Abstract
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.
Highlights
MATERIALS AND METHODSPhylogenetic Tree and Sequence Analysis—The sequence of Rv3868 was downloaded from the Tuberculist site on the World Wide Web
Inactivation of Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 impairs the ability of the pathogen to secrete the virulence factors, their expression itself was unimpaired
Inactivation of a fourth group consisting of Rv3865 and Rv3866 attenuated RD1mediated virulence, the secretion of ESAT-6 and CFP-10 factors was unimpaired
Summary
Phylogenetic Tree and Sequence Analysis—The sequence of Rv3868 was downloaded from the Tuberculist site on the World Wide Web. Titrations were performed at 25 °C by the addition of ATP to 0.6 ml of 50 mM Tris (pH 7.5), 50 mM NaCl, and 5 mM MgCl2 buffer containing different amounts of proteins. The ATPase assays were carried out in structure onto the hexameric D2 domain of NSF NaCl, 0.1% sodium azide, and 5% (v/v) 2H2O was used as inhibitory effects on the activity were observed in the presence reported in earlier experiments by our group [11] The spectra of these factors (data not shown). Dominantly exists as a hexamer at protein concentrations up to Chaperone-like Activity Assay—The assays were carried out ϳ3 mg/ml and elutes at a molecular mass of ϳ380 kDa (Fig. using procedures similar to those described earlier [28] at 43 °C 2A). The protein forms higher order using hen egg white lysozyme (Sigma) and porcine mitochon- oligomers with concomitant reduction in the hexamer populadrial citrate synthase (Sigma) as test substrates
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