Membrane integration of an essential β-barrel protein prerequires burial of an extracellular loop.
Membrane integration of an essential β-barrel protein prerequires burial of an extracellular loop.
复制标题
必需的β-桶蛋白的膜整合需要埋藏细胞外环。
DOI：
10.1073/pnas.1616576114
复制
发表时间：
2017
影响因子：
11.1
通讯作者：
Kahne,DanielE
中科院分区：
文献类型：
--
作者：
Wzorek,JosephS;Lee,James;Tomasek,David;Hagan,ChristineL;Kahne,DanielE
Bam复合物将β-桶蛋白组装到革兰氏阴性菌的外膜（OM）中。这些蛋白质包括具有长的细胞外环的圆柱形β-折叠，并产生孔以允许营养物和废物穿过膜。尽管其功能的重要性，仍然存在一些问题，这些蛋白质是如何组装成OM后，在细胞质中合成和分泌穿过内膜。为了更好地理解这一过程，我们研究了Bam复合物BamA的基本β-桶底物的组装。通过突变该蛋白的β-桶结构域中的保守残基，我们产生了三种装配缺陷型BamA底物，其在周质中的折叠过程的早期停滞。三种缺陷底物中的两种在β链内具有突变，不能与Bam复合物有效结合。第三种底物在保守的细胞外环中含有突变，在组装过程中在BamD上积累，但不能有效地整合到膜中。所有三种基质的组装可以通过人工地将最终成为细胞外环的基质区域拴系到形成的β-桶的内腔来恢复。这些结果意味着折叠过程中的关键步骤涉及新生β-桶壁内部的残基与细胞外环中的残基的相互作用。我们的结论是，β-桶蛋白的膜整合的先决条件是在形成的β-桶内的细胞外环的埋葬。
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.