Aoki Sota, Yoshimoto Shogo, Ishikawa Masahito, Linke Dirk, Lupas Andrei, Hori Katsutoshi
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Aichi 464-8603, Japan.
Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
J Biosci Bioeng. 2020 Apr;129(4):412-417. doi: 10.1016/j.jbiosc.2019.09.022. Epub 2019 Oct 22.
AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.
AtaA是不动杆菌属菌株Tol 5的一种三聚体自转运黏附素，对非生物表面表现出非特异性的高黏附性。为了鉴定AtaA的功能结构域，需要精确设计结构域缺失突变体，以免引起不必要的结构畸变。在此，我们设计并构建了三种类型的AtaA突变体，它们缺失了相同的结构域FGG1。第一种突变体的设计旨在保留夹在缺失区域两侧的卷曲螺旋段中疏水残基的周期性。缺失后，该蛋白正确地展示在细胞表面，并且具有与野生型相同的黏附功能。透射电子显微镜（TEM）成像和圆二色性（CD）光谱表明，其分离的乘客结构域具有与AtaA野生型相同的纤维结构。相比之下，一种设计用于破坏缺失位点处卷曲螺旋周期性的突变体未能到达细胞表面。尽管设计为在卷曲螺旋之间具有柔性连接体的突变体发生了分泌，但细胞的黏附性降低。此外，突变体纤维的TEM成像显示纤维尖端弯曲，并且其CD光谱的变化表明二级结构含量降低。因此，我们成功地在精确缺失一个结构域后，将巨大的同源三聚体纤维结构AtaA天然展示在细胞表面，通过保留其卷曲螺旋周期性维持了正确的折叠状态和黏附功能。这种策略使我们能够构建各种AtaA结构域缺失突变体，而不会产生结构畸变，从而实现完整的功能图谱绘制。
