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J. Biol. Chem., Vol. 277, Issue 24, 21598-21603, June 14, 2002

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Conclusive Evidence That the Major T-cell Antigens of the Mycobacterium tuberculosis Complex ESAT-6 and CFP-10 Form a Tight, 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^*

Philip S. Renshaw, Parthena Panagiotidou^§, Adam Whelan^¶, Stephen V. Gordon^¶, R. Glyn Hewinson^¶, Richard A. Williamson^§, and Mark D. Carr

From the  Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH, ^§ Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, and ^¶ TB Research Group, Department of Bacterial Diseases, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom

Received for publication, February 18, 2002, and in revised form, April 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The proteins ESAT-6 and CFP-10 have been shown to be secreted by Mycobacterium tuberculosis and Mycobacterium bovis cells, to be potent T-cell antigens, and to have a clear but as yet undefined role in tuberculosis pathogenesis. We have successfully overexpressed both ESAT-6 and CFP-10 in Escherichia coli and developed efficient purification schemes. Under in vivo-like conditions, a combination of fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy have shown that ESAT-6 contains up to 75% helical secondary structure, but little if any stable tertiary structure, and exists in a molten globule-like state. In contrast, CFP-10 was found to form an unstructured, random coil polypeptide. An exciting discovery was that ESAT-6 and CFP-10 form a tight, 1:1 complex, in which both proteins adopt a fully folded structure, with about two-thirds of the backbone in a regular helical conformation. This clearly suggests that ESAT-6 and CFP-10 are active as the complex and raises the interesting question of whether other ESAT-6/CFP-10 family proteins (22 paired genes in M. tuberculosis) also form tight, 1:1 complexes, and if so, is this limited to their genome partner, or is there scope for wider interactions within the protein family, which could provide greater functional flexibility?

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tuberculosis is one of the oldest infectious diseases known to mankind (1, 2) and remains one of the most significant bacterial diseases of humans, with about one-third of the world's population infected resulting in ~3 million deaths annually (3-6). The bacteria responsible for tuberculosis belong to the Mycobacterium tuberculosis complex, which is a group of highly related mycobacteria. The complex includes M. tuberculosis, which is responsible for the majority of human tuberculosis, and Mycobacterium bovis, which causes tuberculosis in a range of domesticated and wild animals. The complete sequence of the M. tuberculosis genome was reported about 3 years ago (7) and is believed to contain genes for 3,959 proteins (8, 9). However, we still have relatively little information about which proteins are essential for pathogenesis and even less knowledge of their structures, functions, and mechanisms of action.

The only currently effective vaccine for tuberculosis is a live attenuated strain of M. bovis known as Bacille Calmette-Guérin (BCG);¹ however, despite being one of the most widely used vaccines in the world the molecular basis for the attenuation of M. bovis BCG remains unclear. Recently, genomic hybridization techniques have identified a number of deletions in the genomes of BCG daughter strains; however, only one of these, termed RD1, is deleted consistently from BCG strains but present in all virulent isolates of M. bovis and M. tuberculosis (10, 11). The RD1 deletion contains the genes for nine proteins (Rv3871-Rv3879c), which are clearly implicated in pathogenesis. The genes Rv3874 and Rv3875 code for two sequence-related (25% homology) proteins known as CFP-10 (100 residues) and ESAT-6 (95 residues), respectively. Expression of these two genes has been shown to be coordinately regulated, and both ESAT-6 and CFP-10 are found at low levels in M. tuberculosis and M. bovis culture supernatants (12). In addition, the two proteins are potent T-cell antigens recognized by over 70% of tuberculosis patients (13), which has led to their proposed use as diagnostic reagents for tuberculosis in both humans and animals (14, 15). Preliminary data from a dual knockout of ESAT-6 and CFP-10 in M. bovis appears to indicate that the loss of at least one of these genes results in a significant reduction in the virulence of the engineered M. bovis, which further emphasizes the potential importance of both proteins in tuberculosis pathogenesis and virulence (16). Neither CFP-10 nor ESAT-6 shows any significant sequence similarity with any proteins of known tertiary structure or function. However, they are both members of a large family of mycobacterial proteins found in the M. tuberculosis complex (12, 17), which, in common with CFP-10 and ESAT-6, are found in pairs within the genome, often preceded by members of the PE and PPE gene family (7, 12, 17).

We have produced successfully both CFP-10 and ESAT-6 in Escherichia coli, and in this communication we report the results of detailed structural characterization of both proteins using a combination of fluorescence, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression Vectors-- A pET21a-based expression vector containing the full coding sequence for ESAT-6 was generated by a PCR-based approach and maintained in E. coli BL21(DE3) cells. The ESAT-6 coding sequence was amplified from M. bovis AN5 DNA using appropriate primers and cloned into the NdeI and SalI sites of pET21a. The CFP-10 and CFP-10/ESAT-6 expression vectors were produced similarly by a PCR-based approach, with an artificial bacterial chromosome (Rv414; see Ref. 18) containing the full coding regions for both CFP-10 and ESAT-6 used as a template. The PCR primers used to amplify the CFP-10 coding region were designed to include NcoI and BamH1 restriction sites to allow insertion into pET28a. The larger PCR product containing the coding region for both CFP-10 and ESAT-6 included an NdeI site in place of the NcoI site and was designed to allow the expression of an N-terminal, His₆-tagged variant of CFP-10 following ligation into pET28a. After construction, the integrity of both expression vectors was confirmed by DNA sequencing.

Expression and Purification of ESAT-6-- E. coli BL21(DE3) transformed with the pET21a-based expression vector for ESAT-6 were grown in LB medium containing 100 µg/ml ampicillin. The expression of ESAT-6 was induced in mid-log phase (corresponding to an absorbance at 600 nm of 0.6-0.7) by the addition of isopropyl-1-thio--D-galactopyranoside to 0.45 mM, and the cells were then harvested after 4 h by centrifugation at 7800 × g for 15 min (4 °C). The cell pellets obtained were resuspended and lysed with Bugbuster HT (Novagen) to which was added 0.5 mM EDTA and 100 µM phenylmethylsulfonyl fluoride to inhibit protease activity. The insoluble fraction of the cell lysate, containing the ESAT-6 as inclusion bodies, was recovered by centrifugation (12100 × g for 15 min at 4 °C), and the inclusion bodies were then washed three times in a 50 mM Tris, 10 mM EDTA, and 0.5% (v/v) Triton X-100 buffer adjusted to pH 8.0. After the final wash, the ESAT-6 inclusion bodies were solubilized in 6 M guanidine hydrochloride containing 1 mM EDTA and 100 µM phenylmethylsulfonyl fluoride to give a final ESAT-6 concentration of 0.5 to 1 mg/ml. This solution was dialyzed initially against a 25 mM NaH₂PO₄, 100 mM NaCl, and 1 mM EDTA refolding buffer at pH 6.5 and then into the Q-Sepharose column running buffer consisting of 20 mM Bis-Tris and 1 mM EDTA at pH 6.5. The final purification of ESAT-6 was carried out using a 20-ml Q-Sepharose column to which was applied a stepwise gradient of increasing NaCl concentration. The ESAT-6 was eluted at 150 mM NaCl and was judged to be greater than 95% pure by SDS-PAGE (Invitrogen 4-12% Bis-Tris NuPAGE gel system) and electrospray mass spectrometry.

Expression and Purification of CFP-10-- E. coli cells transformed with the pET28a-based expression vector for CFP-10 were grown in LB medium containing 40 µg/ml kanamycin and were harvested 4 h after induction by isopropyl-1-thio--D-galactopyranoside in mid-log phase. The cell pellets were lysed with Bugbuster HT, as described previously, and the soluble fraction containing CFP-10 was then dialyzed into a 20 mM Tris and 1 mM EDTA column running buffer at pH 8.0. Initial purification of CFP-10 was carried out on a 20-ml Q-Sepharose column pre-equilibrated with the pH 8.0 Tris buffer. The column was washed with a stepwise gradient of increasing NaCl concentration and CFP-10 eluted in the 50 to 75 mM NaCl washes. Fractions containing CFP-10 were pooled, dialyzed against a 20 mM piperazine and 1 mM EDTA buffer at pH 5.8, and then applied to a 20-ml Q-Sepharose column pre-equilibrated with the same piperazine buffer. CFP-10 was eluted from this column in a 50 mM NaCl wash and was judged to be over 95% pure.

Expression and Purification of His-tagged CFP-10-- E. coli cells transformed with the expression vector for hexa-His-tagged CFP-10 were grown in LB medium containing 40 µg/ml kanamycin and were harvested 4 h after induction with isopropyl-1-thio--D-galactopyranoside. The cells were lysed with Bugbuster HT, and the soluble fraction containing His-tagged CFP-10 was then dialyzed into a 20 mM Tris, 100 mM NaCl, and 1 mM EDTA buffer at pH 8.0. The protein was purified by using a 10-ml Ni-NTA column pre-equilibrated with the pH 8.0 buffer, which was washed with a stepwise gradient of increasing imidazole concentration. The His-tagged CFP-10 was eluted in the 50 mM imidazole wash and was found to be at least 95% pure. It should be noted that if His-tagged CFP-10 was prepared from cells grown in medium containing only 20 µg/ml kanamycin then the purified protein was found to contain roughly equal amounts of both His-tagged CFP-10 and ESAT-6.

Circular Dichroism Spectroscopy-- The far UV CD spectra used to determine the secondary structure of ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex were acquired on a Jasco 715 spectrometer. The spectra were collected from protein samples dissolved in a 25 mM NaH₂PO₄ and 100 mM NaCl buffer at pH 6.5, with the protein concentration adjusted to give an absorbance at 280 nm of about 1.0 for a path length of 1 cm. Typically, spectra were recorded from 180 to 250 nm at a scan speed of 20 nm per min, with each spectrum representing the average of 10 accumulations. During acquisition the samples were maintained at a regulated temperature (15 to 40 °C) in a 0.1-mm path length cell.

Fluorescence Spectroscopy-- Intrinsic fluorescence spectra of protein samples were acquired on a PerkinElmer Life Sciences LS50B luminescence spectrometer. The spectra were recorded at 20 °C with excitation at 280 nm and fluorescence monitored from 300 to 450 nm. The final spectra were the average of 10 accumulations collected at a scan rate of 150 nm per min. Typically, the spectra were acquired from 1 µM protein samples dissolved in a 25 mM NaH₂PO₄ and 100 mM NaCl buffer at pH 6.5.

NMR Spectroscopy-- The one-dimensional and 2D ¹H NMR experiments were carried out on 350 µl samples of 0.5 to 1.0 mM ESAT-6, CFP-10, and ESAT-6·CFP-10 complex dissolved in a 25 mM NaH₂PO₄ and 100 mM NaCl buffer at pH 6.5 (10% D₂O). NMR data were acquired on 600-MHz Varian Inova and Bruker Avance spectrometers at temperatures between 15 and 35 °C. The 2D nuclear Overhauser effect spectroscopy (19) and total correlation spectroscopy (20) spectra were recorded with mixing times of 100-150 and 45 ms, respectively, with typical acquisition times of 35 ms in F₁ and 250 ms in F₂.

Fluorescence-based Binding Assays-- Intrinsic fluorescence spectra were collected as described above for a series of samples containing 1 µM CFP-10 and increasing concentrations of ESAT-6 (0 to 2.25 µM). The protein samples were prepared in a 25 mM NaH₂PO₄ and 100 mM NaCl buffer at pH 6.5 and were incubated for 3 h at room temperature before acquiring the fluorescence spectra.

Pull Down Binding Assays-- In a typical pull down binding assay 0.1 µmol of His-tagged CFP-10 was loaded initially onto a 10-ml Ni-NTA column in a 20 mM Tris, 100 mM NaCl, and 1 mM EDTA buffer at pH 8.0, and the column was washed with five column volumes of the buffer. A slight molar excess of ESAT-6 was then applied to the column, and any protein that failed to bind was removed by washing once more with five column volumes of the Tris buffer including 20 mM imidazole. The proteins bound to the column were finally eluted in a 20 mM Tris and 100 mM NaCl buffer at pH 8.0 containing 100 mM imidazole, and the composition of the protein fractions was analyzed by SDS-PAGE.

Protein Denaturation Analysis-- The conformational stability of ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex to denaturation by guanidine hydrochloride was determined by monitoring the change in the wavelength of maximum intrinsic fluorescence emission as a function of guanidine hydrochloride concentration (21). The experiments were carried out on 0.5 to 1.5 µM samples of the proteins dissolved in a 25 mM NaH₂PO₄ and 100 mM NaCl buffer at pH 6.5, which contained between 0 and 2.25 M guanidine hydrochloride. The proteins were incubated with the denaturant overnight at 4 °C and then intrinsic fluorescence spectra were acquired at 10 °C as described previously.

Calculation of Protein Dendrogram-- The neighbor-joining phylogenetic tree for the ESAT-6/CFP-10 family of proteins was calculated using the ClustalX package (22), with the M. tuberculosis and Mycobacterium leprae sequences of CFP-10/ESAT-6 family proteins obtained from the TuberculList and Leproma servers at the Pasteur Institute (www.genolist.pasteur.fr/TuberculList and www.genolist.pasteur.fr/ Leproma).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Expression and Purification-- The expression of ESAT-6 in E. coli resulted in the production of insoluble inclusion bodies of the protein, which were isolated and solubilized in guanidine hydrochloride, and the ESAT-6 was refolded by removal of the denaturant by dialysis. Prior to purification on a Q-Sepharose column the refolded ESAT-6 was found typically to contain three main protein components, which were shown by electrospray mass spectrometry to have masses of 9902.8 ± 1.1 (~75%), 9772.2 ± 1.1 (~20%), and 8807.6 ± 0.9 Da (~5%). The mass values obtained correspond to those expected for full-length ESAT-6 (9903.9 Da), ESAT-6 minus the N-terminal methionine (9772.7 Da), and ESAT-6 with the last 11 C-terminal residues removed (8807.6 Da). If phenylmethylsulfonyl fluoride and EDTA were omitted from the refolding buffers then the majority of the refolded ESAT-6 obtained was present as the C-terminally truncated species, which clearly arises from proteolytic cleavage between Ala-84 and Ser-85. The full-length ESAT-6 and ESAT-6 minus the N-terminal methionine were separated from the C-terminally truncated form by chromatography on Q-Sepharose and once purified were stable for many days at 20 °C. Typical yields for purified ESAT-6 were about 40 mg/liter.

In contrast to ESAT-6, CFP-10 was expressed in E. coli as a soluble product, and the mass determined for the purified protein (10662.2 ± 0.6 Da) corresponds to that expected for CFP-10 after removal of the N-terminal methionine (10662.6 Da). Typical yields of purified CFP-10 were about 20 mg/liter. The His-tagged CFP-10 was also expressed as a soluble protein, and yields of about 10 mg/liter were obtained after purification. The E. coli cells transformed with the dual His-tagged CFP-10/ESAT-6 expression vector were grown initially in the presence of 40 µg/ml kanamycin and under these conditions showed no detectable coexpression of ESAT-6 with His-tagged CFP-10; however, when this was reduced to 20 µg/ml both proteins were detected by SDS-PAGE in roughly equal quantities. In addition, SDS-PAGE analysis of the protein isolated by Ni-NTA affinity chromatography of the lysate from cells grown at the lower kanamycin concentration revealed that the bound material consisted of an equimolar mixture of His-tagged CFP-10 and ESAT-6, which suggested that the proteins formed a stable, 1:1 complex.

Structural Characterization of the Proteins-- Typical intrinsic fluorescence spectra obtained for ESAT-6 (Trp-6, Trp-43, and Trp-58), CFP-10 (Trp-43), and the ESAT-6·CFP-10 complex are shown in Fig. 1. The spectra of both ESAT-6 and CFP-10 are characterized by a fluorescence maximum at about 353 nm at 20 °C, which corresponds to that expected for proteins in which all the tryptophan residues are fully exposed to the aqueous solvent. In contrast, the fluorescence maximum observed for the ESAT-6·CFP-10 complex is blue-shifted to around 342 nm, which indicates that at least one of the four tryptophan side chains has moved to a significantly less polar environment on complex formation, such as the hydrophobic core of the complex.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Intrinsic fluorescence emission spectra obtained for CFP-10 (A), ESAT-6 (B), and the 1:1 ESAT-6·CFP-10 complex (C) at 20 °C. The wavelengths of maximum emission are 353, 353, and 342 nm, respectively, which indicates that the tryptophan residues are fully exposed to the aqueous solvent in both ESAT-6 and CFP-10 but that one or more of the tryptophans become partially or fully buried on formation of the complex.

The far UV circular dichroism spectra shown in Fig 2 are representative of those acquired for ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex. The spectra obtained for both ESAT-6 and the ESAT-6·CFP-10 complex are typical of those seen for proteins with a high helical content, whereas the strikingly different spectrum observed for CFP-10 alone is indicative are a largely unstructured, random coil polypeptide (23, 24). Analysis of these spectra with the CDPro package (25) provided estimates of the secondary structure content for ESAT-6 (56% helix, 7% sheet, 12% turns, and 25% unstructured), CFP-10 (13% helix, 19% sheet, 17% turns, and 51% unstructured), and the complex (66% helix, 4% sheet, 9% turns, and 21% unstructured).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Far UV circular dichroism spectra acquired from solutions of CFP-10 (A), ESAT-6 (B), and the 1:1 ESAT-6·CFP-10 complex (C) at 25 °C. Clearly both ESAT-6 and the ESAT-6·CFP-10 complex have a high helical content, whereas CFP-10 appears to have relatively little regular secondary structure.

Fig. 3 shows the one-dimensional ¹H NMR spectra recorded for ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex. The spectrum observed for the complex contains all the features expected for a folded protein, such as the cluster of high field-shifted methyl signals between 0 and 0.6 ppm and a significant number of resonances from backbone amide groups between 8.5 and 9.5 ppm. In addition, the line widths of the ¹H signals indicate that the complex is a simple ESAT-6·CFP-10 heterodimer with a combined molecular mass of 20.5 kDa. The spectrum of the complex contrasts sharply with that obtained for CFP-10, in which there is no evidence of signals shifted from their random coil chemical shifts and therefore no evidence of any significant folded structure (26). The spectrum obtained for ESAT-6 clearly sits somewhere between that of the complex and CFP-10, with a few ¹H signals clearly shifted from random coil values, such as those from backbone NH groups between 8.5 and 9.3 ppm. Another noticeable feature of the ESAT-6 spectrum is that the ¹H resonances are very broad compared with those of the ESAT-6·CFP-10 complex. This is the opposite of what is expected as the line width of signals from ESAT-6 (9.9 kDa) should be about half of those for the complex (20.5 kDa) because of the 2-fold difference in molecular mass. The broad ¹H signals observed for ESAT-6 are indicative of either protein aggregation or exchange between multiple conformations. In 2D total correlation spectroscopy spectra of ESAT-6 collected under the same conditions at least 15 weak cross-peaks were detected between signals from backbone amide groups, which can only arise as a result of interconversion between multiple conformations in some regions of ESAT-6 (27). In addition, analysis of the 2D nuclear Overhauser effect spectroscopy and total correlation spectroscopy spectra of ESAT-6 suggests that over 70% of the signals from backbone NH groups may be broadened significantly by the exchange between multiple conformations, to the extent that cross-peaks involving these signals are not observed in 2D total correlation spectroscopy spectra.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   One-dimensional 1H NMR spectra recorded for CFP-10 (A), ESAT-6 (B), and the ESAT-6·CFP-10 complex (C). The contrasting broad signals for ESAT-6 and sharp signals for CFP-10 reflect the respective molten globule and random coil states of the two proteins. The spectrum for the complex shows significant dispersion of signals from backbone amide groups (6.5 to 9.5 ppm) and a number of high field-shifted methyl resonances (0 to 0.6 ppm), which are both characteristic features of a folded protein.

The graph shown in Fig. 4 illustrates the effect of increasing guanidine hydrochloride concentration on the wavelength of maximum intrinsic tryptophan fluorescence observed for ESAT-6, CFP-10, and the ESAT-6·CFP-10 complex. The data show clearly that the ESAT-6·CFP-10 complex is stable up to about 0.5 M guanidine hydrochloride and then undergoes a cooperative unfolding reaction with a midpoint at 0.9 M guanidine hydrochloride, which is the type of behavior expected for a folded protein (21) and suggests that protein dissociation and unfolding occur as a single event. In contrast, ESAT-6 does not show a cooperative denaturation curve, which suggests that the protein lacks any stable tertiary structure under native conditions. In the case of CFP-10, the wavelength of maximum fluorescence in the absence of guanidine hydrochloride already corresponds to that expected for a tryptophan fully exposed to an aqueous environment and is therefore insensitive to any unfolding induced by the denaturant.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Typical guanidine hydrochloride-induced denaturation curves for ESAT-6 (), CFP-10 (), and the ESAT-6·CFP-10 complex (). Denaturation of the proteins was followed by monitoring the change in the wavelength of maximum intrinsic fluorescence as a function of increasing denaturant concentration.

Secondary structure predictions were obtained for both ESAT-6 and CFP-10 using the JPRED2 package (28). ESAT-6 is predicted to consist of three helical regions (residues 3 to 18, 23 to 42, and 50 to 86) linked by short loops with an overall helical content of 77%. This is a somewhat higher helical content than the 56% obtained from analysis of CD spectra acquired at 25 °C but very close to the 75% helix indicated by CD spectra recorded at 15 °C. CFP-10 is also predicted to contain only helical secondary structure with five helices (residues 3 to 15, 18 to 22, 29 to 35, 44 to 78, and 87 to 96) joined by short loops, which corresponds to an overall helical content of 70%. This figure contrasts sharply with the predominantly random coil structure indicated for CFP-10 by the CD and NMR analysis.

ESAT-6·CFP-10 Complex Formation-- Fig. 5 illustrates the effect of increasing the molar ratio of ESAT-6 to CFP-10 on the wavelength of maximum intrinsic fluorescence observed for the mixture. The fluorescence maximum initially shifts from about 353 to 342.5 nm, with the shortest wavelength attained at a molar ratio of 1.0, and then shows a steady increase. The data strongly suggest that the two proteins interact to form a tight, 1:1 complex, in which the environment of at least one of the four tryptophan residues is significantly less polar and is therefore consistent with increased folding of one or both proteins.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   A typical example of the change in the wavelength of maximum intrinsic fluorescence observed on increasing the molar ratio of ESAT-6 to CFP-10, which clearly suggests that the two proteins form a tight, 1:1 complex.

The results of SDS-PAGE analysis of a typical ESAT-6 pull down assay using His-tagged CFP-10 bound to a Ni-NTA column as the bait are shown in Fig. 6. ESAT-6 was shown previously not to bind to the Ni-NTA column, and so the data clearly indicate that ESAT-6 binds tightly to His-tagged CFP-10. In addition, the staining intensity observed for the two components of the ESAT-6·CFP-10 complex clearly suggests a stoichiometry of 1:1.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   SDS-PAGE analysis of a typical ESAT-6 pull down assay using His-tagged CFP-10 bound to a Ni-NTA affinity column as bait. Lane 3 corresponds to 128 pmol of the bound protein eluted from the column by imidazole after initially loading 100 nmol of His-tagged CFP-10 followed by an excess of ESAT-6. For comparison lanes 2 and 4 contain 128 pmol of ESAT-6 and CFP-10, respectively. Lane 1 corresponds to a range of low molecular mass marker proteins (Sigma). The data clearly suggest that ESAT-6 and CFP-10 form a tight, 1:1 complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SDS-PAGE analysis of ESAT-6 purified from short term culture filtrates of M. tuberculosis has shown that the secreted protein consists of two major species, which both have the predicted N terminus and run with apparent molecular masses of between 4 and 6 kDa (29). This behavior is very similar to that observed for E. coli-expressed ESAT-6, where mass spectrometry revealed that the higher molecular mass band corresponds to a mixture of full-length ESAT-6 and ESAT-6 minus the N-terminal methionine and that the lower molecular weight species corresponds to ESAT-6 with 11 C-terminal residues removed. The similarity suggests that naturally secreted ESAT-6 is also highly susceptible to proteolytic removal of the 11 C-terminal residues.

The circular dichroism spectra recorded for ESAT-6 indicate clearly that the majority of the protein is in a helical conformation at temperatures below 20 °C, with quantitative analysis of the data suggesting a helical content of about 75%, which is very close to the 77% suggested by secondary structure predictions. The helical structure though is relatively unstable and falls to around 56% at 25 °C and less than 30% at 40 °C. In addition, ESAT-6 shows no resistance to denaturation by guanidine hydrochloride, which suggests that the protein is not folded fully even in the absence of the denaturant. The ¹H NMR data acquired for ESAT-6 clearly indicate that a significant proportion of the protein (possibly as high as 70%) exists in multiple conformations, which interconvert on a time scale that leads to significant broadening of the ¹H NMR signals from ESAT-6 and provides further evidence of structural instability in ESAT-6. Taken together, the spectroscopic data and structural predictions suggest that under in vivo-like conditions ESAT-6 contains a number of regions of regular helical secondary structure but little if any stable tertiary structure, and so the structure of isolated ESAT-6 appears to resemble a molten globule-like state.

In contrast to ESAT-6, the combined features of the circular dichroism, fluorescence, and ¹H NMR spectra of CFP-10 indicate clearly that the protein is an essentially unstructured, random coil polypeptide under in vivo-like conditions. This is somewhat surprising given a predicted helical content of over 70% and also the significant sequence similarity between CFP-10 and ESAT-6 (about 25% homology).

The changes observed in the wavelength of maximum intrinsic tryptophan fluorescence on titrating CFP-10 with ESAT-6, together with the results of ESAT-6 pull down assays using His-tagged CFP-10, indicate clearly that ESAT-6 and CFP-10 form a tight, 1:1 complex. The fluorescence measurements were carried out at a CFP-10 concentration of 1 µM, and the distinct minimum in the wavelength of maximum intrinsic tryptophan fluorescence at an ESAT-6:CFP-10 ratio of 1:1 (Fig. 5) indicates not only that the two proteins form a 1:1 complex but also that at least 90% of the ESAT-6 and CFP-10 are bound together at a concentration of 1 µM, which means that the binding is tight with a dissociation constant for the complex of 1.1 × 10⁸ M or lower.

The spectroscopic and chemical denaturation data obtained for the ESAT-6·CFP-10 complex show clearly that both proteins adopt a stable, fully folded structure in the complex. In recent years it has become clear that this type of behavior, in which a protein or protein domain is only folded fully when bound to its target or partner molecule, is more common than expected initially and may confer a number of functional advantages, including tighter control of the activity of regulatory proteins such as transcription factors and a general mechanism for increasing the specificity of protein-protein and protein-nucleic acid interactions (30, 31). Analysis of the circular dichroism spectra obtained for the ESAT-6·CFP-10 complex suggests that about two-thirds of the polypeptide backbone adopts a regular helical conformation, which is only slightly lower than the helical content expected from secondary structure predictions determined for ESAT-6 and CFP-10. The large number of NH to NH nuclear Overhauser effects seen in 2D nuclear Overhauser effect spectroscopy spectra of the complex also indicates a predominantly helical secondary structure (26) and is entirely consistent with the circular dichroism data.

The adjacent genes for CFP-10 (Rv3874) and ESAT-6 (Rv3875) are cotranscribed and despite lacking a recognizable secretory signal sequence are both found in significant quantities in short term culture filtrates of M. tuberculosis (12, 29). In addition, the work reported here shows clearly that ESAT-6 and CFP-10 only adopt a stable, fully folded structure when they form a tight, 1:1 complex. The expression characteristics of both proteins, together with their structural properties, clearly suggest that the biologically active form of ESAT-6 and CFP-10 will be as the complex.

CFP-10 and ESAT-6 are members of a large family of proteins found in the M. tuberculosis complex, which, in common with CFP-10 and ESAT-6, are generally found in pairs within the genome (7, 12, 17, 32). The phylogenetic tree calculated for the ESAT-6/CFP-10 family proteins identified in the M. tuberculosis (11 pairs) and M. leprae genomes (four pairs and two single genes; see Refs. 7 and 9) reveals that the proteins fall mainly into one of three pairing groups (Fig. 7). Interestingly, only the ESAT-6 and CFP-10 genes are conserved individually in M. leprae (ML0049 and ML0050, respectively), whereas the M. leprae proteins coded by ML2531/ML2532 seem to substitute for both the Rv0287/Rv0288 and Rv3019c/Rv3020c pairs from M. tuberculosis, and another single pair of M. leprae proteins (ML1055/1181 and ML1056/1180) appears to substitute for five pairs of M. tuberculosis proteins (Rv1037c/Rv1038c, Rv1197/Rv1198, Rv1792/Rv1793, Rv2346c/Rv2347c, and Rv3619c/Rv3620c). There are no apparent M. leprae homologues for two pairs of ESAT-6/CFP-10-related genes (Rv3890c/Rv3891c and Rv3904c/Rv3905c) and one pair where there is an M. leprae equivalent for only one (Rv3444c/Rv3445c). The M. leprae genome contains only 1,604 functional protein genes and has been proposed to represent the minimal gene set for a pathogenic mycobacterium (9). The retention of both ESAT-6 and CFP-10 as functional genes in M. leprae reiterates clearly their importance in the lifestyle of mycobacterial pathogens, and similar conservation arguments also suggest a significant role for both Rv0287/Rv0288 and Rv3019c/Rv3020c. This is supported by recent work (13), which showed that, like ESAT-6 and CFP-10, the product of the Rv0287 gene is also secreted by M. tuberculosis cells in culture and is a major T-cell antigen recognized by over 70% of tuberculosis patients.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Phylogenetic tree for the ESAT-6/CFP-10 family of M. tuberculosis proteins (prefixed by Rv) and their M. leprae homologues (prefixed by ML), with major pairing groups highlighted by brackets and labeled. Bootstrap values (%) are indicated for the major branch points in the tree. To generate the family relationships shown in the tree, the protein sequences were aligned initially and then bootstrapped 1,000 times using the PAM 250 amino acid comparison table.

The other members of the ESAT-6/CFP-10 family of proteins are all found as pairs in the genome of M. tuberculosis and are conserved mainly as pairs in M. leprae, which suggests that these pairs of genes will also be regulated coordinately, and the paired protein products form tight, 1:1 complexes and function as heterodimers. In addition, there is the interesting possibility that complex formation between members of the ESAT-6/CFP-10 family of proteins may not be limited to gene partners but could be much more widespread, such that 22 sequences in M. tuberculosis could give rise to many more than 11 functional protein complexes. This clearly suggests a mechanism for enhanced functional flexibility of ESAT-6/CFP-10 family proteins that may be very important for pathogenesis and virulence of members of the M. tuberculosis complex. Perhaps the best known precedence for this type of behavior is the leucine zipper family of transcription factors (c-Jun, c-Fos, cAMP-response element-binding protein (CREB), ATF1, ATF2, etc.), which can form a large number of homo- and heterodimers with distinct functional properties (33, 34). The characterization of the rules governing complex formation between members of the ESAT-6/CFP-10 family, together with the determination of the solution structure of the ESAT-6·CFP-10 complex, is the focus of ongoing work in our laboratory.

	FOOTNOTES

^* This work was supported in part by Grants 047795 and 055394 from the Wellcome Trust, by the award of a Biotechnology and Biological Sciences Research Council studentship (to P. S. R.), and by the Veterinary Laboratories Agency, United Kingdom. M. C. is one of the principle investigators of the M. tuberculosis Structural Genomics Consortium, which is supported by the National Institutes of Health and by the United States Department of Energy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-116-252-3054; Fax: 44-116-223-1503; E-mail: mdc12@le.ac.uk.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M201625200

	ABBREVIATIONS

The abbreviations used are: BCG, Bacille Calmette-Guérin; NMR, nuclear magnetic resonance; Ni-NTA, nickel-nitrilotriacetic acid; 2D, two-dimensional.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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