Section of Molecular Microbiology, Department of Molecular Cell Biology, VU University, 1081 HV Amsterdam, The Netherlands.
J Mol Biol. 2011 Sep 30;412(4):553-67. doi: 10.1016/j.jmb.2011.07.035. Epub 2011 Jul 23.
Autotransporters (ATs) of Gram-negative bacteria contain an N-proximal passenger domain that is transported to the extracellular milieu and a C-terminal β-domain that inserts into the outer membrane (OM) in a β-barrel conformation. This β-domain facilitates translocation of the passenger domain across the OM and has long been considered to be the translocation pore. However, available crystal structures of β-domains show that the β-barrel pore is too narrow for the observed transport of folded elements within the passenger domains. ATs have recently been shown to interact with the β-barrel assembly machinery. These findings questioned a direct involvement of the β-domain in passenger translocation and suggested that it may only target the passenger to the β-barrel assembly machinery pore. To address the function of the β-domain in more detail, we have replaced the β-domain of the Escherichia coli AT hemoglobin protease by β-domains originating from other OM proteins. Furthermore, we have modified the diameter of the β-domain pore. The mutant proteins were analyzed for their capacity to insert into the OM and for surface display of the passenger. Our results show that efficient passenger secretion requires a specific β-domain that not only functions as a targeting device but also is directly involved in the translocation of the passenger to the cell surface.
革兰氏阴性菌的自转运蛋白（ATs）含有一个 N 端的过客域，该域被转运到细胞外环境中，而 C 端的 β 域以 β-桶的构象插入外膜（OM）。这个 β 域有助于过客域穿过 OM 的易位，长期以来一直被认为是易位孔。然而，现有的 β-域晶体结构表明，β-桶孔对于观察到的过客域中折叠元件的运输来说过于狭窄。最近已经表明，ATs 与 β-桶组装机制相互作用。这些发现质疑了 β-域在过客易位中的直接参与，并表明它可能仅将过客靶向到 β-桶组装机制孔。为了更详细地研究 β-域的功能，我们用来自其他 OM 蛋白的 β-域替换了大肠杆菌 AT 血红蛋白蛋白酶的 β-域。此外，我们还改变了 β-域孔的直径。分析了突变蛋白插入 OM 的能力以及过客的表面展示。我们的结果表明，有效的过客分泌需要一个特定的 β-域，该域不仅作为靶向装置起作用，而且还直接参与过客向细胞表面的易位。
