Dynamic basis of lipopolysaccharide export by LptB2FGC

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. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC 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 LptB2FG, 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.

Methods
DEER/PELDOR experiments were conducted on a Bruker Elexsys E580 Q-Band (34 GHz) pulsed ESR spectrometer equipped with an arbitrary waveform generator (SpinJet AWG, Bruker), a 50 W solid-state amplifier, a continuous-flow helium cryostat, and a temperature control system (Oxford Instruments). Measurements were carried out at 50 K using a 10 – 20 µL frozen sample containing 15 – 20% glycerol-d8 in a 1.6 mm quartz ESR tube (Suprasil, Wilmad LabGlass) with a Bruker EN5107D2 dielectric resonator. The phase memory time (TM) measurements were performed with a 48 ns π/2–t–π Gaussian pulse sequence with a two-step phase cycling after incrementing t in 4 ns steps. A dead-time free four-pulse sequence with a 16-step phase cycling (x[x][xp]x) was used for DEER measurements. A 38 ns Gaussian pump pulse (with a full width at half maximum (FWHM) of 16.1 ns) was employed, along with a 48 ns observer pulse (FWHM of 20.4 ns). The pump pulse was placed at the maximum of the echo-detected field-swept spectrum, and the observer pulses were set at 80 MHz lower. Deuterium modulations were averaged by progressively increasing the first interpulse delay by 16 ns over 8 steps.

脂多糖（Lipopolysaccharides, LPS）可赋予革兰氏阴性菌抵御包括抗生素在内的严苛环境胁迫的能力。脂多糖转运（Lipopolysaccharide transport, Lpt）复合物由7种蛋白（A至G）组成，负责跨细胞被膜转运脂多糖。LptB2FG是一类ATP结合盒转运体，能够将脂多糖转运至LptC。但LptB2FG如何将ATP结合与水解过程，与脂多糖向LptC的转运相偶联，目前仍不明确。本研究借助脉冲偶极电子自旋共振光谱学，在胶束和/或蛋白脂质体中观测到了LptB2FG与LptB2FGC的构象异质性。此外，我们通过激光诱导液珠离子解吸质谱法监测了脂多糖的结合与释放过程。在空载状态与钒酸盐捕获状态下，LptF的β-果冻卷结构域均可与LptG及LptC的β-果冻卷结构域稳定结合。胞质侧的ATP结合可别构调控周质侧LptF β-果冻卷结构域的选择性开放。在LptB2FG中，ATP结合会闭合核苷酸结合结构域，引发如结构观测到的第一侧向门塌陷；而作为脂多糖潜在进入位点的第二侧向门，则呈现出异质性构象。LptC的结合可将该门的柔性限制为两种构象，这两种构象大概率对应LptC的螺旋从跨膜结构域中释放或插入其中的状态。本研究结果揭示了LptC跨膜螺旋的动态行为对脂多糖进入门控的调控机制，而其β-果冻卷结构域则锚定在周质中。结合LptF β-果冻卷结构域依赖ATP的长程别构门控效应，这一机制可确保脂多糖跨周质膜的高效且单向转运。

实验方法
双电子-电子共振（DEER/PELDOR）实验在搭载了任意波形发生器（SpinJet AWG，布鲁克（Bruker））、50 W固态放大器、连续流氦低温恒温器以及温控系统（牛津仪器（Oxford Instruments））的布鲁克Elexsys E580 Q波段（34 GHz）脉冲ESR光谱仪上开展。实验于50 K下进行，样品体积为10–20 µL冷冻样品，其中含15–20%氘代甘油（glycerol-d8），置于1.6 mm石英ESR管（Suprasil，威纳德实验室玻璃（Wilmad LabGlass））中，使用布鲁克EN5107D2介质谐振腔。相位记忆时间（T_M）测量采用48 ns的π/2–t–π高斯脉冲序列，在以4 ns为步长递增t后，通过两步相位循环完成采集。DEER测量则采用带有16步相位循环（x[x][xp]x）的无死时间四脉冲序列。实验使用了38 ns的高斯泵浦脉冲（半高全宽（FWHM）为16.1 ns），以及48 ns的观测脉冲（FWHM为20.4 ns）。泵浦脉冲设置在回波检测场扫谱的峰值位置，观测脉冲则设置在比峰值低80 MHz的位置。通过在8个步长内逐步将首个脉冲间延迟增加16 ns，对氘调制信号进行平均处理。