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

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

Dynamic characterisation of the netrin-like domain of human type 1 procollagen C-proteinase enhancer and comparison to the N-terminal domain of tissue inhibitor of metalloproteinases (TIMP)

The backbone mobility of the C-terminal domain of procollagen C-proteinase enhancer (NTR(PCOLCE1)), part of a connective tissue glycoprotein, was determined using (15)N NMR spectroscopy. NTR(PCOLCE1) has been shown to be a netrin-like domain and adopts an OB-fold such as that found in the N-terminal domain of tissue inhibitors of metalloproteinases-1 (N-TIMP-1), N-TIMP-2, the laminin-binding domain of agrin and the C-terminal domain of complement protein C5. NMR relaxation dynamics of NTR(PCOLCE1) highlight conformational flexibility in the N-terminus, strand A and the proximal CD loop. This region in N-TIMP is known to be essential for inhibitory activity against the matrix metalloproteinases and suggests that this region is of equal importance for NTR(PCOLCE1), although the specific functional activity of the NTR(PCOLCE1) domain is still unknown. Dynamics observed within the structural core of NTR(PCOLCE1) that are not observed in N-TIMP molecules suggest that although the two domains have a similar architecture, the NTR(PCOLCE1) domain will show different thermodynamic properties on binding and hence the target molecule could be somewhat different from that observed for the TIMPs. ModelFree order parameters show that NTR(PCOLCE1) has more flexibility than both N-TIMP-1 and N-TIMP-2