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.

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 (T_M) 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][x_p]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.