DOI: 10.5061/dryad.cfxpnvxgd
title: Dynamic basis of lipopolysaccharide export by LptB2FGC

description: 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.
description: 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-d<sub>8</sub> in a
 1.6 mm quartz ESR tube (Suprasil, Wilmad LabGlass) with a Bruker EN5107D2
 dielectric resonator. The phase memory time
 (<em>T<sub>M</sub></em>) measurements were
 performed with a 48 ns π/2–<em>t</em>–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<sub>p</sub>]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.
description: # Dynamic basis of lipopolysaccharide export by LptB2FGC: DEER/PELDOR data
 [https://doi.org/10.5061/dryad.cfxpnvxgd](https://doi.org/10.5061/dryad.cfxpnvxgd) ## Description of the data and file structure These data were recorded on detergent-solubilised or membrane-reconstituted LptB2FG or LptB2FGC complexes. Different double cysteine variants were labeled with MTSL and the interspin distances were determined using DEER/PELDOR spectroscopy. ### Files and variables #### File: eLife\_respository.zip **Description:** Primary DEER/PELDOR data for all the variants. ## Code/Software Files can be viewed and analysed using the DeerLab program - [https://jeschkelab.github.io/DeerLab/](https://jeschkelab.github.io/DeerLab/) or the Comparative DeerAnalyzer - [https://ethz.ch/content/dam/ethz/special-interest/chab/imps/epr-dam/documents/software/deer-15-on/CDAv2Installer_web.exe](https://ethz.ch/content/dam/ethz/special-interest/chab/imps/epr-dam/documents/software/deer-15-on/CDAv2Installer_web.exe)