Biogenesis of bacterial autotransporter proteins

我们一直使用大肠杆菌O157：H7产生的自体转运蛋白ESPP作为模型蛋白来研究自体转运蛋白的生物发生。我们早在几年前就证明了，当ESPP的乘客结构域跨OM转移后，它通过涉及天冬酰胺残基活化的自动催化切割反应释放到细胞外环境中。 在一条主要的调查路线中，我们一直在研究ESPP乘客域跨OM转移的机制。最初有人提出，乘客结构域是通过共价连接的β结构域(自转运体)形成的通道分泌的，但我们从生化和结构研究中获得的结果似乎与这一假说不一致。我们发现，将一个小连接子插入到ESPP乘客结构域中，通过在插入位置附近短暂地拖延易位，有效地创建了一个易位中间体。通过使用定点特定的光交联方法，我们发现靠近失速点的残基与BAMA相互作用，BAMA是催化OM蛋白质组装的异寡聚复合体(BAM复合体)的一种成分，而被捕获在周质中的残基与周质伴侣Sura和Skp相互作用。ESPP-BAMA相互作用是短暂的，只有当乘客域转移停止时才能检测到。这些结果支持这样一个模型，即分子伴侣在乘客结构域分泌之前防止其错误折叠，而Bam复合体在促进β结构域整合到OM中和促进乘客结构域在OM之间的移位方面发挥着重要作用。我们还发现，周质伴侣和Bam复合体的特定成分以时间和空间调节的方式与ESPPβ结构域相互作用。当伴侣Skp最初与整个Beta结构域相互作用时，BAMA、BAMB和BAMD随后与离散的Beta结构域相互作用。BAMB和BAMD与β结构域结合的时间比BAMA更长，因此似乎在组装的后期阶段发挥作用。我们的结果表明，迄今为止神秘的BAMB和BAMD蛋白在自体转运蛋白β结构域以及可能的其他β桶蛋白的膜整合中起着直接的作用。有趣的是，我们还获得了证据，表明β结构域组装的完成受到一种内在的检查点机制的调节，该机制要求完成客体结构域的分泌。 最近，我们一直在研究β结构域在自体转运蛋白生物发生中的作用。多年来，人们已经知道β结构域是客体结构域分泌所必需的，但其确切功能尚不清楚。我们发现ESPP的两个突变(G1066A和G1081D)略微扭曲了β结构域的结构，也推迟了客体结构域易位的启动。定点光交联实验表明，这些突变减缓了Beta结构域插入OM的速度，但不会推迟Beta结构域与Bam复合体的结合。我们的结果表明，β结构域并不是简单地将乘客结构域靶向OM，而是当它到达组装的特定阶段时促进易位。此外，我们的结果提供了证据，证明Bam复合体催化β桶蛋白的膜整合是一个多步骤的过程，这个过程可以被客户蛋白中的微小结构缺陷所干扰。

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. 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. 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. Recently, we have been examining the role of the beta domain in autotransporter biogenesis. It has been known for many years that the beta domain is required for passenger domain secretion, but its exact function has been unclear. We found that two mutations in EspP (G1066A and G1081D) that slightly distort the structure of the beta domain also delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the beta domain into the OM, but do not delay the binding of the beta domain to the Bam complex. Our results demonstrate that the beta domain does not simply target the passenger domain to the OM, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of beta barrel proteins in a multi-step process that can be perturbed by minor structural defects in client proteins.