Abstract
The integration of β-barrel proteins into the bacterial outer membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a β-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli β-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first β-strand of a laterally ‘open’ form of the BamA β-barrel forms a rigid interface with the C-terminal β-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA β-strands forms weaker, conformationally heterogeneous interactions with the first β-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the membrane integration of partially folded β-barrels by a ‘swing’ mechanism.
Highlights
The integration of β-barrel proteins into the bacterial outer membrane (OM) is catalysed by the β-barrel assembly machinery (BAM)
Sec-translocon and maintained in an assembly-competent state by periplasmic chaperones, they are assembled and integrated into the OM in the absence of external energy inputs[13]. This is achieved by the β-barrel assembly machinery (BAM), which in E. coli is composed of a β-barrel protein (BamA) and four accessory lipoproteins (BamBCDE) that associate with the periplasmic POTRA domains of BamA14
Based on evidence that the translocation of fully folded heterologous polypeptides by autotransporters is often limited by their size[38,39], we hypothesised that the fusion of a large protein to the passenger domain would create a molecular ‘knot-in-a-rope’ that arrests assembly after the association of the β-barrel domain with BAM and the initiation of translocation
Summary
The integration of β-barrel proteins into the bacterial outer membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). Integral outer membrane proteins (OMPs) serve multiple critical cellular functions, including nutrient uptake[3], protein secretion[4,5] and adhesion[6] Almost all of these proteins are anchored to the OM by a unique ‘β-barrel’ structure. Sec-translocon and maintained in an assembly-competent state by periplasmic chaperones, they are assembled and integrated into the OM in the absence of external energy inputs[13] This is achieved by the β-barrel assembly machinery (BAM), which in E. coli is composed of a β-barrel protein (BamA) and four accessory lipoproteins (BamBCDE) that associate with the periplasmic POTRA (polypeptide transport-associated) domains of BamA14. BamA is a member of the Omp[85] superfamily, a group of 16-stranded β-barrel proteins that
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