Structural basis of outer membrane biogenesis by the TamAB translocase.

The outer membrane is vital for Gram-negative bacteria, playing crucial roles in colonization, pathogenesis and drug resistance. The translocation and assembly module A and B (TamAB) nanomachinery has been reported to be involved in transport of phospholipids from the inner membrane to the outer membrane, as well as insertion of critical outer membrane proteins. However, the underlying mechanisms remain poorly understood. Here we report cryogenic electron microscopy structures of TamAB in two conformations at resolutions of 3.69 and 3.82 Å. We reveal a hybrid barrel structure formed between the first β-strand of the TamA barrel and the last β-strand of the TamB C-terminal domain, which is folded inside the β-barrel. By integrating structural analysis with functional data, biochemical assays, and molecular dynamics simulations, we identify key residues involved in TamAB interactions and characterize the mechanisms of anterograde phospholipid transport within the continuously beta-helical hydrophobic cavity of TamB. Through disulfide bond crosslinking and functional assays, we reveal that TamA crosslinks with both TamB and Ag43. Additionally, we confirm that the two cryo-EM conformational states of TamAB exist in vivo. While BAM overexpression can compensate for TamAB deletion in Ag43 insertion, it does not rescue phospholipid transport. Given that TamA and TamB orthologs are widely distributed in among bacterial and eukaryotic organisms, our findings have broad implications in cell envelope biogenesis and offer potential avenues for therapeutic development through inhibition.