Arkita Bandyopadhyay
Jawaharlal Nehru University
ABSTRACT
In mycobacteria, various ESX secretion systems are involved in transportation of virulence factors across the lipid rich cell wall. Five paralogous virulence secretion systems (ESX-1 to ESX-5) were identified in mycobacteria and contain conserved EccC enzymes e. g. EccB, EccC, EccD and EccE and all together known as type VII secretion system. Type VII secretion system transport virulence factors of WXG100 superfamily, usually observed as homo or heterodimers and form helix-turn-helix structure. In ESX-1 secretion system virulence factors usually secret in mutually dependent manner and in folded dimeric state.
Recently, low-resolution structure of ESX-5 complex system was determined using cryo electron microscopic technique. Other research group has also determined the structure of ESX-3 system using cryo-electron microscopy. These studies have provided the detailed knowledge about architecture of ESX secretion system and can be used as target for inhibitor development. In addition, structures of other proteins of ESX system e. g. EspB, ESAT-6/CFP-10 complex, T. curvata EccC complex, MycP1, N-terminal domain of EccA1, EccB1 and EccD1 have been determined. The structures of other secreted substrates e. g. EsxG:EsxH and PE-PPE-EspG complexes have been also reported.
M. tuberculosis EccC enzyme contains two polypeptides, (i) a membrane bound Rv3870containing a cytosolic ATPase domain and (ii) Rv3871containing two cytosolic ATPase domains. The Rv3870enzyme (747 residues, Mw~ 80.9 kDa) contains two transmembrane domains, a DUF domain (Domain of unknown function) and single P-loop type NTPase domain belong to FtsK/SpoIIIE family. The Rv3871enzyme contains (Mw~ 64.5 kDa, 591 residues) two P-loop type NTPases domains belong to FtsK/SpoIIIE family. ATPase activity of EccC enzymes rendered to the ‘Arginine finger’, which upon oligomerization enters into active site of next
monomer and forms an active complex. The Rv3870 is a key enzyme and its mutation exhibited defect in mycobacterial growth during acute phase of mouse infection.
In current study, we have analyzed the structure and ATPase activity of NTPase domain of Rv3870 (ΔRv3870, 442-747 residues). The ΔRv3870 residues involved in ATP+Mg2+ binding was identified and their roles in ATPase activity were identified using mutant enzymes. Binding and ATPase activity analysis of ΔRv3870 was performed in presence of Rv3871 enzyme. Low
resolution structure of ΔRv3870 was determined using small angle X-ray scattering technique and enzyme dynamics involved in ATP+Mg2+ recognition was determined using dynamics simulation. Our data explains the structure and mechanism of ΔRv3870enzyme, which will contribute significantly in inhibitor development against M. tuberculosis.
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