Structural and biochemical analysis of ATPase activity and EsxAB substrate binding of M. tuberculosis EccCb1 enzyme

The EccC enzyme of M. tuberculosis ESX-1 system is a promising target for antivirulence drug development. The EccC enzyme comprises two polypeptides (i) EccCa1, a membrane bound enzyme having two ATPase domains D2 & D3 (ii) cytosolic EccCb, which contains two ATPase domains. In current study, we have analyzed the low-resolution structure of EccCb1, performed ATPase activity and EsxAB substrate binding analysis. The EccCb1 enzyme eluted as oligomer from size exclusion column and small angle X-ray scattering analysis revealed the double hexameric structure in solution. The EccCb1 enzyme showed catalytic efficiency ( k cat /K M )∼ 0.020±0.005 μM -1 min -1 , however ∼ 3.7 fold lower than its D2 and ∼1.7 fold lower than D3 domains respectively. The D2 and D3 domains exhibited the ATPase activity and mutation of residues involved in ATP+Mg 2+ binding have yielded 56-94% reduction in catalytic efficiency for both D2 and D3 domains. The EccCb1 binds the EsxAB substrate with K D ∼ 11.4±3.4 nM via specific groove located at C-terminal region of D3 domain. ATP binding to EccCb1 enhanced the EsxAB substrate binding by ∼ 3 fold, indicating ATPase energy involvement in EsxAB substrate translocation. We modeled the dodecameric EccCb1+EsxAB+ ATP+Mg 2+ complex, which showed the binding pockets involved in ATP+Mg 2+ and EsxAB substrate binding. The enzyme dynamics involved in ATP+Mg 2+ and EsxAB substrate recognition were identified and showed the enhanced stability of EccCb1 enzyme as a result of ligand binding. Overall, our structural and biochemical analysis showed the low-resolution structure and mechanism involved in ATPase activity and EsxAB substrate binding and dynamics involved in EsxAB substrate and ATP+Mg 2+ recognition. Overall, our structural and biochemical data on EccCb1 will contribute significantly in development of antivirulence inhibitors, which will prevent virulence factor secretion by M. tuberculosis ESX-1 system.