Structural characterisation of CFP-10 and ESAT-6, potent T-cell antigens of the Mycrobacterium tuberculosis complex : implications for pathogenesis and virulence

Currently, the only effective vaccine against tuberculosis is a live attenuated strain of Mycobacterium bovis known as BCG, however, the molecular basis for the attenuation remains unclear. Recently, a number of deletions in the genomes of BCG daughter strains have been identified. One of these (RD1) contains the genes for 9 proteins (Rv3871 - Rv3879c), two of which, Rv3874 and Rv3875 code for two sequence related proteins, known as CFP-10 and ESAT-6 respectively, that are clearly implicated in pathogenesis.;Analysis using a range of spectroscopic techniques have shown that ESAT-6 contains up to 70% helical secondary structure, but little if any stable tertiary structure and exists in a molten globule-like state. In contrast, CFP-10 has been found to form a largely unstructured, random coil polypeptide. An exciting discovery is that CFP-10 and ESAT-6 form a tight, 1:1 complex, in which both proteins adopt a stable fully folded structure with over 75% of the backbone in a regular helical conformation. This clearly suggests that CFP-10 and ESAT-6 function as the complex.;High quality heteronuclear NMR spectra have been acquired from uniformly 15N/13C labelled CFP-10 bound to unlabelled ESAT-6, which have allowed the determination of the secondary structure of CFP-10 in the complex. The protein consists of 2 long helices (residues A8 - Q40 and A46 - G80) linked by a short turn/loop, which together form a helix-turn-helix hairpin structure. The N- (7 residues) and C-termini (20 residues) appear to be disordered and flexible. The sequence similarity between CFP-10 and ESAT-6 suggests that the two proteins will adopt similar topologies and together with NMR chemical shift data indicating the relative orientations of the two proteins in the complex, suggests that the complex will contain a four-helix-bundle with the two helix-turn-helix aligned anti-parallel to each other as seen in the E. coli protein ROP.