Biogenesis of bacterial autotransporter proteins

我们一直在使用由E. coli O 157：H7中的EspP蛋白作为研究自转运蛋白生物合成的模型蛋白。几年前，我们表明，EspP乘客结构域易位后，通过自催化裂解反应，涉及激活的天冬酰胺残基的OM，它被释放到细胞外环境。 在一个主要的调查路线，我们一直在研究的机制，其中EspP乘客域是跨OM易位。最初提出乘客结构域通过共价连接的β结构域形成的通道分泌（因此命名为自转运蛋白），但我们从生化和结构研究中获得的结果似乎与这一假设不一致。我们发现，插入一个小的接头到EspP乘客结构域有效地创建一个易位中间体，通过瞬时停止易位附近的插入位点。通过使用位点特异性光交联方法，我们发现，邻近失速点的残基与BamA相互作用，BamA是催化OM蛋白组装的异源寡聚复合物（BAM复合物）的组分，并且被困在周质中的残基与周质伴侣SurA和Skp相互作用。EspP-BamA的相互作用是短暂的，只能检测到乘客结构域易位停止时。这些结果支持一种模型，其中分子伴侣在其分泌之前防止乘客结构域的错误折叠，并且BAM复合物在促进β结构域整合到OM中和乘客结构域跨OM的易位中起主要作用。最近，我们发现，周质分子伴侣和特定的组件的BAM复杂的相互作用与EspP β结构域在时间和空间上调节的方式。虽然伴侣蛋白Skp最初与整个β结构域相互作用，但BamA、BamB和BamD随后与离散的β结构域区域相互作用。BamB和BamD保持与β结构域结合的时间比BamA长，因此似乎在组装的后期发挥作用。我们的研究结果表明，迄今为止神秘的BamB和BamD蛋白发挥了直接的作用，在膜整合的自转运蛋白β结构域和可能的其他β桶蛋白。有趣的是，我们还获得了β结构域组装完成受内在检查点机制调控的证据，该机制需要完成乘客结构域分泌。 在第二条调查线，我们一直在研究乘客域分泌的能量学。虽然乘客域分泌似乎不使用ATP，这种反应的能量来源是未知的。我们发现EspP乘客结构域的有效分泌需要C-末端17 kD乘客结构域片段的稳定折叠。因为易位在C-至-N-末端方向上进行，所以这是暴露在细胞表面上的乘客结构域的第一区段。我们发现，干扰C-末端片段折叠的突变不影响其跨OM的易位，但损害乘客结构域的其余部分的分泌。有趣的是，对动力学折叠突变体的检查强烈表明，17 kD片段在细胞外空间折叠。我们的研究结果提供了第一个直接证据，证明蛋白质的矢量折叠可以作为布朗棘轮，驱动其易位穿过生物膜。

We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. We showed several years ago that after the EspP passenger domain is translocated across the OM it is released into the extracellular milieu through an autocatalytic cleavage reaction that involves the activation of an asparagine residue. In one major line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. It was originally proposed that the passenger domain is secreted through a channel formed by the covalently linked beta domain (whence the name autotransporter), but results that we obtained from both biochemical and structural studies appear to be inconsistent with this hypothesis. We found that the insertion of a small linker into the EspP passenger domain effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. By using a site-specific photocrosslinking approach we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues that are trapped in the periplasm interact with the periplasmic chaperones SurA and Skp. The EspP-BamA interaction was short-lived and could only be detected when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain prior to its secretion and the Bam complex plays a major role in facilitating both the integration of the beta domain into the OM and the translocation of the passenger domain across the OM. Recently, we found that periplasmic chaperones and specific components of the Bam complex interact with the EspP beta domain in a temporally and spatially regulated fashion. While the chaperone Skp initially interacted with the entire beta domain, BamA, BamB and BamD subsequently interacted with discrete beta domain regions. BamB and BamD remained bound to the beta domain longer than BamA and therefore appeared to function at a later stage of assembly. Our results suggest that the hitherto enigmatic BamB and BamD proteins play a direct role in the membrane integration of autotransporter beta domains and possibly other beta barrel proteins. Interestingly, we also obtained evidence that the completion of beta domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In a second line of investigation, we have been examining the energetics of passenger domain secretion. Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. We found that efficient secretion of the EspP passenger domain requires the stable folding of a C-terminal 17 kD passenger domain segment. Because translocation proceeds in a C-to-N-terminal direction, this is the first segment of the passenger domain that is exposed on the cell surface. We found that mutations that perturb the folding of the C-terminal segment do not affect its translocation across the OM, but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the 17 kD segment folds in the extracellular space. Our results provide the first direct evidence that the vectorial folding of a protein can act as a Brownian ratchet that drives its translocation across a biological membrane.