structural characterization of bacterial secretion channels

一个大的乘客域被一个小得多的β域转移的过程目前还不清楚。下面描述的是已经提出的解释乘客域易位的三个模型。在一种模型中，乘客结构域的c端以易位后的构象折叠到周质中的β结构域孔中。折叠前的β结构域随后被插入到OM中，乘客结构域通过一种协调一致的机制在OM中运输，该机制可能涉及Omp85，一种促进OM蛋白质整合和组装的重要蛋白质。该模型的一个优点是，它避免了在没有外部能源的情况下，需要通过一个相对较小的桶孔来转移一个或多个乘客域。第二个易位模型关注的是乘客域的不寻常结构，它们似乎都包含β -螺线管基序。一旦一小部分到达细胞表面，这些基序可以通过在OM的细胞外侧折叠来提供易位所需的能量。在这个模型中，一个短发夹包含乘客域的c端，位于桶状孔内，其尖端突出到细胞外空间。在发夹顶端折叠，然后将乘客区域的其余部分拉过孔。第三种模型是基于对IgA蛋白酶的β结构域在大肠杆菌中产生蛋白质时形成多聚环状结构的观察。中心空腔直径约为20a，并被假定为运输多个乘客域。

The process of translocating a large passenger domain by a much smaller beta-domain is currently not understood. Described below are three models that have been proposed to explain passenger domain translocation. In one model, the C-terminus of the passenger domain is folded into the beta-domain pore in the periplasm in a post-translocation conformation. The prefolded beta-domain is then inserted into the OM and the passenger domain is transported across the OM by a concerted mechanism that possibly involves Omp85, an essential protein that promotes OM protein integration and assembly. An advantage of this model is that it circumvents the need for one or more passenger domains to be translocated through a relatively small barrel pore in the absence of an external energy source. A second translocation model focuses on the unusual architecture of passenger domains, which all appear to contain beta-solenoid motifs. These motifs could supply the energy needed for translocation by folding on the extracellular side of the OM once a small portion has reached the cell surface. In this model, a short hairpin comprising the C-terminus of the passenger domain is positioned inside the barrel pore with its tip protruding into the extracellular space. Folding at the tip of the hairpin would then pull the rest of the passenger domain through the pore. A third model is based on the observation that the beta-domain of IgA protease forms multimeric ring-like structures when the protein is produced in E. coli. The central cavity is about 20 A in diameter, and was postulated to transport multiple passenger domains. A major focus of this project is EspP, a classical autotransporter associated with diarrheagenic strains of E. coli. It belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporters, whose passengers encode serine proteases that cleave various mammalian proteins. Biochemical studies have indicated that EspP is a monomer. Once the EspP passenger domain is translocated across the OM, it is cleaved from the membrane embedded beta-domain between two asparagine residues (N1023/N1024) and released from the cell surface. The Asn/Asn cleavage site defines the boundary of the EspP passenger domain (residues 56-1023) and beta-domain (residues 1024 1300). Although the passenger domain contains a serine protease motif located at residues 261-264, this motif is not used to cleave the two domains. Our goals for this project are to solve crystal structures of the pre- and post-cleavage forms of one or more autotransporters and to design experiments to probe substrate translocation across the outer membrane. The following work was accomplished in 2007: Structure determination of a bacterial autotransporter: To learn what happens to the beta-domain after cleavage and release of the passenger domain, we determined the crystal structure of the native beta-domain of EspP at 2.7 A resolution. This is the first structure of an autotransporter beta-domain post-cleavage, and it consists of a monomeric 12-stranded beta-barrel with its N-terminal 15 residues inserted into the barrel lumen from the periplasmic side. In agreement with a recently proposed autocatalytic cleavage mechanism, residues implicated in cleavage are located deep inside the beta-barrel, in a region of EspP that would be embedded in the OM. Interestingly, the structure suggests that two discrete conformational changes occur after cleavage and release of the passenger domain, one that confers increased stability on the beta-domain and another that restricts access to the barrel pore. Our structure does not support an oligomeric translocation model, but rather a model in which a single beta-barrel facilitates the translocation of a single passenger domain to the extracellular surface. Currently, we are attempting to solve the structure of EspP in its pre-cleavage conformation. Several mutants whose passenger domains are translocated to the extracellular space but are not cleaved will be purified for crystallization trials. A pre-cleavage structure will reveal additional details of the cleavage mechanism and allow us to attempt structure-based mutagenesis to test the proposed mechanisms of passenger translocation.