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。在一个主要的调查路线，我们一直在研究的EspP乘客域是跨外膜（OM）易位的机制。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（两个细胞膜之间的空间）中的残基与伴侣SurA和Skp相互作用。EspP-BamA相互作用是短暂的，只有当乘客结构域易位停止时才能检测到。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。我们还发现，周质分子伴侣和特定的组件的BAM复合物相互作用的EspP β结构域在时间和空间调节的方式。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。 在第二系列的研究中，我们一直在研究乘客结构域分泌的能量学。由于周质缺乏ATP，驱动乘客域分泌的能量来源尚不清楚。流行的模型假设细胞外空间中乘客结构域的矢量折叠促进单向分泌。我们使用的嵌合蛋白组成的受体结合结构域（RD）的百日咳杆菌腺苷酸环化酶毒素CyaA融合的C-末端的EspP来测试这一假设。RD是一种高度酸性的重复多肽，在缺乏钙的情况下本质上是无序的。令人惊讶的是，我们发现RD部分在保持未折叠构象时有效分泌。此外，我们发现中和或逆转酸性氨基酸簇的电荷会阻止改变残基附近的易位。这些结果挑战了矢量折叠模型，并结合对天然乘客结构域序列的分析，提供了净负电荷在驱动易位反应中起重要作用的证据。 最近，我们能够使用纯化的组分在体外重建EspP的组装。我们发现Bam复合物和SurA对于促进EspP β结构域整合到蛋白脂质体中以及乘客结构域易位到囊泡的内腔中都是必要且足够的。该反应不需要ATP或其他外源性能源。因为乘客结构域易位在C-至-N-末端方向上进行，所以我们能够检查具有不同长度乘客结构域的N-末端截短的EspP衍生物的组装。有趣的是，我们发现组装的速率与乘客结构域的长度无关。这一观察结果表明，β结构域的膜整合是自转运蛋白组装的限速步骤。我们还发现，一个拷贝的BAM复合物纳入纳米盘是足以催化EspP组装。

We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. In one major line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the outer membrane (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 are 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 (the space between the two cell membranes) interact with the chaperones SurA and Skp. The EspP-BamA interaction is short-lived and can only be detected when passenger domain translocation is 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. We also 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 interacts with the entire beta domain, BamA, BamB and BamD subsequently interact with discrete beta domain regions. BamB and BamD remain bound to the beta domain longer than BamA and therefore appear 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. In a second series of studies we have been examining the energetics of passenger domain secretion. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the passenger domain in the extracellular space facilitates unidirectional secretion. We used a chimeric protein composed of the receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C-terminus of EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety is efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalls translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, taken together with an analysis of naturally occurring passenger domain sequences, provide evidence that a net negative charge plays a significant role in driving the translocation reaction. Recently, we were able to reconstitute the assembly of EspP in vitro using purified components. We found that the Bam complex and SurA are both necessary and sufficient to promote the integration of the EspP beta domain into proteoliposomes and the translocation of the passenger domain into the lumen of the vesicles. The reaction does not require ATP or other exogenous energy source. Because passenger domain translocation proceeds in a C-to-N-terminal direction, we were able to examine the assembly of N-terminally truncated EspP derivatives that have different length passenger domains. Interestingly, we found that the rate of assembly is independent of passenger domain length. This observation suggests that the membrane integration of the beta domain is the rate-limiting step in autotransporter assembly. We also found that a single copy of the Bam complex incorporated into nanodiscs is sufficient to catalyze EspP assembly.