Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM

Abstract

The β-barrel assembly machinery (BAM) is a ∼203 kDa complex of five proteins (BamA–E), which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating' motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

“…Consistent with this model, engineered disulfide bonds that prevent the opening of the putative lateral gate are lethal in E. coli (17). Disulfide locking of the BamA b-barrel, however, does not effectively block OMP assembly in vitro (16,18,19). Molecular dynamics simulations (20, 21) and other studies (22) that suggest that the BamA β-barrel causes thinning of the adjacent membrane have led to an alternative model in which the Bam complex catalyzes OMP assembly by creating a local deformation of the lipid bilayer.…”

“…The complex has the shape of a top hat in which the BamA bbarrel forms the crown and the POTRA domains together with the lipoproteins form a brim (14,15). Based on the structure, it has been proposed that the rotation of the ring-like structure formed by the periplasmic elements of the Bam complex leads to the opening of an unstable junction between the first and last β-strands of the BamA b-barrel ("lateral gate") and thereby promotes the insertion of OMPs into the lipid bilayer in a stepwise fashion (14)(15)(16). The lipoprotein ring would create a protective environment that shields OMPs from repulsive interactions with periplasmic lipid head groups.…”

“…Although the structure of the Bam complex was recently solved by both X-ray crystallography and cryo-EM (13)(14)(15)(16), the mechanism by which it catalyzes the integration of b-barrel proteins into the OM remains poorly understood. The complex has the shape of a top hat in which the BamA bbarrel forms the crown and the POTRA domains together with the lipoproteins form a brim (14,15).…”

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

“…The overall structure was found in a bent L-shaped conformation, closely resembling that of POTRA3-5 of E. coli BamA (26,39,40). All three POTRA domains contained the conserved βααββ fold observed in other POTRA domains with relatively good structural alignment (23,47,48). Interestingly, the previously observed ∼40-residue insertion within Toc75 POTRA2 (17) overlaps with an extended α-helix encompassing residues 275-296, which we refer to as the P2-helix.…”

“…Recent X-ray crystallography and cryo-EM studies reported the structures of several Omp85 family members, including FhaC, TamA, and BamA (28,(36)(37)(38)(43)(44)(45)(46)(47). Given the conservation of key features, these structures serve as models for Toc75, albeit with low sequence identity (7%, 7%, and 8%, respectively).…”