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

Abstract

Transmembrane {beta}-barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the {beta}-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 {beta}-barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "{beta}-signal" motif of EspP to correctly orient {beta}-strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable "hybrid-barrel" intermediates in which membrane integrated {beta}-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 {beta}-sheets progressively fold towards BamA to form a {beta}-barrel. Along with in vivo experiments that tracked {beta}-barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate {beta}-barrel folding.