Department of Physics, Freie Universität Berlin, Berlin, Germany.
Institute of Physical and Theoretical Chemistry, Goethe Universität Frankfurt, Frankfurt, Germany.
Elife. 2024 Oct 7;13:RP99338. doi: 10.7554/eLife.99338.
Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptBFG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptBFG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptBFG and LptBFGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptBFG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.
脂多糖 (LPS) 赋予革兰氏阴性细菌抵抗恶劣条件的能力，包括抗生素。脂多糖转运 (Lpt) 复合物由七种蛋白 (A-G) 组成，将 LPS 穿过细胞包膜输出。LptBFG 形成一个 ATP 结合盒转运蛋白，将 LPS 转移到 LptC。LptBFG 如何将 ATP 结合和水解与 LPS 转运到 LptC 偶联仍不清楚。我们使用脉冲偶极电子自旋共振光谱法在胶束和/或蛋白脂质体中观察到 LptBFG 和 LptBFGC 的构象异质性。此外，我们使用激光诱导液珠离子解吸质谱监测 LPS 的结合和释放。LptF 的 β- 果冻卷结构域在apo 和钒酸盐捕获状态下都与 LptG 和 LptC 的 β- 果冻卷结构域稳定相互作用。细胞质侧的 ATP 结合与选择性打开周质 LptF β- 果冻卷结构域的变构偶联。在 LptBFG 中，ATP 结合关闭核苷酸结合结构域，导致如结构中观察到的第一侧门的崩塌。然而，第二个侧门，形成 LPS 的假定入口位点，表现出异构构象。LptC 结合限制了此门的灵活性，使其呈现两种构象，可能代表 LptC 的螺旋要么从跨膜结构域中释放出来，要么插入其中。我们的结果揭示了 LPS 入口门通过 LptC 跨膜螺旋的动态行为进行调节，而其 β- 果冻卷结构域则锚定在周质中。这与长程 ATP 依赖性变构门控 LptF β- 果冻卷结构域相结合，可能确保 LPS 在周质中有效且单向的转运。
