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Structural basis for bacterial lipoprotein relocation by the transporter LolCDE

Nature Structural & Molecular Biology volume 28, pages 347–355 (2021)Cite this article

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Abstract

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2–3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.

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Fig. 1: Lipoprotein transport by the Lol pathway in vivo and in vitro.
Fig. 2: Cryo-EM structures of LolCDE captured in different states.
Fig. 3: Lipoprotein sorting and binding.
Fig. 4: Conformational changes in the NBDs of LolCDE.
Fig. 5: Mechanism of a cycle of lipoprotein transport by LolCDE.

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Data availability

Electron microscope density maps and atomic models have been deposited in the EMDB and PDB, respectively, with accession codes EMD-11882 and PDB 7ARH (lipoprotein-bound LolCDE), EMD-11887 and PDB 7ARM (lipoprotein-bound LolCDEA), EMD-11885 and PDB 7ARK (AMP-PNP-bound LolCDE with closed NBD), EMD-11884 and PDB 7ARJ (AMP-PNP-bound LolCDE with open NBD), EMD-11886 and PDB 7ARL (ADP-bound LolCDE), and EMD-11883 and PDB 7ARI (apo LolCDE). Source data are provided with this paper.

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Acknowledgements

We thank Y. Q. Wei and B. R. Dong for supporting the project and C. Ma for his help with protein purification. This work was supported by grants from the National Key Research and Development Program of China (2017YFA0504803 and 2018YFA0507700 to X. Zhang) and the National Natural Science Foundation of China (31900039 to X.T., 32000844 to S.C. and 81971974 to H.D.), the Fundamental Research Funds for the Central Universities (2018XZZX001-13 to X. Zhang), the 1.3.5 Project for Disciplines Excellence of West China Hospital, Sichuan University (ZYYC20021) and Sichuan Science and Technology Program (2018TJPT0015 and 2018JY0094 to H.D).

Author information

Author notes

  1. These authors contributed equally: Xiaodi Tang, Shenghai Chang, Ke Zhang, Qinghua Luo.

Authors and Affiliations

  1. State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan, China

    Xiaodi Tang, Ke Zhang, Qinghua Luo, Ting Wang, Wen Qiao, Chongrong Shen, Zhibo Zhang, Xiaofeng Zhu, Xiawei Wei & Haohao Dong

  2. Department of Biophysics and Department of Pathology of Sir Run Run Shaw Hospital, Zhejiang University, School of Medicine, Hangzhou, Zhejiang, China

    Shenghai Chang, Chen Wang & Xing Zhang

  3. Center of Cryo Electron Microscopy, Zhejiang University, Hangzhou, Zhejiang, China

    Shenghai Chang, Chen Wang & Xing Zhang

  4. Zhejiang Laboratory for System and Precision Medicine, Zhejiang University Medical Center, Hangzhou, Zhejiang, China

    Shenghai Chang, Chen Wang & Xing Zhang

  5. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei, China

    Zhengyu Zhang

  6. College of Life Science, Sichuan University, Chengdu, Sichuan, China

    Xiaofeng Zhu

  7. Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, UK

    Changjiang Dong

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  1. Xiaodi Tang
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Contributions

H.D. and X.T. conceived and designed the experiments. X.T., H.D. and Zhengyu Zhang made the constructs for protein expression. X.T., K.Z., Zhengyu Zhang, Q.L. and W.Q. expressed and purified the proteins. K.Z., Q.L., T.W., W.Q., C.S., Zhibo Zhang, X.W. and X. Zhu performed the mutagenesis, ATPase activities, the transport assays, cell-based assays and surface plasmon resonance analysis. X.T., H.D., K.Z., Q.L. and W.Q. prepared the samples. S.C., C.W. and X. Zhang undertook data collection, processing of electron microscopy data and structure constitution. H.D., X.T. and C.D. carried out model building and refinement. H.D. and X.T. wrote the manuscript and X. Zhang, S.C. and C.D. revised the manuscript.

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Correspondence to Changjiang Dong, Xing Zhang or Haohao Dong.

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Peer review information Nature Structural & Molecular Biology thanks Alessandra Polissi and Markus Seeger for their contribution to the peer review of this work. Florian Ullrich and Anke Sparmann were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Activity analysis of purified LolCDE complex.

a, Purified LolCDE was assessed for ATPase activity in detergent or liposomes at different ATP concentrations. b, c, The relative ATPase activities of LolCDE were measured in the presence of AMP-PNP (b) or ADP (c). d, In vivo cellular viability of the LolCDE silencing strain (HD200313) was studied. The LolCDE gene in HD200313 is controlled under an araBAD promotor that can only be induced in the presence of arabinose (method). Insertion of a leaky LolCDE expressing plasmid was used to test mutations in the LolCDE gene. e, ATPase activity determination of LolCDE with insertion of tags. f, LolCDE in liposomes of E. coli polar lipids were treated with or without proteinase K to assess the reconstitution orientations by SDS-PAGE. Data in d, f are representative results from n = 3 independent experiments. Data in a-c, e represent mean ± s.d. (n = 3 independent experiments). Statistics for panels a-c, e are available as Source Data. Uncropped images for panel d, f are available as Source Data.

Source data

Extended Data Fig. 2 Image-processing workflow for lipoprotein-bound LolCDE.

a, Cryo-EM microscope and selected two-dimensional class averages of cryo-EM particle images of lipoprotein-bound LolCDE. b, Scheme of three-dimensional classification, refinement of cryo-EM particle images, and the final 3D reconstitution of lipoprotein-bound LolCDE. c, Gold-standard FSC curves of the final cryo-EM maps of lipoprotein-bound LolCDE. d, The overall cryo-EM maps of lipoprotein-bound LolCDE are colored according to the local resolution. e, Cryo-EM maps with the atom model for individual transmembrane helices of LolC and LolE and the lipoprotein.

Extended Data Fig. 3 Image-processing workflow for AMP-PNP-bound LolCDE in closed and open NBD conformation.

a, Typical cryo-EM microscope and selected two-dimensional class averages of cryo-EM particle images of AMP-PNP-bound LolCDE. b, Scheme of three-dimensional classification, refinement of cryo-EM particle images and final 3D reconstitution of AMP-PNP-bound LolCDE with closed LolD at 4.1Å and open LolD at 3.2Å. c, Gold-standard FSC curves of the final cryo-EM maps of AMP-PNP-bound LolCDE.

Extended Data Fig. 4 Image-processing flowchart for apo LolCDE and lipoprotein bound-LolCDEA.

a, Typical cryo-EM microscope images of apo LolCDE and lipoprotein bound-LolCDEA. b, Selected two-dimensional class averages of cryo-EM particle images of apo LolCDE and lipoprotein bound-LolCDEA. c, Scheme of three-dimensional classification, refinement of cryo-EM particle images and the final 3D reconstitution of apo LolCDE at 3.4Å and lipoprotein bound-LolCDEA at 3.6Å. d, Gold-standard FSC curves of the final cryo-EM maps of apo LolCDE and lipoprotein bound LolCDEA .

Extended Data Fig. 5 Comparison of cryo-EM LolCDE structure to other known structures.

a, Lipoprotein-bound LolCDE (yellow) is superimposed to apo MacB (purple) (PDB code:5GKO). b, c, Dimerized conformations of LolCDE (orange) (b) and MacB (pink) (PDB 5LG7) (c) are superimposed to their apo state. d, PD of LolC is superimposed to crystal structure of LolC periplasmic domain (gray) (PDB code: 6F3Z). e, Superimposition between PDs of LolC (yellow) and LolE (green). f, Superimposition between PDs of LolC (yellow) and MacB (purple). The PD of MacB does not have the hook.

Extended Data Fig. 6 Topologic arrangement of LolC and LolE and surface representation of LolCDE.

a, Topology of LolC (yellow) showing the secondary structure arrangement. b, Topology of LolE (green) showing the secondary structure arrangement. c, Surface representation of lipoprotein-bound LolCDE. d, Cartoon representation of lipoprotein bound-LolCDE, rotated 180° along y-axis from c. e, Surface representation of apo LolCDE. f, Cartoon representation of apo LolCDE, rotated 90° along y-axis from e.

Extended Data Fig. 7 Conformational changes of LolCDE in different states.

a, Structural superimposition of apo LolCDE (cyan) to lipoprotein-bound LolCDE (yellow). b, Rotation of 90° from the left panel along y-axis. c, Structural superimposition of ADP-bound LolCDE (blue) to lipoprotein-bound LolCDE (yellow). d, Structural superimposition of AMP-PNP-bound LolCDE (un-dimerized LolD) (orange) to lipoprotein-bound LolCDE (yellow). e, Structural superimposition of AMP-PNP-bound LolD dimerized LolCDE (purple) to lipoprotein-bound LolCDE (yellow). f, Structural superimposition of four lipoprotein-bound structures, showing the same conformation regardless of nucleotide bindings.

Extended Data Fig. 8 Lipoprotein recognition in the central channel of LolCDE.

a, Residues of LolC and LolE showing interactions with the triacyl tails of the bound lipoprotein. Lipoprotein is shown in stick. b, Effects to lipoprotein binding upon mutations of lipoprotein peptide binding residues of LolC (F51D, L55D and D362A) and LolE (Y260E and R263A). c, Functional assays of mutants of lipoprotein binding residues. d, Protein leaky expression level control of the mutants. e, Periplasmic domains of LolC and LolE showing interactions of LolE with the peptide of the bound lipoprotein. f, Silver stain of purified LolCDE, showing a major band of E. coli lipoprotein Lpp along with protein LolCDE. g, MALDI-TOF/TOF/TOF mass spectrometry identification of Lpp peptide. Data in b-d, f are representative results from n = 3 independent experiments. Uncropped images for panels c, f are available as Source Data. Mass spectrometry raw data for g are available as Source Data.

Source data

Extended Data Fig. 9 Effect of nucleotide binding mutants of LolD.

a, b, Atomic interaction between the NBD of LolD and TMD of LolC (a) or LolE (b). c, Stabilization of the LolCDE complex was determined upon LolD mutations. d, Nucleotide (ADP) binding residues are shown at the interface of the NBDs of LolCDE. e, Size-exclusion chromatography of purified wild-type LolCDE and mutants of LolD. f, SDS-PAGE of purified LolCDE and mutants. g, ATPase activity was determined upon single mutations on LolD (K48A, E171A or S147E) in detergent or liposome. Data in c, e and f are representative results from n = 3 independent experiments. Data in g represent mean ± s.d. (n = 3 experiments). Statistics for panel g are available as Source Data.

Source data

Extended Data Fig. 10 Analysis of LolA binding to LolCDE.

a–d, SPR senorgram of lipoprotein-bound (a,c) or apo (b,d) LolA to lipoprotein-bound (a,b) or apo (c,d) LolCDE. e, Mean of KD values from three independent experiments with error bars ±SD, **p<0.01. f, Western-blot of analytes of LolA and LolCDE showing lipoprotein-bound or apo states. g, In the lipoprotein-bound LolCDEA structure, LolA docks on the lateral gate LolC-TM2/LolE-TM1 that is opposite to the lipoprotein-bound lateral gate LolC-TM1/LolE-TM2. h-i, Modelling LolA (light blue, h or wheat, i) in the AMP-PNP bound NBD-closed (h) or apo (i) LolCDE structures using the crystal structure of PD of LolC/LolA complex (PDB 6F3Z). j, Superimposition of lipoprotein-bound LolCDEA (red), modelled AMP-PNP bound LolCDEA (light blue) and modelled apo-LolCDEA (wheat) to compare the distinct positions of LolA related to LolCDE. Data in a-d are representative results from n = 3 independent experiments. Data in e represent mean ± s.d. (n = 3 independent experiments). Statistics for panels a-e are available as Source Data. Uncropped images for panel f are available as Source Data.

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Supplementary information

Source data

Source Data Fig. 1

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Statistical source data.

Source Data Extended Data Fig. 1

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Mass spectrometry raw data.

Source Data Extended Data Fig. 8

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Statistical source data.

Source Data Extended Data Fig. 9

Uncropped blots.

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Statistical source data.

Source Data Extended Data Fig. 10

Uncropped blots.

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Tang, X., Chang, S., Zhang, K. et al. Structural basis for bacterial lipoprotein relocation by the transporter LolCDE. Nat Struct Mol Biol 28, 347–355 (2021). https://doi.org/10.1038/s41594-021-00573-x

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