Department of Chemistry, Bar Ilan University, Ramat Gan 52900, Israel.
Departments of Life Sciences, National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
Biochim Biophys Acta Biomembr. 2019 Dec 1;1861(12):183054. doi: 10.1016/j.bbamem.2019.183054. Epub 2019 Sep 2.
BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29-121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical 'tip motif'. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1-287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.
BteA 是一种 69kDa 的细胞毒性蛋白，是经典博德特氏菌（百日咳和相关哺乳动物呼吸道疾病的病原体）中 III 型分泌系统（T3SS）效应物。与其他介导细胞毒性的效应物一样，BteA 利用其多功能的 N 端结构域靶向宿主膜中的富含磷脂酰肌醇（PI）的微域。尽管它们的结构相似，但 T3SS 效应物表现出可变的膜相互作用模式，目前仅有限的结构信息可用于 BteA 膜靶向结构域及其功能的分子机制。在这里，我们采用结构方法的协同组合，确定了该功能结构域的结构，并揭示了介导其与膜相互作用的关键分子决定因素。BteA 的 29-121 个残基形成一个拉长的四螺旋束，与两个较短的垂直螺旋紧密结合，其中第二个螺旋在关键的“尖端基序”中覆盖该结构域。BteA 螺旋束之前的一个柔性区域包含结合其同源伴侣 BtcA 所需的特征性β基序。我们表明 BteA 靶向含有 PI(4,5)P 的脂蛋白纳米盘，并通过跨越其前两个螺旋的广泛正电荷表面与可溶性 PI(4,5)P 类似物结合，并且这种相互作用对于 PI(3,5)P 较弱，对于 PI(4)P 则被消除。我们通过使用小角 X 射线散射（SAXS）观察 BteA 诱导的含有 PI(4,5)P 的磷脂双层结构的变化，证实了这种膜靶向模型。我们还通过使用量热法、荧光法和 SAXS 方法扩展了这些结果到更大的 BteA 结构域（残基 1-287），证实了其与双层的相互作用。这种对 BteA 介导的膜靶向结构基础的新观点是全面了解博德特氏菌细胞毒性以及广泛的病原体与各自宿主相互作用的重要一步。
