Mechanistic insights into TAM-mediated OMP assembly in Gram-negative bacteria.

Abstract

The β-barrel assembly machinery (BAM) is essential for outer membrane protein (OMP) biogenesis in Gram-negative bacteria and represents a validated target for antibiotic development. Its evolutionary relative, the translocation and assembly module (TAM), is thought to assemble OMPs but through a mechanism that remains poorly understood. Here, we present cryo-electron microscopy structures of Escherichia coli TamA and TamAB, capturing multiple conformational states. These structures reveal that the TamB C-terminal tail engages the TamA β-barrel to open its lateral gate, forming a transient hybrid β-interface and activating an extended AsmA-like periplasmic conduit that may facilitate substrate transport. Consistent with this mechanism, NanoLuc Binary Technology (NanoBiT)-based folding assays demonstrate that TamAB promotes OMP assembly more efficiently than TamA alone, with activity dependent on both lateral-gate opening and hybrid-interface formation. Together, our results define a substrate-mimetic priming mechanism that sets TAM apart from BAM and establish a structural framework for understanding OMP biogenesis.