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Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG

Nature Structural & Molecular Biology volume 24, pages 469–474 (2017)Cite this article

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Abstract

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

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Figure 1: Overall structure of the LPS transporter LptB2FG from P. aeruginosa.
Figure 2: Relative ATPase activities of LptB, LptB2FG and LptB2FGC.
Figure 3: Attributes of the V-shaped LPS-binding pocket in LptB2FG.
Figure 4: LptB dimer in LptB2FG and its interactions with LptF and LptG.
Figure 5: A working model for LPS extraction by LptB2FG.

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Acknowledgements

The authors thank Z. Liu, X. Zhang and H. Wu for valuable discussions and critically reading the manuscript. The authors also thank N. Ruiz (Department of Microbiology, The Ohio State University, Columbus, Ohio, USA) for generously providing the lptFG-depleted NR1113 E. coli strain. The diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF, China) and National Center for Protein Science Shanghai (NCPSS, China). This work was supported by grants from the National Natural Science Foundation of China (31625009 to Y.H.), the Ministry of Science and Technology (2016YFA0500404 and 2013CB910603 to Y.H.) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020302 to Y. H.).

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Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Qingshan Luo, Xu Yang, Shan Yu, Huigang Shi, Le Xiao, Chuanqi Sun, Xinzheng Zhang & Yihua Huang

  2. University of Chinese Academy of Sciences, Beijing, China

    Qingshan Luo, Huigang Shi, Le Xiao, Chuanqi Sun, Xinzheng Zhang & Yihua Huang

  3. Institute of Bio-analytical Chemistry, School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing, P.R.China

    Kun Wang & Min Zhou

  4. Discovery Biology, Albany Molecular Research Inc., Buffalo, New York, USA

    Guangyu Zhu

  5. National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Tingting Li & Dianfan Li

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  1. Qingshan Luo
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Contributions

Y.H. supervised the project. Q.L., X.Y., S.Y., H.S., K.W., L.X. and T.L. performed the experiments. Q.L. and Y.H. collected diffraction data. Y.H., Q.L. and G.Z. built the model and refined the structure. Y.H., C.S., G.Z., X.Z., D.L. and M.Z. contributed to manuscript preparation. Y.H. and Q.L. wrote the manuscript. All authors contributed to data analysis.

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Integrated supplementary information

Supplementary Figure 1 Chemical structure of LPS and LPS biogenesis in Gram-negative bacteria.

a. Chemical structure of LPS. LPS molecule consists of Lipid A, core oligosaccharide and O-antigen. The polar part of Lipid A is negatively-changed due to the presence of two phosphate groups. b. Ra-LPS molecule is approximately 32Å in height and 28 Å×12 Å in the other two dimensions. The dimensions of an Ra-LPS is based on the crystal structure of TLR4/MD-2/Ra-LPS complex (PDB ID: 3FXI). c. LPS biogenesis in Gram-negative bacteria. After flipped to the IM outer leaflet by MsbA, LPS is extracted from IM, transported cross the periplasm and finally inserted in the OM by the LptABCDEFG transenvelope complex. LptB2FG is a quaternary ABC transporter.

Supplementary Figure 2 The electron density maps of LptB2FG.

a. Stereo views (cross-eyed) of the 2Fo-Fc electron density map for the complete LptB2FG complex structure at 3.46Å. b. The 2Fo-Fc electron density maps of representative regions of the TMDs of LptFG (TM1-LptF and TM1-LptG) are shown. Selenomethionine residues (in red) and bulky residues are used as makers for guiding model building. c-d. Validation of side-chain register of the nucleotide-free LptB2FG transporter. Anomalous electron density maps define selenomethionine (contour level: 3.0σ) in (c) and Pt sites (contour level: 4.5σ) in (d). In (c), anomalous density was observed for 28 out of 32 selenomethionines of the complete LptB2FG complex.

Supplementary Figure 3 Domain organization of the LptB2FG complex.

a. Domain organization of the LptB2FG complex. The two ATPase domains (LptB) in cytoplasm are colored in cyan and green. The TMD domains of LptF and LptG, each containing six transmembrane helices, are colored in violet and yellow, respectively. The two periplasmic β-jellyroll domains of LptF and LptG that stem from TM3 and TM4 of LptF(G) are colored in grey. The two coupling helices of LptF and LptG connecting TM2 and TM3 of LptF(G) in cytoplasm are highlighted in blue. b. Overlay of the TMD of LptF with that of LptG. LptF and LptG are colored in violet and yellow, respectively.

Supplementary Figure 4 Sequence alignment of LptF homologs and residues selected for functional analysis in the structure.

a. Sequence alignment of LptF homologs from five representative Gram-negative bacterial strains. b. Conserved hydrophobic and positive residues of LptF lining the “V”-shaped cavity selected for mutational studies. Conservation of LptF residues in different Gram-negative homologs is shown in ENDscript. Secondary structures are numbered within the respective domains. Conserved residues lining the inner surface of the “V”-shaped cavity were selected for mutational analyses are highlighted. The labeled residue types and numbers in both alignments correspond to those in E. coli.

Supplementary Figure 5 Sequence alignment of LptG homologs and residues selected for functional analysis in the structure.

a. Sequence alignment of LptG homologs from five representative Gram-negative bacterial strains. b. Conserved hydrophobic and positive residues of LptG lining the “V”-shaped cavity selected for mutational studies. Conservation of LptG residues in different Gram-negative homologs is shown in ENDscript. Secondary structures are numbered within the respective domains. Conserved residues lining the inner surface of the “V”-shaped cavity were selected for mutational analyses are highlighted. The labeled residue types and numbers in both alignments correspond to those in E. coli.

Supplementary Figure 6 Mutagenesis study of the conserved residues that line the inner surface of the “V”-shaped cavity in the TMDs of LptF and LptG.

The growth phenotypes of the lptFG-depleted E. coli strain NR1113 transformed with various hydrophobic-to-hydrophilic LptG_Ec mutants (a) and LptF_Ec mutants (b) on LB agar plates containing 0.1% L-arabinose and 50 μg ml−1 kanamycin. The growth phenotypes of the lptFG-depleted E. coli strain NR1113 transformed with positive-to-negative mutations in the absence of L-aribinose (c), mutant protein expression levels (d) and the growth phenotypes in the presence of 0.1% L-arabinose (e). Mutational analyses of residues from the coupling helices of LptF_Ec and LptG_Ec on LB agar plates containing 0.1% L-arabinose and 50 μg ml−1 kanamycin (f). All labeled residue types and numbers correspond to those of LptF_Ec and LptG_Ec. In the presence of 0.1% L-arabinose, the lptFG-depleted E. coli strain NR1113 transformed with WT, vector control and various mutants all grew well similar to that of WT. In the absence of L-arabinose, the lptFG-depleted E. coli strain NR1113 transformed with pQLink-Kan vector and plasmids encoding wild-type (WT) LptFG were used as negative control and positive control, respectively. All the complementation assays were repeated at least three times and a representative result is shown.

Supplementary Figure 7 Three representative ABC exporters in their inward-facing or outward-facing conformational states.

The nucleotide-free LptB2FG transporter (a), the heterodimeric TM287-TM288 exporter in the apo state (PDB code: 4Q4H) (b) and the heterodimeric nucleotide-free human sterol ABCG5-ABCG8 exporter (PDB code: 5DO7) (c) in inward-facing conformational state; the homodimeric AMP-PNP-bound Sav1866 exporter (PDB code: 2ONJ) (d) in outward-facing conformational state.

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Luo, Q., Yang, X., Yu, S. et al. Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG. Nat Struct Mol Biol 24, 469–474 (2017). https://doi.org/10.1038/nsmb.3399

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