University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Corrensstr. 48, 48149 Münster, Germany.
University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Corrensstr. 48, 48149 Münster, Germany.
J Mol Biol. 2024 Oct 15;436(20):168749. doi: 10.1016/j.jmb.2024.168749. Epub 2024 Aug 20.
Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a β-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the β- and the β-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the β-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the β-strand with the β-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the β-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the β-signals of classical autotransporter proteins and outer membrane β-barrel proteins in general. We therefore propose that the β-strand of YeeJ acts as its β-signal and that the assembly of the YeeJ β-barrel is driven by its C-terminal β-strand, like in most other autotransporter proteins, despite its inverted domain organization.
细菌外膜蛋白通过β-桶状易位结构域呈现具有各种功能的载体结构域。YeeJ 是大肠杆菌 MG1655 的一种自转运蛋白。与大多数其他自转运蛋白不同，其载体结构域位于易位结构域的 C 末端。由于这种倒置的结构域组织，YeeJ 属于自转运蛋白的 Ve 型。为了研究 YeeJ 的组装，测量了异源 mCherry 载体结构域的荧光来定量其组装。基于与 YeeJ 相似的 119 个序列的 AlphaFold2 模型，生成了 Ve 型自转运蛋白的β-和β-链的序列保守性 logo。然后，分析了这些链中的突变对 YeeJ 组装的影响。β-链的 N 端芳香族氨基酸的突变不影响易位结构域的组装和载体结构域的显示。同样，用β-链交换β-链也不会损害自转运蛋白融合蛋白的组装。β-链的 C 端芳香族氨基酸的突变强烈削弱了 mCherry 载体结构域的表面显示。该氨基酸以前已被证明是经典自转运蛋白和一般外膜β-桶蛋白的β-信号的必需特征。因此，我们提出 YeeJ 的β-链作为其β-信号，并且 YeeJ β-桶的组装由其 C 末端的β-链驱动，就像大多数其他自转运蛋白一样，尽管其结构域组织倒置。
