Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer,Journal of the American Chemical Society

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Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2020-07-02 , DOI: 10.1021/jacs.0c02362
Sejeong Lee ₁ , Nicholas G Housden ₂ , Sandra A Ionescu ₁ , Matthew H Zimmer ₁ , Renata Kaminska ₂ , Colin Kleanthous ₂ , Hagan Bayley ₁

Affiliation

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

中文翻译：

通过被动蛋白穿过 OmpF 孔蛋白三聚体的孔进行跨膜表位传递

革兰氏阴性细菌外膜 (OM) 中的三聚体孔蛋白是营养物质和抗生素被动扩散到细胞中的管道。孔蛋白在 OM 中形成的狭窄通道也被细菌素利用，通过一个鲜为人知的过程转移到细胞中。在这里，使用平面脂质双层中的单通道电记录结合蛋白质工程，我们解释了内切核酸酶细菌素 ColE9 的固有非结构化 N 端易位结构域 (IUTD) 通过 OmpF 被动导入穿过大肠杆菌 OM 的机制. 我们表明输入主要由 OmpF 孔与 IUTD 内的结合表位的弱相互作用主导，这些表位在方向上有偏向，导致超过 60 个氨基酸穿过 OmpF 的 2 个亚基。单分子动力学分析表明，IUTD 从 OmpF 的细胞外侧进入并易位到周质中，多肽链在此处旋转以进入相邻的亚基，只有其中一些分子从第二个亚基中弹出最终重新进入形成稳定的复合体之前。这些紧密相连的转运/结合过程产生了一个基本上不可逆的钩状组装，它限制了 OmpF 三聚体的两个亚基之间的输入激活肽表位。只有这些分子中的一些在最终重新进入形成稳定的复合体之前从第二个亚基中弹出。这些紧密相连的转运/结合过程产生了一个基本上不可逆的钩状组装，它限制了 OmpF 三聚体的两个亚基之间的输入激活肽表位。只有这些分子中的一些在最终重新进入形成稳定的复合体之前从第二个亚基中弹出。这些紧密相连的转运/结合过程产生了一个基本上不可逆的钩状组装，它限制了 OmpF 三聚体的两个亚基之间的输入激活肽表位。

更新日期：2020-07-02

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