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?
Renshaw
Philip S
PS
Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH, United Kingdom.
Panagiotidou
Parthena
P
Whelan
Adam
A
Gordon
Stephen V
SV
Hewinson
R Glyn
RG
Williamson
Richard A
RA
Carr
Mark D
MD
D000941:Antigens / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009163:Mycobacterium bovis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D010277:Parasympathomimetics / Q000494:pharmacology; D010802:Phylogeny; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014466:Ultraviolet Rays
D000941:Antigens; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D010277:Parasympathomimetics; D006639:Histidine; D019791:Guanidine