Ieva Raffaele, Skillman Kristen M, Bernstein Harris D
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0538, USA.
Mol Microbiol. 2008 Jan;67(1):188-201. doi: 10.1111/j.1365-2958.2007.06048.x. Epub 2007 Nov 27.
Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'beta-domain' that forms a beta-barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the beta-domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the beta-domain pore. By examining the accessibility of tobacco etch virus protease sites and single-cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre-translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP beta-domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre-formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.
细菌自转运蛋白由一个通过未知机制转运到细胞外空间的N端“乘客结构域”和一个在外膜中形成β桶的C端“β结构域”组成。最近的研究表明，完全组装好的自转运蛋白具有一种不寻常的结构，其中一个小的乘客结构域片段穿过由β结构域形成的孔。然而，尚不清楚这种构象是在乘客结构域易位之前形成的，还是由乘客结构域通过β结构域孔的易位导致的。通过检测烟草蚀纹病毒蛋白酶切割位点和大肠杆菌O157:H7自转运蛋白EspP的乘客结构域中单个半胱氨酸残基在蛋白质生物合成不同阶段的可及性，我们鉴定出一种新型的转运前中间体，其拓扑结构类似于完全组装好的蛋白质。在细胞分级分离实验中，这种中间体在周质中被分离出来。数据强烈表明，EspP的β结构域和一个嵌入的多肽片段作为一个单一的预形成单元整合到外膜中。数据还提供了间接证据，表明至少一些外膜蛋白在膜整合之前就获得了相当多的三级结构。
