Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis.
Rv3868, a conserved hypothetical protein of the ESAT-6 secretion system of Mycobacterium tuberculosis, is essential for the secretion of at least four virulence factors. Each protein chain is approximately 63 kDa and assembles into a hexamer. Limited proteolysis demonstrates that it consists of two domains joined by a linker. The N-terminal domain is a compact, helical domain of approximately 30 kDa and apparently functions to regulate the ATPase activity of the C-terminal domain and the oligomerization. The nucleotide binding site is situated in the C-terminal domain, which exhibits ATP-dependent self-association. It is also the oligomerization domain. Dynamic fluorescence quenching studies demonstrate that the domain is proximal to the C terminus in the apoprotein and exhibits a specific movement upon ATP binding. In silico modeling of the domains suggests that Arg-429 of a neighboring subunit forms a part of the binding site upon oligomerization. Mutational analysis of binding site residues demonstrates that the Arg-429 functions as the important "sensor arginine" in AAA-ATPases. Protein NMR experiments involving CFP-10 and activity assays rule out a general chaperone-like function for Rv3868. On the other hand, ATP-dependent "open-close" movements of the individual domains apparently enable it to interact and transfer energy to co-proteins in the ESX-1 pathway.
Luthra
Amit
A
Molecular and Structural Biology Division, Central Drug Research Institute, P.O. Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India.
Mahmood
Anjum
A
Arora
Ashish
A
Ramachandran
Ravishankar
R
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases