Wzorek Joseph S, Lee James, Tomasek David, Hagan Christine L, Kahne Daniel E
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):2598-2603. doi: 10.1073/pnas.1616576114. Epub 2017 Feb 21.
The Bam complex assembles β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. These proteins comprise cylindrical β-sheets with long extracellular loops and create pores to allow passage of nutrients and waste products across the membrane. Despite their functional importance, several questions remain about how these proteins are assembled into the OM after their synthesis in the cytoplasm and secretion across the inner membrane. To understand this process better, we studied the assembly of an essential β-barrel substrate for the Bam complex, BamA. By mutating conserved residues in the β-barrel domain of this protein, we generated three assembly-defective BamA substrates that stall early in the folding process in the periplasm. Two of the three defective substrates, which harbor mutations within β-strands, fail to associate productively with the Bam complex. The third substrate, which harbors mutations in a conserved extracellular loop, accumulates on BamD during assembly, but does not integrate efficiently into the membrane. The assembly of all three substrates can be restored by artificially tethering a region of the substrate, which ultimately becomes an extracellular loop, to the lumen of the forming β-barrel. These results imply that a critical step in the folding process involves the interaction of residues on the interior of the nascent β-barrel wall with residues in one of the extracellular loops. We conclude that a prerequisite for membrane integration of β-barrel proteins is burial of the extracellular loops within the forming β-barrel.
Bam复合物将β-桶状蛋白组装到革兰氏阴性菌的外膜（OM）中。这些蛋白由带有长细胞外环的圆柱形β-折叠片组成，并形成孔道以允许营养物质和代谢废物穿过膜。尽管它们在功能上很重要，但关于这些蛋白在细胞质中合成并跨内膜分泌后如何组装到外膜中仍存在几个问题。为了更好地理解这一过程，我们研究了Bam复合物的一种必需β-桶状底物BamA的组装。通过突变该蛋白β-桶状结构域中的保守残基，我们生成了三种组装缺陷型BamA底物，它们在周质中的折叠过程早期就停滞了。三种缺陷型底物中的两种，其β-链内存在突变，无法与Bam复合物有效结合。第三种底物，其保守细胞外环中存在突变，在组装过程中积累在BamD上，但不能有效整合到膜中。通过将底物的一个区域（最终成为细胞外环）人工连接到正在形成的β-桶状结构的内腔，可以恢复所有三种底物的组装。这些结果表明，折叠过程中的一个关键步骤涉及新生β-桶状壁内部的残基与其中一个细胞外环中的残基相互作用。我们得出结论，β-桶状蛋白膜整合的一个先决条件是将细胞外环埋藏在正在形成的β-桶状结构中。
