Structural analysis of M. tuberculosis EccC1 and its complex with EsxAB virulence factor using X-ray crystallography,...

M. tuberculosis ESX-1 system secretes virulence factors into host macrophages during infection, however, the mechanism of secretion is currently unknown. Here, we have determined the crystal structure of MtbEccCb1-D2 protein (Leu34-Ser313 residues, Mw ∼ 31.4 kDa) in complex with ATPγS and Mg²⁺, which adopts a classical Ftsk/SpoEIII type fold. The EccCb1-D2 showed two melting temperatures, Tm1 at 37.64 ± 0.08 °C and Tm2 at 65.85 ± 0.12 °C, during the unfolding pathway. Modeled ∆EccC1 and ∆EccC1 + EsxAB hexamers showed a channel (∼34 Å) involved in EsxAB (∼29 Å) translocation toward the inner membrane. At the entrance gate of the channel, the LxxxMxF motif of the EsxB export arm binds to the substrate binding pocket of the EccCb1-D3 protein. Inside the channel, the PL-1 and PL-2 pore loops, close to the α7-helix and the loop between β8-β9 strands in EccCa1-D1, EccCb1-D2, and EccCb1-D3 may be involved in EsxAB factor translocation. Stability, fluctuation, and compactness parameters in 100 ns dynamics simulation analysis showed the highest flexibility in ΔEccCa1, ∆EccC1, and ∆EccC1 + EsxAB hexamers and stability in ΔEccCb1 hexamer. Our EccCb1-D2 structure and dynamics simulation analysis on four modeled systems have revealed the mechanism involved in EsxAB translocation, a key target for the development of antivirulence inhibitors against M. tuberculosis.