Iadanza Matthew G, Higgins Anna J, Schiffrin Bob, Calabrese Antonio N, Brockwell David J, Ashcroft Alison E, Radford Sheena E, Ranson Neil A
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Mount Preston Street, Leeds LS2 9JT, UK.
Nat Commun. 2016 Sep 30;7:12865. doi: 10.1038/ncomms12865.
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.
β桶组装机器（BAM）是一种由五种蛋白质（BamA - E）组成的约203 kDa的复合体，对大肠杆菌的生存至关重要。BAM通过一种尚不清楚的机制促进β桶蛋白在外膜中的折叠和插入。目前的几种模型表明，BAM通过BamA的β桶的“侧向门控”运动发挥作用。在此，我们展示了分辨率为4.9 Å的BamABCDE复合体的冷冻电镜结构。该结构处于侧向开放构象，表明门控独立于BamB结合。我们描述了整个复合体的构象变化以及BamA、B、D和E之间的相互作用，还有去污剂胶束，这些表明了BAM与脂质双层之间的通讯。最后，使用改进的重组方案和功能测定，我们表明对于外膜蛋白OmpT，体外有效折叠需要BAM中的侧向门控。
