Molecular basis for cell surface architecture and evolution of bacterial flagellin by structure and unction analysis of the protein

同化藻酸盐的鞘氨醇单胞菌。菌株A1通过细胞表面褶皱分子的重排形成一个口状凹坑，并通过凹坑结合一种大分子海藻酸盐，该凹坑作为胞外多糖的集中器。在藻酸盐存在下，P5(40 KDa)和P6(31 KDa)这两个蛋白在细胞表面诱导表达，表明它们是形成凹坑的原因。本研究分析了P5和P6的结构和功能。一种p6干扰物在藻酸盐介质中表现出显著的生长抑制作用，p5和p6的双重破坏导致生长失败，表明这些鞭毛蛋白同源物可能是细胞存活所必需的。P6-干扰物的细胞表面与野生菌株Al的不同之处在于凹坑的形成是不完整的，细胞表面结构从褶皱的…改变。网络的结构更有条理。表面等离子体共振生物传感器分析表明，这两种蛋白都能高亲和力地结合海藻酸盐(解离常数Kd〓~nM)。根据这些结果，定位于细胞表面的p5和p6可以被认为是外部藻酸的受体。p5由N-/C-末端α(α1+α2)结构域和中央B结构域组成。由于缺失α_1结构域的P5突变体不具有与藻酸盐结合的能力，因此α_1结构域是与藻酸盐结合所必需的。与沙门氏菌鞭毛蛋白P5相比，α(α1+α2)结构域结构相似，表明细菌鞭毛蛋白中常见的藻酸盐结合能力是由于结构域保守所致。另一方面，P5的β结构域与位于基板和尾部纤维之间的铰链区的T4噬菌体蛋白Gp11的指状域结构相似。指状区与基板蛋白GP10的结合对于尾部纤维在细菌细胞表面的附着是重要的。Finger结构域与另一个蛋白质显示出亲和力，表明P5β结构域作为锚与菌株Al细胞表面分子相互作用，并有助于藻酸盐结合结构域与外部环境的定向。较少

Alginate-assimilating Sphingomonas sp. strain Al forms a mouth-like pit through the rearrangement of cell surface pleat molecules and incorporates a macromolecule alginate through the pit The pit functions as a concentrator for extracellular polysaccharides. Two proteins, p5 (40 kDa) and p6 (31 kDa), are inducibly expressed on the cell surface in the presence of alginate, suggesting that they are responsible for the formation of the pit In this study, the structure and function of p5 and p6 have been analyzed.Both of p5 and p6 are similar to bacterial flagellins, although strain Al forms no flagella. A p6-disruptant shows significant growth retardation in the alginate medium, and double disruption of p5 and p6 results in growth failure, indicating that these flagellin homologs might be essential for cell viability. The cell surface of the p6-disruptant differs from that of wild-strain Al in that the formation of the pit is incomplete and the cell surface structure changes from a pleat … More structure to a network one. Surface plasmon resonance biosensor analysis indicated that both proteins specifically bind alginate with high affinity (dissociation constant Kd〓〜 nM). Based on these results, p5 and p6 localized on the cell surface can be identified as a receptor for external alginate.p5 consists of N-/C-terminal α (α1 + α2) domain and central B domain. Since p5 mutant lacking α_1 domain showed no alginate-binding ability, α_1 domain is essential to bind to alginate. In comparison of p5 with the Salmonella flagellated flagellin, the structure of α (α1 + α2) domain is similar each other, indicating that the alginate-binding ability commonly observed in bacterial flagellins is due to a domain conserved. On the other hand, p5 β domain is structurally similar to the finger domain of T4 phage protein gp11 located in the hinge region between the base plate and tail fiber. The binding of the finger domain to the base plate protein gp10 is important for the attachment of the tail fiber on the bacterial cell surface. The finger domain shows an affinity with the other protein, suggesting that p5 β domain functions as an anchor interacting with the strain Al cell surface molecules and contributes to the orientation of alginate-binding a domain to the external milieu. Less