One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS)1. The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface2. Of the seven Lpt proteins, the LptD–LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane3,4. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD–LptE from Shigella flexneri at 2.4 Å resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded β-barrel formed by LptD. Importantly, the secondary structures of the first two β-strands are distorted by two proline residues, weakening their interactions with neighbouring β-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD–LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.
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The authors thank Y. Shan for discussions, H. Wu, J. Deisenhofer, Y. Jiang, N. Huang, Z. Zhou, G. Li, Z. Liu and members of the Huang group for critically reading the manuscript. The diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF, China) and Beijing Synchrotron Radiation Facility of China (BSRF, China). This work was supported by grants from the Ministry of Science and Technology (2012CB917302 and 2013CB910603 to Y.H.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB080203 to Y.H. and X.C.Z.) and the National Natural Science Foundation of China (31170698 to Y.H.).
The authors declare no competing financial interests.
Extended data figures and tables
a, Chemical structure of LPS. LPS is composed of three modules: lipid A, a core oligosaccharide and a highly variable O-antigen constituted of repeating oligosaccharide units. The core is covalently linked to lipid A and can be further divided into inner and outer core. The inner core contains at least one residue of 3-deoxy-d-manno-oct-2 ulosonic acid (Kdo) linking to lipid A. b, Ra LPS is about 32 Å in height and 28 Å × 12 Å in the other two dimensions. The size of Ra LPS is based on the crystal structure of the TLR4/MD-2/Ra LPS complex (PDB ID 3FXI).
Extended Data Figure 2 Sequence alignment of LptD proteins from five representative Gram-negative bacterial species.
Secondary structures of LptD from S. flexneri (SfLptD) are labelled at the top of the sequence alignment. Strands that consist of the β-barrel of LptD are numbered. Residues of the two distorted β-strands (β1 and β2), L4 and L8 are boxed. Identical hydrophobic residues, positively-charged residues and negatively-charged residues are highlighted in yellow, blue and red, respectively. Three proline residues Pro 231, Pro 246 and Pro 261 in SfLptD are conserved in different species. SfLptD, StLptD, PaLptD, KpLptD and AbLptD represent LtpD sequences from S. flexneri, Salmonella enteric Typhimurium str. LT2, P. aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, respectively.
Extended Data Figure 3 Two pairs of disulphide bonds are important for the stability and folding of LptD.
a, Non-reducing SDS–PAGE showing the purified LptD mutant proteins containing different Cys-to-Ser mutations. CCCC indicates that amino acids present at positions 31, 173, 724 and 725 in LptD are cysteine residues. LptD with mutation to Ser at different positions in the LptD sequence are co-expressed with LptE–His. b, Reducing SDS–PAGE showing LptD mutant proteins containing different Cys-to-Ser mutations. Samples were reduced by addition of 10 mM β-mercaptoethanol in the SDS–PAGE loading dye. All samples were boiled for 10 min at 95 °C. The degradation bands that appear below wild-type LptD were confirmed to be LptD fragments by mass spectrometry. All data shown are representative of at least three independent experiments.
The three sequences show very low sequence identity, but have similar secondary structures and folds. The three LPS-binding residues in LptA and LptD_NT are boxed (‘×’ indicates residues that are invisible in the crystal structure).
a, Electrostatic surface of the LptD–LptE complex (side view). The hydrophobic girdle indicates the membrane-bound region, and this was verified by the presence of the expected aromatic belts. b, c, Periplasmic view of the interior surface properties of LptD–LptE complex (b) and that of LptD–LptE complex with LptD_NT domain removed (c). The interior surface of the complex is fairly hydrophilic with evenly distributed hydrophobic and hydrophilic residues lining inside.
a–c, Superposition of SfLptE with NmLptE (a), NeLptE (b) and SoLptE (c). d, Structure-based sequence alignment of SfLptE with NeLptE, NmLptE and SoLptE. SfLptD, NmLptE, NeLptE and SoLptE are coloured in magenta, orange, cyan and green, respectively. SfLptE, NeLptE, NmLptE and SoLptE represent LptE orthologues from S. flexneri, N. europaea, N. meningitides and S. oneidensis, respectively. Secondary structure elements are labelled on the top. The primary structures of these LptE orthologues have very low sequence identity, but their secondary structure distribution and fold are quite similar.
Extended Data Figure 7 Electron density maps showing detergent molecules in the LptD_NT groove and residues at the LPS exit portal.
a, Electron density maps (2Fo − Fc) showing the presence of two detergent molecules in the groove of LptD_NT. b, Stereo view of electron density map (2Fo − Fc) of residues at the potential LPS exit portal. The contour level for maps is 1.0σ.
Extended Data Figure 8 Residue E242 of LptD forms a polar-interaction network within strands β1 and β2.
Residue E242 of LptD forms a salt-bridge interaction with K234 and a few hydrogen bonds with residues of strands β1 and β2.
LptD_NT relays LPS from LptA. Portion of LptD_NT of LptD likely resides in the outer membrane, and the LPS-conduit of the β-jellyroll is open to lipid phase (Fig. 1c). At the interface of LptD_NT domain and the β-barrel of LptD, the hydrophobic portion of LPS may directly enter into the lipid phase, whereas the hydrophilic moiety of LPS may enter into the barrel of LptD. The LPS molecules are inserted into the outer leaflet of the outer membrane upon dislocation of strands β1 and β2. LptE is a lipoprotein, and probably binds to the hydrophilic head groups of LPS in the process.
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Qiao, S., Luo, Q., Zhao, Y. et al. Structural basis for lipopolysaccharide insertion in the bacterial outer membrane. Nature 511, 108–111 (2014). https://doi.org/10.1038/nature13484
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