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。ETEC菌株的EM研究观察到两种不同形式的CFA/I菌毛，一种是螺旋状的细丝，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。这也表明CfaC可能存在脯氨酸异构酶活性。我们的研究结果证实了在没有明显的一级序列相似性的情况下，第5类毛被和第1类毛之间存在很强的结构相似性。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。我们的模型支持细菌利用抗原变异来逃避宿主免疫反应的观点，因为占据CfaB和相关5类pilins的预测表面暴露面的残基比pilins蛋白的其余部分显示出更高的遗传序列变异性。R181位于CfaEad远端的上表面，先前已知对结合很重要，位于带正电的凹区中，周围有一簇残基，这些残基在5类纤维粘附素中高度保守，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。最近，我们还确定了CfaA的晶体结构，CfaA是组装CFA/I薄膜所必需的伴侣成分。

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. EM studies of ETEC strains observed two-distinct forms of CFA/I pili, a helical filament 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. This also suggested the existence of a possible prolinyl isomerase activity for CfaC. Our findings affirm the strong structural similarities seen between Class 5 fimbriae and Class 1 pili 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. Our model supports the notion that bacteria use antigenic variation to evade host immune response 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. 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. Recently, we have also determined the crystal structure of CfaA, the chaperone component that is essential for assembly of CFA/I fimbriae.