tailieunhanh - Báo cáo khoa học: Mycobacterium tuberculosis H37Rv ESAT-6–CFP-10 complex formation confers thermodynamic and biochemical stability

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 tuberculosisH37Rv, 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. | ềFEBS Journal Mycobacterium tuberculosis H37Rv ESAT-6-CFP-10 complex formation confers thermodynamic and biochemical stability Akshaya K. Meher1 Naresh Chandra Bal1 Kandala V. R. Chary2 and Ashish Arora1 1 Molecular and StructuralBiology CentralDrug Research Institute Lucknow India 2 Department of ChemicalScience Tata Institute of FundamentalResearch Mumbai India Keywords association constant ESAT-6-CFP-10 complex limited proteolysis lipid-protein interactions thermal unfolding Correspondence A. Arora Molecular and StructuralBiology CentralDrug Research Institute Lucknow 226 001 India Fax 91 522 223405 Tel 91 522 261 2411 18 ext. 4329 E-mail ashishcdri@ Received 4 January 2006 revised 30 January 2006 accepted 6 February 2006 doi 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 virulence. In this study we explored the thermodynamic and biochemical changes 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 X 107 M-1 and kcal-mol-1 respectively. Strikingly the thermal unfolding of the ESAT-6-CFP-10 heterodimeric complex was completely reversible with a Tm of C and DH of 69 kcal-mol-1. Mixing of ESAT-6 and CFP-10 at any temperature below the Tm 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 .