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Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB

Nature Structural & Molecular Biology volume 28, pages 81–91 (2021)Cite this article

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Abstract

The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3–4.1 Å and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA–OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development.

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Fig. 1: PL transport between OM and IM proteoliposomes by the Mla pathway.
Fig. 2: Architectures of PE- and ADP-bound MlaFEDB complexes.
Fig. 3: MlaFEDB recognition of PE.
Fig. 4: Structural and functional characterization of MlaD.
Fig. 5: Structural and functional characterization of MlaF.

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Data availability

Electron microscopy density maps and atomic models have been deposited in the EMDB and PDB, respectively, with accession codes EMD-11547 and PDB 6ZY2 (apo MlaFEDB complex); EMD-11548 and PDB 6ZY3 (PE-bound MlaFEDB complex); EMD-11555 and PDB 6ZY9 (AMP-PNP-bound MlaFEDB complex); and EMD-11549 and PDB 6ZY4 (ADP-bound MlaFEDB complex). 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 Y. Zhang for advice on cryo-sample preparation. This work was supported by grants from the National Key Research and Development Program of China (2017YFA0504803 and 2018YFA0507700 to X. Zhang and 2017YFC0840100 and 2017YFC00840101 to H.D.) and the Fundamental Research Funds for the Central Universities (2018XZZX001-13) to X. Zhang; the National Natural Science Foundation of China (31900039 to X.T. and 81971974 to H.D.); the National Clinical Research Centre for Geriatrics, West China Hospital (Z2018B01) to H.D.; Laboratory and Equipment Management, Zhejiang University (SJS201814) to S.C.; and a Wellcome Trust investigator award (WT106121MA) to C.D.

Author information

Author notes

  1. These authors contributed equally: Xiaodi Tang, Shenghai Chang, Wen Qiao.

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, China

    Xiaodi Tang, Wen Qiao, Qinghua Luo, Yuejia Chen, Ke Zhang, Ting Wang, Zhibo Zhang, Changbin Zhang, Xiaofeng Zhu, Xiawei Wei & Haohao Dong

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

    Shenghai Chang, Zhiying Jia & Xing Zhang

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

    Shenghai Chang, Zhiying Jia & Xing Zhang

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

    Shenghai Chang, Zhiying Jia & Xing Zhang

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

    James Coleman & Changjiang Dong

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

    Xiaofeng Zhu

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  1. Xiaodi Tang
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Contributions

H.D. and X.T. conceived and designed the experiments. X.T. and H.D. generated the constructs for protein expression. X.T., W.Q., Q.L. and K.Z. expressed and purified the proteins. Y.C., W.Q., Q.L., T.W., K.Z., Z.Z., C.Z., X.W., X. Zhu and J.C. performed mutagenesis and the ATPase and transport assays. X.T., H.D., W.Q., Q.L. and K.Z. prepared the samples. S.C., Z.J. and X. Zhang undertook data collection, processed electron microscopy data and performed structure determination. 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.

Corresponding authors

Correspondence to Changjiang Dong, Xing Zhang or Haohao Dong.

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Peer review information Anke Sparmann was the primary editor 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 Determination of ATPase and PL transport activities of MlaFEDB.

a, Coomassie brilliant blue staining of the purified MlaFEDB on SDS-PAGE. b, The relative ATPase activities of purified MlaFEDB in liposomes or detergent. c, d, The relative ATPase activities of MlaFEDB in the presence of ADP (c) or AMP-PNP (d). e, f, SDS-PAGE analysis of MlaA-OmpF (e) or MlaFEDB (f) constitution ratio and orientation ratio in proteoliposomes of E. coli polar lipids or POPC with or without proteinase K treatment. g, FRET scan of PL transport assay using MlaA-OmpF wildtype or mutant MlaA(∆Asn41-Phe42)-OmpF in retrograde direction. h, Thin-layer chromatogram (TLC) showing transported PLs from IM or OM proteoliposomes to apo-MlaC. i, SDS-PAGE analysis of proteins involved in the transported system of Fig. g. j, FRET scan of PL transport assay containing IM proteoliposomes and apo-MlaC only in the presence or absence of ATP and MgCl2. k, l, FRET scan of PL transport assay using OM proteoliposomes containing lipid A in different ratios to PLs for anterograde (k) and retrograde direction (l). m, Loading control of lipids A and MlaA protein by western blot. Data in a, e-m are representative results from n = 3 independent experiments. Data in b-d, g, j-l represents mean ± s.d. (n = 3 independent experiments). Uncropped images for all gels are available online. Source data for panels b-d and g, j-l are available in Supplementary Data Set 1.

Source data

Extended Data Fig. 2 Flowchart for cryo-EM single-particle data processing of PE bound MlaFEDB.

a, A micrograph of the single particles after drift correction and dose-weighting, 2D classifications, 3D classification, selections and 3D refinement. b, Angular distribution of the cryo-EM particles included in the final 3D reconstruction. c, Cryo-EM map coloured by local resolutions. d, Gold-standard FSC curves of the final EM maps. e, Values are plotted for the model versus the final map (FSC average, black), for the model that was refined into the first half-map and FSC calculated either for the same map (model vs first half-map, red) or for the second half-map (model vs second half map, blue). f, Cryo-EM density with the atomic model for TM1-TM5 and elbow helix of MlaE. The PE density from C1 (yellow) and C2 (blue) full maps are compared, which shows symmetry due to additive signal of two possible binding gestures of PE in either of the MlaE subunit.

Extended Data Fig. 3 Interactions of MlaE to the peripasmic MlaD and the cytoplasmic MlaF.

a, Cartoon representation of PE-bound MlaFEDB complex. The colour scheme is the same as the Fig. 2. Each MlaE is surrounded by three TM segments from three adjacent MlaD subunits. b, MlaE interacts with the periplasmic domain of MlaD through the periplasmic loop 1 and 2. The interacting residues are labelled. c, The coupling helix residues of one MlaE unit interact with residues located at the groove of one MlaF unit. d, Two MlaD TM segments interact with the elbow helix of MlaE. e, The third MlaD TM segment interacts with TM1 and TM3 of MlaE.

Extended Data Fig. 4 Comparison of PE, ADP, AMP-PNP bound MlaFEDB structures to the apo structure of MlaFEDB.

The apo MlaFEDB is shown in green. The PE, ADP and AMP-PNP are shown in sphere. a, PE bound MlaFEDB complex is superimposed to the apo MlaFEDB complex. PE-bound MlaFEDB complex is coloured in blue. b, ADP-bound MlaFEDB structure is superimposed to the apo MlaFEDB. ADP-bound MlaFEDB is coloured in yellow. c, AMP-PNP bound MlaFEDB structure is superimposed to the apo MlaFEDB. AMP-PNP bound MlaFEDB is in orange. d, Cartoon representation of MlaFE structure of the complex.

Extended Data Fig. 5 PL binding residues in the cavity of MlaE.

a, Cartoon representation of apo MlaFEDB. b, 90° rotation along the y-axis relative to the left panel. c, Cytoplasmic view of apo MlaFEDB. d, Leaky expression of pTRC99a_MlaFEDCB in E. coli MlaE null strain, with or without MlaE mutations V77D, Y81E, E98R. e, PL transport assay by TLC showing transported PL to apo-MlaC using wildtype MlaFEDB and mutant MlaFE(Y81E)DB or MlaFE(E98R)DB constituted IM proteoliposomes in the absence of ATP. f, SDS-PAGE analysis of proteins involved in the PL transport assay of Fig e. Data in d-f are representative results from n = 3 independent experiments. Uncropped images for panels d-f are available as online.

Source data

Extended Data Fig. 6 Interactions of MlaB to MlaF using AMP-PNP bound structure of MlaFEDB.

a, Cartoon representation of structure of the AMP-PNP bound MlaFEDB complex. b, 90° rotation along the y-axis relative to the left panel. c, Structure of MlaB showing the STAS domain, consisting of three α-helices and four β-strands. d, The interactions between one MlaB and one MlaF subunit. e, 90° rotation along the y-axis relative to the left panel. f, SDS-PAGE analysis of proteins of wildtype and MlaF binding residue mutants MlaFEDB(W29E), MlaFEDB(Y88E) and MlaFEDB(T52A). g, ATPase activity of Mutant MlaFEDB(T52A) in both detergent and liposomes. h, FRET scan of PL transport using mutant MlaFEDB(T52A) or wildtype MlaFEDB IM proteoliposomes in the presence of ATP and MgCl2 for retrograde direction. Data f, h are representative results from n = 3 independent experiments. Data in g, h presents mean ± s.d. (n = 3 independent experiments). Uncropped images for panel f are available online. Source data for panels g and h are available in Supplementary Data Set 1.

Source data

Extended Data Fig. 7 Functional residues characterization of MlaD.

a, Coomassie brilliant blue staining of purified Mla proteins with MlaD truncations. Purified MlaFEB was incubated with TM truncated MlaD(ΔTM) or full length MlaD to form complex in vitro, which were purified using affinity columns and analyzed on SDS-PAGE along with wild-type MlaFEDB and MlaFEB. b, The relative ATPase activities of wild-type, MlaFEB and in vitro formed MlaFEDB were measured in detergent and liposomes. Soluble MlaD(ΔTM) was added into MlaFEB constructed systems and its effect to ATPase activity was also analyzed. c, FRET scan of PL transport assay using wildtype MlaFEDB or MlaFEB complex incubated with truncated MlaD(ΔTM) or full length MlaD for retrograde direction. Soluble MlaD(ΔTM) was added into MlaFEB constructed systems and its effect to transport activity was also analyzed. d, FRET scan of PL transport assay using wildtype MlaFEDB or MlaFEDB mutants MlaFED(ΔL143-G153)B for retrograde direction. e, Leaky expression of pTRC99a_MlaFEDCB containing MlaD mutation L143E, I147E, F150E or Y152E. f, SDS-PAGE analysis of purified MlaFEDB complexes with MlaD mutation I143E, I147, F150E or Y152E. The mutants MlaD I143E, I147E and F150E lost the SDS-resistance hexameric form. Data are representative results from n = 3 independent experiments. Data in b represents mean ± s.d. (n = 4 independent experiments). Data in c, d present mean of triplicates ± s.d. (n = 3). Uncropped images for panel a, e and f are available online. Source data for panels c and d are available in Supplementary Data Set 1.

Source data

Extended Data Fig. 8 Effect of nucleotide binding mutants of MlaF on ATPase activity and in vitro PL transport.

a, The size-exclusion chromatogram of purified MlaFEDB complexes with MlaF single catalytic mutants of F16A, R18A, K47A, E170A and H203A. The mutants were eluted almost at the same time as the wild-type MlaFEDB, indicating that the mutants have the intact structure as the wild-type. b, The relative ATPase activities of MlaFEDB wild-type and mutants in detergent and liposomes. c, SDS-PAGE analysis of purified wildtype and the mutants. d, FRET scan of PL transport using MlaFEDB mutant IM proteoliposomes containing single mutation on MlaF K47A, E170A or H203A, wildtype OM proteoliposomes and MlaC for retrograde direction. The catalytic and ATP binding residue mutants abolished retrograde transport of PLs. e, Amino acid sequence alignment of MlaF and LptB. MlaF and LptB have 24.54% amino acid identity. The C-terminal tail of MlaF is longer than that of LptB. f, Dimeric MlaF is superimposed into the dimeric LptB. MlaF has a long C-terminal tail that interacts with the other MlaF molecule, but LptB does not. Data in a, c and d are representative results from n = 3 independent experiments. Data in b represents mean ± s.d. (n = 3 independent experiments). An uncropped image for panel c is available online. Source data for panels b and d are available in Supplementary Data Set 1.

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Extended Data Fig. 9 Functional interacting residues between MlaB and MlaF.

a, Residues from the C-terminal tail of MlaF show interactions with the opposite MlaF and MlaB. b, SDS-PAGE analysis of purified MlaFEDB wildtype or mutant containing single mutation of the signature motif residues mutants E144A, S146S, R151A of MlaF, and C-terminal residues Y256D and H262D of MlaF. c, The relative ATPase activity of MlaFEDB mutants in detergent and in liposomes. d, FRET scan of PL transport assay using wildtype MlaFEDB or mutant IM proteoliposomes, wildtype OM proteoliposome and MlaC for retrograde direction. e, Cellular sensitivity to chlorpromazine by the mutant MlaF(Y256D)EDB or MlaF(H262D)EDB, showing no cellular effect. f, Leaky expression of pTRC99a_MlaFEDCB carrying mutations on C-terminal residues. g, SDS-PAGE analysis of purified MlaFEDB and mutant with MlaF being truncated at the C-terminal tail MlaF(ΔI247-S269)EDB. h, Leaky expression of pTRC99a_MlaFEDCB carrying mutations on signature motif residues. Data in b, d-h are representative results from n = 3 independent experiments. Data in c represents mean ± s.d. (n = 3 independent experiments). Uncropped images are available online. Source data for panels c and d are available in Supplementary Data Set 1.

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Extended Data Fig. 10 Structures of reported ABC transporters in apo and AMP-PNP bound states.

a, Human ABCA1 lipid exporter, apo ABCA1 (PDB code: 5XJY). The ABCA1 has limited structural similarity to MlaFEDB (Dali server search with a Z score of 10). b, E. coli vitamin B12 importer, apo BtuCDF (PDB code: 2QI9), BtuCDF in complex with AMP-PNP (PDB code: 4FI3). BtuCDF binding AMP-PNP causes conformational changes. c, E. coli lipopolysaccharide transporter, apo LptB2FGC (PDB code: 6S8N), LptB2FGC in complex with AMP-PNP (PDB code: 6S8G). LptB has some structural similarity to MlaF. LptB2FGC binding AMP-PNP causes significant conformational changes. d, human ABCG2 multidrug transporter, apo (PDB code: 6MIJ), ABCG2 E211Q mutant in complex with ATP (PDB code: 6HZM). ATP binding ABCG2 causes conformational changes. e, human mitochondrial ABC transporter ABCB10, apo (PDB code: 3ZDQ), in complex with AMP-PNP (PDB code: 4AYW).

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Supplementary Dataset 1

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Tang, X., Chang, S., Qiao, W. et al. Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB. Nat Struct Mol Biol 28, 81–91 (2021). https://doi.org/10.1038/s41594-020-00532-y

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