Wollmann Petra, Zeth Kornelius, Lupas Andrei N, Linke Dirk
Max Planck Institute for Biochemistry, Department Membrane Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany.
Int J Biol Macromol. 2006 Aug 15;39(1-3):3-9. doi: 10.1016/j.ijbiomac.2005.11.009. Epub 2006 Jan 6.
Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA, represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-rod-anchor architecture. Whereas their head and rod domains may be of heterologous origin, their anchor domains are homologous and display the properties of autotransporters. Conflicting topology models exist for these membrane anchors. Here, we describe the expression and purification of the membrane anchor of YadA from Yersinia enterocolitica for structural biology experiments. We expressed YadA-M in the Escherichia coli outer membrane. After solubilization and purification, it is a trimer of extreme stability. Using protein FTIR and secondary structure analysis, we show that the anchor is a beta-barrel, but contains a helical part at its N-terminus. We have crystallized the protein under various conditions and present X-ray data to 3.8 A resolution.
非菌毛黏附素，如耶尔森菌属的YadA、莫拉克斯菌属的UspA1和A2、嗜血杆菌属的Hia和Hsf，或巴尔通体属的BadA，是致病性变形菌黏附宿主的一类重要分子。它们形成具有头-杆-锚结构的三聚体表面结构。虽然它们的头部和杆部结构域可能来自异源，但它们的锚定结构域是同源的，并具有自转运蛋白的特性。关于这些膜锚定结构存在相互矛盾的拓扑模型。在这里，我们描述了用于结构生物学实验的小肠结肠炎耶尔森菌YadA膜锚定结构的表达和纯化。我们在大肠杆菌外膜中表达了YadA-M。经过溶解和纯化后，它是一种具有极高稳定性的三聚体。通过蛋白质傅里叶变换红外光谱和二级结构分析，我们表明该锚定结构是一个β桶，但在其N端含有一个螺旋部分。我们在各种条件下使该蛋白质结晶，并给出了分辨率为3.8埃的X射线数据。
