Structural studies of fimbriae of enterotoxigenic E. coli (ETEC)

CfaE的一种变体，供体链补充CfaE (dscCfaE)，包含一个c端发夹连接体，随后是主要毛状亚基CfaB的n端衍生的前19个氨基酸残基，被纯化至均匀性。dscfae蛋白易结晶并测定其结构。dscfae分子由两个大小大致相等的结构域组成。CfaE的n端结构域被称为粘附素结构域（CfaEad），在残基A23至D200的结构中表示。它由一个反平行β -片（表1）和一个混合β -片（表2）组成。c端结构域紧跟着短三残基连接体（K201-G202-N203），介导粘附亚基附着到菌膜主体上。因此，它被称为pilin结构域（CfaEpd）。pilin结构域折叠成β -三明治，其拓扑结构使人联想到粘附素结构域。CfaEad和CfaEpd的β -结构都显示出类似于具有9条β链的v型Ig折叠的拓扑结构。为了了解主要亚基和次要亚基是如何组装成CFA/I菌膜的，我们进一步设计了在大肠杆菌中表达的供体链互补CfaEB复合物（dscCfaEB）结构。对重组dscCfaEB蛋白进行纯化和结晶。测定了CfaEB配合物的晶体结构，不仅提供了主要亚基CfaB的结构信息，还提供了主要亚基和次要亚基之间连接的几何形状信息。除了CfaEB配合物外，我们还确定了主要亚基配合物CfaBB和CfaBBB的晶体结构，为构建与纯化CFA/I菌毛的EM重建相一致的CFA/I菌毛模型提供了基础。R181位于CfaEad的上表面，与CfaEpd远端，先前已知对结合很重要，位于带正电的凹区中，被一簇在5类毛粘附素中高度保守的残基包围，包括来自三个不同环的残基（即B-C， D-E和F-G环）。因此，这个口袋似乎是一个带负电荷的唾液化受体可能结合的合适位置。为了确认该结构域的作用，我们将R181相邻的R67突变为丙氨酸（dscCfaE/R67A）并进行纯化。珠状吸附的dscCfaE/R67A不能凝集人红细胞，这与我们之前对dscCfaE/R181A突变体的研究结果相似。这些结果暗示由这两个残基锚定的口袋作为假定的受体结合域。为了确定R181附近的单个残基在血凝中的作用，我们在质粒pMAM2中引入了位点特异性突变到CfaE中，该质粒编码CFA/I的所有组分，并指导CfaE单位点突变的突变菌的表面表达。介绍了12个这样的突变，这些突变涉及的残基要么是不变的（完全保守的），要么是5类ETEC毛粘连蛋白的亚类特异性。所有带正电的残基（R181, R182, R67）都是受体结合和聚集在一起形成带正电中心所必需的。带正电的结合袋中心被一带亚类特异性残基所包围。这些残基的突变显示出与红细胞相互作用的改变，其中一些对人类a型或牛型红细胞表现出歧视性行为。我们首次阐明了来自定植因子抗原I （CFA/I）菌毛的ETEC主要毛蛋白亚基CfaB的原子结构。这些数据用于构建两种形态的CFA/I纤维的模型，这两种形态都是在体内观察到的，一种是螺旋状的纤维，它通常是组装成螺旋状的，另一种是延伸的、未缠绕的构象。模型和确证的突变数据表明，脯氨酸异构化参与了CFA/I螺旋和扩展形式之间的转化。我们的研究结果证实了5类菌毛（来自主要引起胃肠道疾病的细菌）和1类菌毛（来自引起泌尿系统、呼吸道和其他感染的细菌）在没有显著一级序列相似性的情况下具有很强的结构相似性。他们还认为，不同种类的菌毛类型之间的形态和生化差异提供了结构专门化，促进了每种细菌病原菌在其首选宿主微环境中的生存。最后，我们提出了细菌利用抗原变异来逃避宿主免疫反应的结构证据，因为占据CfaB和相关5类pilins的预测表面暴露面的残基比其余的pilins蛋白显示出更高的遗传序列变异性。最近，我们还确定了CfaA的晶体结构，这是对CFA/I晶膜组装至关重要的伴侣成分。由伴侣-引导通路（CUP）组装的菌毛需要质周伴侣来协助亚基折叠，维持其稳定性，并护送它们到达生物组装位点。到目前为止，根据其F1和G1 β -链之间的亚基相互作用环的长短，CUP伴侣蛋白被分为FGS和FGL两个家族。CfaA是产肠毒素大肠杆菌（ETEC）菌毛定植因子抗原I （CFA/I）组装的伴侣，ETEC是旅行者和幼儿腹泻的原因。本研究通过对CfaA的晶体结构和突变分析，揭示了其独特的结构和功能特征，从而提出CfaA及其密切相关的伴侣蛋白是一个独立的家族。由该伴侣蛋白家族特有区域的突变引起的表型变化提供了对其功能的深入了解。此外，区分CfaA与FGL和FGS伴侣家族的那些区域似乎影响其同源亚基和引导蛋白在菌毛组装过程中的相互作用。尽管对毛膜组装的能量学缺乏详细的了解，但人们认为，在亚基重折叠期间，质周伴侣蛋白与毛蛋白亚基形成二元配合物，从而起到了动力学和能量陷阱的作用，从而阻止了亚基的聚集，并随后推动它们的聚合成有序的螺旋组装。CFA/I菌群是产肠毒素大肠杆菌第5类菌群的原型，它的组装是由一种新分类的周质伴蛋白CfaA辅助的。在这项工作中，我们表明，与其他细菌菌毛的毛蛋白亚基不同，CfaB， CFA/I的主要毛蛋白亚基，在没有伴侣的情况下是稳定的，并且能够在体内和体外自发地折叠和聚合成环三聚体。我们进一步表明，伴侣蛋白CfaA能够动态捕获CfaB，形成亚稳态复合物。突变对CfaA和CfaB异源二聚体的稳定导致了复合物的晶体结构，揭示了CfaA和CfaB通过供体链互补和间隙介导的锚定进行独特的相互作用。突变表明，供体链互补控制着伴侣-亚基复合物的稳定性，而亚基c端的间隙介导的锚定似乎有助于亚基的再折叠。令人惊讶的是，伴侣-亚基复合物的过度稳定导致了纤维膜组装的延迟，而复合物的不稳定导致了没有纤维膜的组装。因此，CfaA的主要功能是作为一个精细的动力学陷阱，以避免通路外亚基自聚合，从而产生能量有利的三聚体，并可以作为CFA/I菌毛组装的驱动力，代表了5类菌毛组装所特有的能量景观。

A variant of CfaE, donor strand complemented CfaE (dscCfaE), containing a C-terminal hairpin linker followed by the first 19 amino acid residues derived from the N-terminus of the major fimbrial subunit CfaB was purified to homogeneity. The dscCfaE protein was readily crystallized and the structure was determined. The dscCfaE molecule consists of two domains of roughly equal size. The N-terminal domain of CfaE is referred to as the adhesin domain (CfaEad) and is represented in the structure from residues A23 to D200. It consists of one anti-parallel beta-sheet (Sheet 1) and one mixed beta-sheet (Sheet 2). The C-terminal domain immediately follows the short three-residue linker (K201-G202-N203) and mediates attachment of the adhesive subunit to the main body of the fimbria. It is therefore termed the pilin domain (CfaEpd). The pilin domain folds into a beta-sandwich with a topology reminiscent of the adhesin domain. Both CfaEad and CfaEpd beta-structures display a topology that resembles the v-type Ig fold with nine beta-strands. In order to understand how the major and minor subunits are assembled into a CFA/I fimbria, we further engineered the donor strand complemented CfaEB complex (dscCfaEB) construct, which was expressed in E. coli. The recombinant dscCfaEB protein was purified and crystallized. The crystal structure of the CfaEB complex was determined, providing structural information on not only the major subunit CfaB, but also the geometry of the connection between the major and minor subunit. In addition to the CfaEB complex, we also determined crystal structures for the major-major subunit complexes CfaBB and CfaBBB, providing a basis for constructing a model of CFA/I pilus consistent with EM reconstructions of purified CFA/I pilus. Located at the upper surface of CfaEad distal to the CfaEpd, R181, which was previously known to be important for binding, is found in a positively charged depression and surrounded by a cluster of residues that are highly conserved in the Class 5 fimbrial adhesins, including residues from three different loops (i.e., B-C, D-E, and F-G loops). This pocket thus appears to be a suitable location to which a negatively charged sialylated receptor might bind. To confirm the role of this domain, R67, which is adjacent to R181, was mutated to alanine (dscCfaE/R67A) and purified. Bead-adsorbed dscCfaE/R67A failed to agglutinate human erythrocytes, similar to our previous findings for the dscCfaE/R181A mutant. These results implicate the pocket anchored by these two residues as the putative receptor-binding domain. To determine the role in hemagglutination of individual residues in the neighborhood of R181, we introduced site-specific mutations into CfaE in the plasmid pMAM2, which encodes all components of the CFA/I and directs surface expression of mutant fimbriae with single site mutations of CfaE. Twelve such mutations involving residues that are either invariant (fully conserved) or are subclass-specific for Class 5 ETEC fimbrial adhesins were introduced. All positively charged residues (R181, R182, R67) are absolutely required for receptor binding and cluster together to form a positively charged center. The positively charged center of the binding pocket is surrounded by a band of subclass-specific residues. Mutations of those residues display altered interactions with red cells and several show discriminatory behavior to either human type-A or bovine red cell species. We elucidated, for the first time, atomic structures of an ETEC major pilin subunit, CfaB from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for two morphological forms of CFA/I fimbriae that are both observed in vivo, the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between the helical and extended forms of CFA/I fimbriae. Our findings affirm the strong structural similarities seen between Class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and Class 1 pili (from bacteria that cause urinary, respiratory and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Lastly, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related Class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein. More recently, we have also determined the crystal structure of CfaA, the chaperone component that is essential for assembly of CFA/I fimbriae. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 beta-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with mutational analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provides insight into their function. Moreover, those regions that distinguish CfaA from the FGL and FGS chaperone families appear to influence interactions with their cognate subunits and usher proteins during pilus assembly. Despite the lack of detailed understanding with respect to the energetics of fimbria assembly, periplasmic chaperones are thought to serve both as kinetic and energy traps by forming binary complexes with pilin subunits during subunit refolding, which prevents subunits from aggregating and subsequently drives their polymerization into ordered helical assemblies. The assembly of CFA/I fimbriae, an archetype in the family of class 5 fimbriae from enterotoxigenic E. coli, is assisted by the periplasmic chaperone CfaA, which belongs to a newly classified group of periplasmic chaperones. In this work, we show that unlike pilin subunits of other bacterial fimbriae, CfaB, the major pilin subunits of CFA/I, are stable in the absence of the chaperone and are able to spontaneously refold and polymerize into cyclic trimers both in vivo and in vitro. We further show that chaperone CfaA is able to kinetically trap CfaB to form a metastable complex. Stabilization of the CfaA and CfaB heterodimer by mutations led to the crystal structure of the complex, revealing distinctive interactions between CfaA and CfaB through donor-strand complementation and cleft-mediated anchorage. Mutagenesis indicated that donor-strand complementation controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus appears to assist in subunit refolding. Surprisingly, over-stabilization of the chaperon-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA functions predominantly as a kinetic trap fine-tuned to avoid off-pathway subunit self-polymerization, which results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly.