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

CfaE的一个变体，给予体链互补CfaE(DscCfaE)，含有一个C-末端的发夹接头，紧随其后的是主要菌毛亚基CFAB的N-末端的前19个氨基酸残基。DscCfaE蛋白易于结晶并确定其结构。DscCfaE分子由两个大小大致相等的结构域组成。CfaE的N-末端结构域被称为粘附素结构域(CfaEad)，表示在残基A23到D200的结构中。它由一个反平行的测试页(表1)和一个混合的测试页(表2)组成。C-末端结构域紧跟在短的三个残基连接子(K201-G202-N203)之后，介导粘附亚单位与菌毛主体的附着。因此，它被称为Pilin结构域(CfaEpd)。Pilin结构域折叠成一个β-三明治，其拓扑结构使人想起粘附素结构域。CfaEad和CfaEpdβ-结构都显示出类似于v型Ig折叠的拓扑结构，其中有9条β-链。为了了解主亚基和次亚基是如何组装成CFA/I菌毛的，我们进一步设计了供体链互补的CfaEB复合体(DscCfaEB)，并在大肠杆菌中进行了表达。对重组dscCfaEB蛋白进行了纯化和结晶。测定了CfaEB络合物的晶体结构，不仅提供了关于主亚单位CFAB的结构信息，而且提供了关于主亚单位和次亚单位之间连接的几何形状的结构信息。除了CfaEB复合体外，我们还测定了主亚基复合体CfaBB和CfaBBB的晶体结构，为构建与纯化的CFA/I菌毛的EM重建一致的CFA/I菌毛模型提供了基础。对ETEC菌株的EM研究观察到两种不同形式的CFA/I菌毛，一种是螺旋丝，另一种是延伸的、未缠绕的构象。模拟和确证突变数据表明，在CFA/I菌毛的螺旋形式和扩展形式之间的转换过程中涉及到脯氨酸异构化。这也表明CfaC可能存在一种脯氨基异构酶活性。我们的发现证实了在没有显著的初级序列相似性的情况下，5类菌毛和1类菌毛之间存在着强烈的结构相似性。他们还指出，菌毛类型之间的形态和生化差异，无论其类别如何，都提供了结构特化，促进了每种细菌致病类型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类毛孔蛋白表面的残基显示出比毛孔蛋白其余部分更高的遗传序列变异性。R181位于CfaEad的上表面，位于CfaEpd的远端，先前已知对结合很重要，它位于正电荷凹陷中，被一簇在5类菌毛粘附素中高度保守的残基包围，包括来自三个不同环(即B‘-C、D’-E和F-G环)的残基。因此，这个口袋似乎是一个合适的位置，一个带负电荷的唾液酸化受体可能会与之结合。为了证实该结构域的作用，将与R181相邻的R67突变为丙氨酸(dscCfaE/R67A)并进行纯化。吸附在微珠上的dscCfaE/R67A不能凝集人红细胞，这与我们之前对dscCfaE/R181A突变体的发现类似。这些结果暗示这两个残基锚定的口袋可能是受体结合结构域。为了确定R181附近单个残基在血凝中的作用，我们在载体pMAM2的CfaE中引入了位点特异性突变，该突变编码CFA/I的所有成分，并通过CfaE的单位点突变来指导突变菌毛的表面表达。介绍了12个涉及5类ETEC菌毛粘附素不变(完全保守)或亚类特异性残基的突变。所有带正电的残基(R181、R182、R67)都是受体结合所必需的，并聚集在一起形成一个带正电的中心。结合口袋的正电荷中心被一条亚类特异性残基带包围。这些残基的突变显示出与红细胞相互作用的改变，其中几个显示出对人类A型或牛红细胞物种的歧视行为。最近，我们还测定了CFAA的晶体结构，CFAA是组装CFA/I菌毛所必需的伴侣成分。

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.