Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

Abstract

Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machine (BAM) via an unexplained process that occurs without known external energy sources. Here we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved β signal motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable hybrid-barrel intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold towards BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

“…This study provided evidence that BamA forms a hybrid barrel with an incoming OMP through a stable interaction between β1 and the C-terminal β strand of the client (a segment that contains the conserved 'β signal' motif GXXϕXϕ, where ϕ is an aromatic amino acid) (13) and a more dynamic interaction between β15/ β16 and N-terminal β-strands of the client (28). Consistent with the biochemical data, a high-resolution structure of the same assembly intermediate bound to the Bam complex determined by cryo-EM confirmed both the interaction of BamA β1 with the β signal and the extreme dynamics of the interaction on the other side of the hybrid barrel (29).…”