The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

The folding of outer membrane proteins (OMPs) is catalyzed by the beta barrel assembly machinery (BAM). Here, structural and functional analyses of BAM stabilized in distinct conformations elucidate the roles of lateral gate opening and interactions of BAM with the lipid bilayer in OMP assembly. The folding of beta-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the beta-barrel assembly machinery (BAM). How lateral opening in the beta-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.