Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen–deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the β-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the β-barrel and β-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.
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Data supporting the findings of this study are available from the corresponding authors upon reasonable request. HDX-MS raw data and the HDX data tables have been deposited to the ProteomeXchange Consortium via the PRIDE60 partner repository with the dataset identifier PXD021743.
The structural models employed in this study are accessible through the PDB (https://www.rcsb.org/) under accession nos. 5IV9 (KpLptDE), 5XO4 (thanatin), 2R1A (EcLptA), 6GD5 (EcLptA–thanatin complex) and 4Q35 (SfLptDE). Source data are provided with this paper.
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Research in C.V.R.’s laboratory is supported by a Medical Research Council program grant (MR/N020413/1), a European Research Council Advanced Grant ENABLE (695511) and a Wellcome Trust Senior Investigator Award (104633/Z/14/Z). Research in P.J.S.’s laboratory is funded by Wellcome (208361/Z/17/Z), the MRC (MR/S009213/1) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER and JADE, granted via the UK High-End Computing Consortium for Biomolecular Simulation (HECBioSim). P.J.S. acknowledges Athena at HPC Midlands+, funded by the EPSRC under grant no. EP/P020232/1 and the University of Warwick Scientific Computing Research Technology Platform for computational access. F.F. holds a SABS CDT studentship supported by the EPSRC and MRC (EP/L016044/1). J.B.S. is supported by the Oxford interdisciplinary DTP and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/M011224/1). We also thank S. Roy (University of Oxford) for his help with HDX-MS data analysis and rendering.
C.V.R. is a co-founder and consultant at OMass Therapeutics. The other authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
LptDE (5 μM, left) in 0.5% (w/v) C8E4 was mixed with 10 μM Re-LPS (a, orange adducts), POPG (b, red adducts) or CDL (c, magenta adducts). The protein-lipid mixture was then supplemented with increasing concentrations of n-nonyl-β-D-glucopyranoside (NG). Detergent addition decreases POPG and CDL binding, but has no pronounced effect on Re-LPS.
a, Mass spectra recorded for solutions of LptDE (5 μM) with increasing concentrations of thanatin (green adducts). b, Mass spectra of Re-LPS-bound LptDE supplemented with 1 μM thanatin (as described in Fig. 1d) with a focus on the 16+ charge state showing the presence of the 2:1 Re-LPS:thanatin complex, particularly at higher Re-LPS concentration. c, Quantification of the total amount of Re-LPS bound to LptDE in the absence or in the presence of thanatin (related to Fig. 1d). Error bars represent s.d. (n = 3). d, Thanatin-first nMS analysis: LptDE (5 μM) was initially mixed with thanatin and then supplemented with Re-LPS (10 μM).
68 peptides covering 70.5% of LptD sequence a, and 17 peptides covering 87.4% of LptE b, were identified following digestion with immobilized pepsin.
Mass spectra are shown for apo-LptDE, LPS, thanatin or LPS + thanatin states. Two binomial isotopic envelopes produced the best fit for the spectra yielding low- (green) and high-mass (light blue) populations. The sums of the two binomial distributions are shown in red. a, Peptide 66-92. b, Peptide 105-115. c, Peptide 116-19. d, Peptide 119-129. e, Peptide 130-141. f, Peptide 171-179.
Extended Data Fig. 5 Monoexponential fitting of the high-mass population in peptides showing EX1 kinetics.
Extracted relative abundances of high- mass populations plotted as a function of labeling time and fitted to a single exponential function with a variable intercept and plateau (to account for the lack of saturation seen in most peptides) to obtain the rate of translation from the low-mass population to the high-mass population (kop) and the half-life of the low mass population (t1/2). The apo state is indicated in purple, LPS-bound state in orange, thanatin-bound state in green, and LPS + thanatin state in maroon. Standard deviations are plotted as error bars (nbiological = 2; ntechnical = 3) but are in some instances too small to be visible. In the case of thanatin-bound state and LPS + thanatin state kinetic values could not be extracted because of poor fitting (the increase of high-mass population is incremental given the slow kinetics). In these cases, the dotted line is included only for visual guidance.
a, Average percentage occupancy of Re-LPS contacts made with the β-taco with a 4 Å cut-off. These contacts have been compared to those made by the detergent in the SfLptD structure (PDB ID: 4Q35; marked by an asterisk) and found that 90% of residues which interact with detergent. b, Top: Kernel density estimate (KDE) of the center of mass (COM) position between the five replicates indicate minimal diffusion over the course of the simulation. Middle: Partial density of Re-LPS across the β-taco demonstrating consistent and rigid contact across the domain, this implies that the Re-LPS bound within the β-taco does not have enough simulation time to sample the entire soluble domain in different binding poses, which would account for the discrepancy in deprotected coverage across this domain when compared to HDX-MS (Supplementary Fig. 15). Bottom: reference cartoon structure of β-taco to compare with above plot axes.
a, Average Re-LPS contacts mapped onto cartoon representations of LptDE. b, Residues interacting with the three Re-LPS lipids referenced in Fig. 2c (Re-LPS(1-3)) are shown in stick representation. c, C-terminal strand of the β-taco is in contact with the bilayer (Supplementary Fig. 15c). d, Ile186 and Phe187 are in transient contact with the Re-LPS lipids engaged with the lateral putative exit gate of the β-barrel. The open gate causes Re-LPS lipids (shown in yellow and pink) to be laterally pulled into the β-barrel sinking them into the inner leaflet of the and allowing the tails to interact with the C-terminal strand of the β-taco. This may suggest how Re-LPS gets laterally extruded into the OM during the translocation process.
Top: snapshot of the key lipid interactions between Re-LPS and the lateral gate peptide (232-251). Bottom: Plots measuring the distance from the geometric center of the peptide (232-251) to the geometric center of the lipids. Data plotted as the average of five repeats throughout the course of the simulation, standard deviation shaded gray.
a, Sequence alignment between KpLptD and EcLptA. Red color indicates conserved residues, yellow indicates highly similar residues. Circles indicate crucial residues for LptA-thanatin interaction. Blue color represents van-der-Waals interactions while green represents ionic bridges and H-bonds. b, Structure alignment of LptD (PDB ID: 5IV9) with apo-LptA (PDB ID: 2R1A). The RMSD is equal to 1.48 Å over 115 Cα.
a, RMSD of thanatin docked to the bottom (i) of the β-jellyroll in its closed conformation compared to being docked to the top (ii) where standard deviation is colored gray. b, Right: schematic showing the orientation of the β-taco vector drawn through Cα of residues Asp33 and Pro190, where x, y displacement is shown as red arrows. Right: x,y displacement of the β-taco vector mapped relative to the origin 0.0, 0.0 (defined as the original position of β-taco in the crystal structure) comparing the conformation space sampled of the apo-β-taco against when thanatin is docked to the (i) bottom and (ii) top of the β-taco.
Supplementary Figs. 1–18 and Tables 1–5.
Morph of LptDE complex. Morph between the closed conformation of the complex and the open conformation of the complex, with disulfide bonds between cysteines at the β-taco (Cys7, Cys149) and the β-barrel (Cys696, Cys697) shown as spheres.
HDX-MS data used to create deuterium uptake plots for apo and LPS-bound protein and to map the difference in relative deuterium uptake (LPS - apo) onto LptD structure.
HDX-MS data used to create deuterium uptake plots for apo, thantin-bound and LPS + thanatin bound protein and to map the difference in relative deuterium uptake (ligand bound - apo) onto LptD structure.
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Fiorentino, F., Sauer, J.B., Qiu, X. et al. Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide. Nat Chem Biol 17, 187–195 (2021). https://doi.org/10.1038/s41589-020-00694-2