP-10 complex FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS 1453 were observed for the segments Ala14-Ala15-Ser16 (1.187 p.p.m.), Ala17-Ile18 (1.200 p.p.m.), Ser24-Ile25 (0.934 p.p.m.), Leu28-Leu29-Asp30 (0.897 p.p.m.), Glu31-Gly32-Lys33-Gln34-Ser35-Leu36 (0.745 p.p.m.), Leu39-Ala40-Ala41-Ala42-Trp43 (0.808 p.p.m.), and Glu64-Leu65-Asn66 (1.415 p.p.m.). Values in paren- theses are the chemical shift of the side chain protons of CFP-10 with which backbone amide proton of ESAT-6 show the NOE. Figure 10C shows the NOE between the amide protons for the segment Leu39 to Trp43 from ESAT-6 to the side chain proton of CFP-10. Strongest NOEs were observed for the residues Leu29, Gly32, Ala41 and Leu65. On the basis of this, four point mutants L29D, G32D, A41D and L65D of ESAT-6 were generated. We studied complex formation between ESAT-6 mutants and CFP-10 by CFP-10 pull-down assays and CD spectroscopy. In parallel, we also studied the interaction of ESAT-6 mutants with Myr 2 PtdCho membranes by CD spectroscopy. SDS ⁄ PAGE of the CFP-10 pull-down assay is shown in Fig. 11A. Two prominent low-molecular- mass bands corresponding to untagged CFP-10 and A B Fig. 11. Study of complex formation between ESAT-6 mutants and CFP-10. (A) A SDS ⁄ 15% polyacrylamide gel showing results of CFP-10 pull-down assay. LMW, low-molecular-mass protein marker. The rest of the lanes show purified ESAT-6 or ESAT-6 mutants and Ni ⁄ NTA eluate (see Experimental procedures). (B) Far-UV CD spectra of CFP-10 (h), ESAT-6 mutants (n) and 1 : 1 mixture of ESAT-6 mutant and CFP-10 (s) recorded in phosphate buffer, pH 6.5, at 25 °C. Stability of ESAT-6–CFP-10 complex A. K. Meher et al. 1454 FEBS Journal 273 (2006) 1445–1462 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... events starting from the expression of ESAT-6 and CFP-10 to their functional activity Overall, complex formation results in structural changes, enhanced thermodynamic and biochemical stability, and loss of binding to phospholipid membranes These features of complex formation are likely to determine the physiological role of CFP-10, ESAT-6 and ⁄ or the complex in vivo Our study provides the essential... lysis, and consequently in M tuberculosis virulence Secretion of ESAT-6 and CFP-10 is dependent on an intact Esx-1 system and Esx-1 associated protein EspA and is essential for both virulence and specific T-cell response Recently, Brodin et al [11] have shown that M tuberculosis H37Rv mutants with mutations of ESAT-6 that prohibit complex formation with CFP-10, for example L28A ⁄ L29S, W43R, and G45T,... of complex formation between members of the Mycobacterium tuberculosis complex CFP-10 ⁄ ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility FEMS Microbiol Lett 238, 255–262 Brodin P, de Jonge MI, Majlessi L, Leclerc C, Nilges M, Cole ST & Brosch R (2005) Functional analysis of ESAT-6, the dominant T-cell antigen of Mycobacterium tuberculosis, ... 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6-CFP-10 complex Implications for pathogenesis and virulence J Biol Chem 277, 21598–21603 Renshaw PS, Lightbody KL, Veverka V, Muskett FW, Kelly G, Frenkiel TA, Gordon SV, Hewinson RG, Burke B, Norman J, et al (2005) Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and. .. Rasmussen PB, Rosenkrands I, Andersen P & Gicquel B (1998) A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecularmass culture filtrate protein (CFP-10) Microbiology 144, 3195–3203 Renshaw PS, Panagiotidou P, Whelan A, Gordon SV, Hewinson RG, Williamson RA & Carr MD (2002) Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form... position ‘a’ and ‘d’ of the four helices (the N-terminal and C-terminal helices of ESAT-6 and CFP-10) are hydrophobic and form the interface between the two proteins, residues at position ‘e’ and ‘g’ are generally polar and are responsible for specificity of interactions between the neighboring helices, whereas residues at positions ‘b’, ‘c’ and ‘f’ are at the outer surface of the helix and cannot possibly... reversible thermal unfolding of a complex formed between a molten globule and an inherently unstructured protein CD and NMR experiments show that the molecular steps involved in unfolding of the complex were retraced on refolding Further, a mixing experiment shows that complex formation between ESAT-6 and CFP-10 can take place at any temperature below the Tm of the complex This strongly reflects the... Williams A, Griffiths KE, Marchal G, Leclerc C & Cole Stability of ESAT-6–CFP-10 complex 3 4 5 6 7 8 9 10 11 12 ST (2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis Nat Med 9, 533–539 van Pinxteren LA, Ravn P, Agger EM, Pollock J & Andersen P (2000) Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10 Clin Diagn Lab Immunol 7, 155–160 Stanley... 37 °C Therefore, complex formation probably provides higher stability to ESAT-6 and CFP-10 towards intracellular proteases It is also very interesting to note that the flexible C-terminus of CFP-10 in the ESAT-6–CFP-10 complex, which has recently been shown to be responsible for specific binding to the surface of monocytes and macrophages [9], is quite susceptible to trypsin A similar stability profile... ESAT-6 and CFP-10 need to be determined more accurately Renshaw et al [8] have previously estimated the dissociation constant (Kd) of the ESAT-6–CFP-10 complex to be 1.1 · 10)8 m or lower, based on intrinsic tryptophan fluorescence studies We used ITC to accurately determine the association constant KB (KB ¼ 1 ⁄ Kd) and also the thermodynamic parameters DH, DS, and DG associated with complex formation . Mycobacterium tuberculosis H37Rv ESAT-6–CFP-10 complex formation confers thermodynamic and biochemical stability Akshaya K. Meher 1 , Naresh Chandra Bal 1 , Kandala V. R. Chary 2 and Ashish. increase in a-helical content and enhanced thermal stability. Overall, complex formation resulted in structural changes, enhanced ther- modynamic and biochemical stability, and loss of binding to phospholipid membranes (e), and 1 : 1 ESAT-6–CFP-10 complex formed by mixing equimolar proteins at 25 °C(n), and equimolar ESAT-6 and CFP-10 mixed together at 25, 30, 35, 40, 45, 50 and 55 °C(d). Stability of ESAT-6–CFP-10