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

Abstract

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-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 revealed 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 new insights into the role of BAM structure and lipid dynamics in OMP folding.

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

“…Alternatively, the conformational changes observed in the surface loops of BamA in the B/CO-state relative to our other structures (e.g., L4, 6, and 7) may be required for the folding of β-barrels more generally. Indeed, this finding may explain why the function of BAM is strongly inhibited when BamA L4 is bound by the bactericidal antibody fragment Fab1 which presumably prevents this conformational cycling 20 (White et al, 2021). It is important to note that none of our reconstructions exhibited the twisted interface that results in a hybrid-barrel with a "W-shaped" cross-section observed in the BAM-BamAΔL1 structure (Tomasek et al, 2020).…”

“…The structural dynamics that occur as OMPs transition from an incompletely folded state to a fully folded β-barrel remain unclear. However, available evidence suggests that OMP βsignals may be recognized by BAM and that the unusual conformational malleability of BAM (particularly BamA) may facilitate the folding process (Doerner and Sousa, 2017;Doyle and Bernstein, 2019;Hagan et al, 2015;Iadanza et al, 2016;Kaur et al, 2021;Lundquist et al, 2018;Noinaj et al, 2014;Tomasek et al, 2020;White et al, 2021). Interestingly, BamA does not contain a canonical β-signal at its C-terminus, but instead has a "kinked" structure that causes its terminal residues to move dynamically and generate a unique unstable β-seam (that forms hydrogen-bonds poorly) (Lundquist et al, 2018;Noinaj et al, 2013).…”

“…Because the interactions between densely packed β-barrels and LPS molecules results in a rigid structure in which protein diffusion is low (Rassam et al, 2015;Rojas et al, 2018;Ursell et al, 2012), the mechanism by which β-barrels are folded into the OM is even more puzzling. A recent study showed that the BAM lipoproteins can alter membrane fluidity (albeit in synthetic bilayers) and thereby potentially facilitate β-barrel integration (White et al, 2021). Intriguing molecular dynamics simulations have also raised the possibility that the unique 'wedge-shaped" aromatic girdles of the BamA β-barrel might thin the OM to reduce the energy required for assembly (Liu and Gumbart, 2020;Noinaj et al, 2013).…”