Nesper Jutta, Hill Chris M D, Paiment Anne, Harauz George, Beis Konstantinos, Naismith James H, Whitfield Chris
Department of Microbiology, University of Guelph, Guelph, Ontario N1G2W1, Canada.
J Biol Chem. 2003 Dec 12;278(50):49763-72. doi: 10.1074/jbc.M308775200. Epub 2003 Sep 30.
The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57-66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. WzaHis6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of WzaHis6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the WzaHis6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.
荚膜多糖（CPS）组装的后期步骤及其向细菌细胞表面的转运尚不清楚。先前已证明Wza蛋白是在大肠杆菌血清型K30表面形成原型1型荚膜结构所必需的（Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57 - 66）。Wza是一种保守的外膜脂蛋白，形成采用环状结构的多聚体，并且多项证据表明这些结构在荚膜聚合物跨外膜输出中起作用。Wza以天然形式和带有C端六聚组氨酸标签的形式进行纯化。WzaHis6被酰化且在荚膜组装中具有功能，尽管与天然Wza蛋白相比其效率略有降低。将纯化的多聚体重新组装到脂质中后获得了WzaHis6的有序二维晶体。对负染晶体进行电子显微镜观察和傅里叶滤波显示平均外径为8.84 nm、平均中心腔直径为2.28 nm的环状多聚体。单颗粒分析得到了估计分辨率为3 nm的投影结构，支持WzaHis6包含八个相同亚基的结构。一种Wza的衍生物（Wza*），其中原始信号序列被OmpF的信号序列取代，表明Wza天然酰化的N端对于正常多聚体结构的形成及其进行CPS组装的能力至关重要，但对于将Wza靶向到外膜并非必需。在存在Wza*的情况下，CPS在周质中积累，但在细胞表面未检测到。完整细胞的化学交联表明形成了一种至少包含Wza和内膜酪氨酸自激酶Wzc的跨膜复合物。
