structural characterization of bacterial secretion channels

细菌分泌通道的结构特征

• 批准号: 7593557
• 负责人: Susan Buchanan
• 金额: $ 38.6万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593557
• 关键词: Agreement; Architecture; Asparagine; Bacterial Adhesins; Biochemical; Bordetella pertussis; C-terminal; Caliber; Cell surface; Cells; Cleaved cell; Crystallization; Depth; Energy Supply; Energy-Generating Resources; Enterobacteriaceae; Escherichia coli; Extracellular Space; Family; Goals; Gram-Negative Bacteria; IgA-specific serine endopeptidase; Iron; Learning; Lipids; Mediating; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular Conformation; Mutagenesis; N-terminal; Peptide Signal Sequences; Positioning Attribute; Process; Proteins; Proteolysis; Resolution; Rest; Serine Protease; Shigella flexneri; Side; Site; Structure; Surface; Testing; Virulence; Work; base; beta barrel; cell motility; design; extracellular; monomer; mutant; periplasm; pertactin; research study; size

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.

通过小得多的测试域转移大的乘客域的过程目前还不清楚。下面描述的是已经提出的三种解释乘客域转移的模型。在一种模型中，客体结构域的C末端以易位后的构象折叠到周质中的β结构域孔中。然后将预先折叠的β结构域插入OM中，并通过一种可能涉及Omp85的协调机制将乘客结构域运送到OM中，Omp85是一种促进OM蛋白质整合和组装的重要蛋白质。这种模型的一个优点是，在没有外部能源的情况下，它避免了通过相对较小的桶孔转移一个或多个客域的需要。第二个易位模型关注的是乘客域的不同寻常的结构，这些域似乎都包含贝塔螺线管基序。一旦一小部分到达细胞表面，这些基序可以通过在OM的胞外侧折叠来提供转运所需的能量。在这个模型中，包含乘客结构域C-末端的短发夹被定位在管状毛孔内，其尖端突出到细胞外空间。在发夹的尖端折叠，然后会将乘客域的其余部分拉过毛孔。第三个模型是基于观察到当蛋白在大肠杆菌中生产时，IgA蛋白酶的β结构域形成多聚环状结构。中心空腔的直径约为20A，并被假设为运输多个乘客区域。 该项目的一个主要焦点是ESPP，这是一种与致泻性大肠杆菌菌株相关的经典自体转运蛋白。它属于肠杆菌科丝氨酸蛋白酶自身转运体(SPATE)家族，其乘客编码丝氨酸蛋白酶，可裂解多种哺乳动物蛋白质。生化研究表明，ESPP是一种单体。一旦ESPP的客体结构域被转移到OM上，它就会从嵌入在两个天冬酰胺残基(N1023/N1024)之间的膜上被切割下来，并从细胞表面释放出来。ASN/ASN裂解位点定义了ESPP乘客结构域(残基56-1023)和β结构域(残基1024 1300)的边界。虽然Passenger结构域包含位于261-264残基的丝氨酸蛋白酶基序，但该基序不用于切割这两个结构域。 我们这个项目的目标是解决一个或多个自体转运蛋白的分裂前和分裂后形式的晶体结构，并设计实验来探测底物跨外膜的运输。2007年完成了以下工作： 细菌自身转运蛋白的结构测定： 为了了解客体结构域切割和释放后β结构域的变化，我们在2.7A分辨率下测定了ESPP天然β结构域的晶体结构。这是第一个自身转运蛋白β结构域的切割后结构，它由一个12链的单体β桶组成，其N端15个残基从周质侧插入管腔中。与最近提出的自动催化切割机制一致，参与切割的残基位于β-桶的深处，位于嵌入OM的ESPP区域。有趣的是，该结构表明，在Passenger结构域被切割和释放后，发生了两个离散的构象变化，一个增加了β-结构域的稳定性，另一个限制了进入桶状孔的途径。我们的结构不支持寡聚易位模型，而是一个单一的β-桶促进单个客体结构域到细胞外表面的易位模型。 目前，我们正在尝试解决ESPP裂解前构象的结构问题。几个客体结构域被转移到细胞外空间但没有被切割的突变体将被提纯，用于结晶试验。预切割结构将揭示切割机制的更多细节，并允许我们尝试基于结构的突变来测试所提出的乘客易位机制。