FIG 4.

FliA_Td interacts with FlgM_Td. (A) Protein-protein docking analysis of FliA_Td and FlgM_Td. The homology models of FliA_Td and FlgM_Td were first generated using the cocrystal structure of A. aeolicus s²⁸/FlgM complex (PDB code 1rp3), and then protein-protein docking analysis was conducted on the ClusPro server, with FliA_Td shown as a molecular surface and FlgM_Td shown as a backbone ribbon. (B) Coimmunoprecipitation (co-IP). This assay was performed using T. denticola whole-cell lysates and a polyclonal antibody against FliA_Td (αFliA_Td). The final co-IP eluates were probed using anti-FlgM_Td. A deletion mutant of flgM_Td (ΔflgM) was used as a negative control. (C to F) Copurification of N-terminal His-tagged FliA_Td (His-FliA_Td) and C-terminal FLAG-tagged FlgM_Td (FlgM_Td-FLAG). The two tagged proteins and their corresponding site-directed mutated or truncated proteins were coexpressed in the E. coli BL21 Star(DE3) strain and then purified using FLAG beads. The resulting samples were subjected to SDS-PAGE (C and E), followed by immunoblotting analysis (D and F) using either anti-FLAG (αFlag) or anti-His (αHis) antibodies. In panels C and D, in addition to the wild-type FliA_Td protein (lane 1), three site-directed mutated recombinant proteins were included: E221D (lane 2), V231E (lane 3), and a double mutation of E221D andV231E (lane 4). In panels E and F, lane 1 shows the wild-type FlgM_Td protein and lane 2 shows a truncated FlgM_Td protein without the H4′ helix (79 to 93 aa).