structural characterization of bacterial secretion channels

细菌分泌通道的结构特征

• 批准号: 8148751
• 负责人: Susan Buchanan
• 金额: $ 43.66万
• 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/8148751
• 关键词: Agreement; Architecture; Asparagine; Bacterial Adhesins; Biochemical; Bordetella pertussis; C-terminal; Caliber; Cell surface; Cells; Cleaved cell; Crystallization; Data; Energy Supply; Energy-Generating Resources; Enterobacteriaceae; Escherichia coli; Extracellular Space; Family; Gene Structure; Goals; Gram-Negative Bacteria; IgA-specific serine endopeptidase; Iron; Learning; Length; Lipids; Manuscripts; Mediating; Membrane; Membrane Proteins; Microbial Biofilms; Modeling; Molecular Conformation; Mutagenesis; N-terminal; Peptide Signal Sequences; Phase; Positioning Attribute; Process; Protein translocation; Proteins; Proteolysis; Publications; Publishing; Resolution; Rest; Serine Protease; Shigella flexneri; Side; Site; Solutions; Structure; Surface; Testing; Virulence; Work; Writing; base; beta barrel; cell motility; design; electron density; extracellular; improved; insight; monomer; mutant; periplasm; pertactin; research study

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. This model is considered unlikely for the majority of autotransporters. 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. Structure determination of a bacterial autotransporter To learn what happens to the β-domain after cleavage and release of the passenger domain, we determined the crystal structure of the native β-domain of EspP at 2.7 resolution in 2007. This is the first structure of an autotransporter β-domain post-cleavage, and it consists of a monomeric 12-stranded β-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 β-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 β-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 β-barrel facilitates the translocation of a single passenger domain to the extracellular surface. During the past two years, we have been working on the structure of EspP in its pre-cleavage conformation. Several mutants whose passenger domains are translocated to the extracellular space but are not cleaved have been cloned and expressed. By varying lengths of passenger domain in these constructs, we optimized crystallization conditions and have recently collected high resolution diffraction data on three EspP mutants that translocate the passenger but do not cleave it. We are currently building models into the electron density, but already it is clear that some aspects of the proposed cleavage mechanism will be revised through this work. We aim to solve another two or three mutant structures in the coming months and publish a comprehensive analysis of the pre-cleavage state, including cleavage mechanism and passenger release, in 2010. A pre-cleavage structure will also allow us to attempt structure-based mutagenesis to test between the proposed mechanisms of passenger translocation. As of August 2010, all four mutant structures have been fully refined and we are currently writing a manuscript for publication. The new structures give us additional insight into the cleavage mechanism, but we still do not know exactly how the passenger domain is tranlocated. To learn more about protein translocation, we initiated a new autotransporter project which focuses on proteins related to EspP but having a different gene organization. Whether these proteins have the same structure and function the same way, is currently unclear. To date we have 3.5 data on one such protein and we are working to improve the resolution and obtain phases for structure solution.

目前尚不清楚将大乘客域转位为小得多的β域的过程。 下面描述的是为解释乘客域易位而提出的三个模型。在一种模型中，过客结构域的 C 末端以易位后构象折叠到周质中的 β 结构域孔中。然后将预折叠的 β 结构域插入 OM 中，并通过可能涉及 Omp85（一种促进 OM 蛋白整合和组装的必需蛋白）的协调机制将过客结构域运输穿过 OM。该模型的一个优点是，它避免了在没有外部能源的情况下通过相对较小的桶孔易位一个或多个过客结构域的需要。第二个易位模型侧重于乘客域的不寻常结构，这些域似乎都包含 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。在此模型中，包含过客结构域 C 末端的短发夹位于桶孔内，其尖端突出到细胞外空间。然后，在发夹尖端折叠会将乘客结构域的其余部分拉过孔。第三个模型基于这样的观察：当 IgA 蛋白酶在大肠杆菌中产生时，其 β 结构域会形成多聚环状结构。中央空腔的直径约为 20 A，假设可传输多个乘客域。对于大多数自动运输车来说，这种模型被认为不太可能。 该项目的一个主要焦点是 EspP，一种与致腹泻大肠杆菌菌株相关的经典自转运蛋白。它属于 SPATE（肠杆菌科丝氨酸蛋白酶自转运蛋白）自转运蛋白家族，其乘客编码可切割各种哺乳动物蛋白质的丝氨酸蛋白酶。生化研究表明EspP是单体。一旦 EspP 过客结构域跨 OM 易位，它就会从两个天冬酰胺残基 (N1023/N1024) 之间的膜嵌入 β 结构域中裂解，并从细胞表面释放。 Asn/Asn 切割位点定义了 EspP 过客结构域（残基 56-1023）和 beta 结构域（残基 1024-1300）的边界。尽管过客结构域包含位于残基 261-264 的丝氨酸蛋白酶基序，但该基序不用于切割这两个结构域。 我们该项目的目标是解决一种或多种自转运蛋白裂解前和裂解后形式的晶体结构，并设计实验来探测跨外膜的底物易位。 细菌自转运蛋白的结构测定 为了了解裂解和释放过客结构域后 β 结构域发生了什么，我们于 2007 年以 2.7 分辨率测定了 EspP 天然 β 结构域的晶体结构。这是自转运蛋白 β 结构域裂解后的第一个结构，它由单体 12 链 β 桶组成，其 N 端 15 个残基从 周质侧。与最近提出的自催化裂解机制一致，参与裂解的残基位于 β-桶深处，即嵌入 OM 的 EspP 区域。有趣的是，该结构表明在乘客结构域裂解和释放后发生了两种离散的构象变化，一种改变了β结构域的稳定性，另一种则限制了进入桶孔。我们的结构不支持寡聚易位模型，而是支持单个β-桶促进单个过客结构域易位到细胞外表面的模型。 在过去的两年中，我们一直致力于EspP 裂解前构象的结构研究。一些载客结构域易位至细胞外空间但未被切割的突变体已被克隆和表达。通过改变这些构建体中过客结构域的长度，我们优化了结晶条件，并且最近收集了三个 EspP 突变体的高分辨率衍射数据，这些突变体使过客结构易位但不裂解它。我们目前正在建立电子密度模型，但很明显，所提出的裂解机制的某些方面将通过这项工作进行修改。我们的目标是在未来几个月内解决另外两个或三个突变结构，并于 2010 年发表对预裂解状态的全面分析，包括裂解机制和乘客释放。预裂解结构还将使我们能够尝试基于结构的诱变，以测试拟议的乘客易位机制。截至 2010 年 8 月，所有四种突变体结构均已完全完善，我们目前正在撰写手稿以供出版。新的结构让我们对裂解机制有了更多的了解，但我们仍然不知道乘客结构域是如何易位的。 为了了解有关蛋白质易位的更多信息，我们启动了一个新的自转运蛋白项目，该项目重点关注与 EspP 相关但具有不同基因组织的蛋白质。目前尚不清楚这些蛋白质是否具有相同的结构和相同的功能。迄今为止，我们已经拥有一种此类蛋白质的 3.5 个数据，我们正在努力提高分辨率并获得结构解决方案的相。