title: Characterization of EccA3, a CbbX family ATPase from the ESX-3 secretion pathway of M. tuberculosis.
abstract: EccA family proteins are conserved components of ESX secretion pathways in M. tuberculosis H37Rv. Here, we report the characterization of EccA3 (Rv0282), a CbbX family AAA (ATPases Associated with diverse cellular Activities) protein from the ESX-3 pathway that is required for in vitro growth of mycobacteria, secretion of virulence factors, and acquisition of iron and zinc. EccA3 is a thermostable ATPase with a molecular weight of ~68kDa. It exists as a dodecamer in the apo form and associates as a hexamer in the presence of ATP. Its C-terminal region consists of a CbbX-like AAA-domain while the N-terminal region contains a tetratricopeptide repeat (TPR) domain with lower homology to other EccA-type proteins. Further, the C-terminal domain functions as the oligomerization domain and also exhibits ATPase activity. Mutational analysis, steady state kinetics and molecular docking studies identify R573 as the important 'sensor arginine' and R505 as an 'arginine finger' in EccA3. Dynamic fluorescence quenching experiments suggest that the N-terminal domain moves closer to the C-terminal domain upon ATP-binding. The ATP-dependent 'open-close' relative movements of the two domains might help EccA3 interaction and secretion of essential virulence factors.
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, UP, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Gaur
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, UP, India.
authors.forename: Vijay Kumar
authors.initials: VK
authors.lastname: Sharma
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, UP, India.
authors.forename: Sonal
authors.initials: S
authors.lastname: Shree
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, UP, India.
authors.forename: Niyati
authors.initials: N
authors.lastname: Rai
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, UP, India. Electronic address: r_ravishankar@cdri.res.in.
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D006868:Hydrolysis; D062105:Molecular Docking Simulation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: AAA proteins; CbbX protein family; ESX secretion system; EccA protein; Type VII secretion system