Structures of folding intermediates on BAM show diverse substrates fold by a conserved mechanism

The outer membrane of Gram-negative bacteria contains a wide variety of integral β-barrel proteins. Here, we study the conserved β-barrel assembly machine which is responsible for folding and inserting these membrane β-barrels. By determining the structures of a BamA substrate barrel trapped in progressively folded states, we uncover features that are maintained throughout the machine’s folding process. By determining an additional structure of an LptD substrate during its folding, we are able to generalize our observations to different substrates. Together, these structures of barrel assembly machine (BAM’s) two essential β-barrel substrates folding on the machine suggest how BAM can fold a diverse set of integral membrane β-barrels by a conserved mechanism. These observations could enable strategies to combat Gram-negative pathogens. The outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria contain β-barrel membrane proteins that are assembled by conserved multisubunit machines. In bacteria, the β-barrel assembly machine (BAM) folds over a hundred compositionally different substrates into barrels that vary greatly in size. Some larger barrels require globular proteins to plug the barrel lumen. How a single machine can assemble such different barrels is unknown. Here we report three structures representing progressively folded stages of a 16-stranded barrel engaged with BAM, as well as the structure of a late-stage folding intermediate of a 26-stranded substrate folding around its soluble lipoprotein plug on BAM. We find that BAM catalyzes folding of these substrates by a uniform mechanism in which BAM undergoes major distortions to accommodate the nascent barrel.