Abstract
The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane β-barrel proteins (OMPs) that are essential interchange portals of materials1,2,3. All known OMPs share the antiparallel β-strand topology4, implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial β-barrel assembly machinery (BAM) to initiate OMP folding5,6; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly.
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Data availability
Electron microscopy density maps and atomic models have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB, respectively, with accession codes EMD-33763 and 7YE4 (BAM–pair1+4-EspP), EMD-16137 and 8BNZ (BAM–pair1-EspP), EMD-33765 and 7YE6 (BAM–pair2-EspP), and EMD-16138 and 8BO2 (BAM–pair3-EspP). Previously reported structures (5D0Q, 5D0O, 6V05, 5EKQ, 7TT5, 7TT7, 7RI4, 2QOM and 3SLJ) were used as reference for structural comparative analysis.
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Acknowledgements
We thank Y. Q. Wei and B. R. Dong for their continuous support, encouragement and advice, C. Ma at Zhejiang University School of Medicine for his assistance in protein purification and Z. Su at SKLB Duyu High Performance Computing Centre of Sichuan University for help in figure preparation. This work was partially supported by the National Key Research and Development Program of China (2018YFA0507700 to X. Zhang, 2021YFA1301900 and 2021YFA1301203 to H.D., 2017YFA0504803 to X. Zhang, and 2021YFA1201201 and 2021YFF1200404 to R.Z.); the National Natural Science Foundation of China (31900039 and 32170029 to X.T., 32000844 to S.C., 81971974 to H.D. and U1967217 to R.Z.); the National Center of Technology Innovation for Biopharmaceuticals (NCTIB2022HS02010 to R.Z.); the Fundamental Research Funds for Central Universities (226-2022-00043 and 226-2022-00192 to R.Z.); the 1.3.5 Project for Disciplines Excellence of West China Hospital, Sichuan University (ZYYC20021 to H.D.); the National Independent Innovation Demonstration Zone Shanghai Zhangjiang Major Projects (ZJZX2020014 to R.Z.); the Starry Night Science Fund at Shanghai Institute for Advanced Study of Zhejiang University (SN-ZJU-SIAS-003/006/009 to R.Z.); and BirenTech Research (BR-ZJU-SIAS-001 to R.Z.); as well as grants from Laboratory and Equipment Management Department, Zhejiang University (SYB202132 to S.C.).
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H.D. and X.T. conceived and designed the experiments. R.Z. and K.C.C. designed the molecular models and simulations. X.T., H.D., C.S. and Q.L. made the constructs for protein expression. X.T., C.S., Q.L. and Z.Z. expressed and purified the proteins. C.S. and Q.L. did the mutagenesis, the transport assays and the cell-based assays. C.S., Q.L. and Z.Z. prepared the samples. S.C. and X. Zhang undertook data collection, processed electron microscopy data and performed structure construction. R.Z., K.C.C. and T.X. performed the MD simulations. X.W., X. Zhu, G.L. and B.L. analysed the experimental data and prepared the figures. H.D., X.T. and C.D. did the model building and refinement. H.D. and X.T. wrote the manuscript. X. Zhang, R.Z., S.C. and C.D. revised the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Analysis of the purified BAM-EspP proteins containing cysteine pair1+4, pairs1, pair2 and pair3.
a, Scheme of outer membrane protein (OMP) transport and assemble from the cytoplasm to the outer membrane via BAM. Newly synthesized OMP is translocated across the IM through SecYEG to the periplasm, where chaperons SurA and Skp carry the nascent OMP to BAM for assembly. b, Schematic diagram of recombinant BamABCDE and maltose-binding protein (MBP) fused EspP protein containing β-barrel and short passenger peptide (upper panel). Schematic BAM-EspP complex showing positions of cysteine pair mutations (lower panel). Cysteine pair 1: BamA(G431C)-EspP(N1293C), cysteine pair 2: BamA(N427C)-EspP(R1297C), cysteine pair 3: BamA (S425C)-EspP(S1299C) and cysteine pair 4: BamA(G781C)-EspP(A1043C). c-j, SDS-PAGE (c-f) and SEC profile (g-j) of purified BAM-EspP proteins by size exclusion chromatography using a Superdex200 Increase 10/300 column (void volume≈4.8ml) in the absence or presence of DTT. The results shown are representative data of experiments n = 3. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Assessment of disulfide bond formations of cysteine pair1, pair2 and pair3.
In vivo disulfide bond formations of the BamA cysteine mutants N431C (a), N427C (b) and S425C (c) and the EspP cysteine mutants R1297C, N1295C, R1297C and S1299C. The crosslinked BamA-MBPEspP, the immature MBPEspP and mature EspP were detected by anti-Flag for EspP (upper two blots) and anti-Myc for BamA (lower two blots) in the absence and presence of DTT. Non-specific band is indicated with *. The results shown are representative data of experiments n = 3. For source data, see Supplementary Fig. 1.
Extended Data Fig. 3 Physiological assessment of the crosslinked BAM-EspP complexes.
a–d, In vivo folding state of the crosslinked BAM-EspP by cysteine pair1 (a), pair1+4 (b), pair2 (c) and pair3 (d) were monitored over 90 mins. MBP-EspP intermediate, mature EspP and BamA were detected by western blots under reducing conditions. EspP mutant of the C-terminal loop deletion (del S1281-D1289 of ECL6) was used as a positive control. Anti-RpoB immunoblotting was used as loading control. The results shown are representative data of experiments n = 3. e–h, The 200-ns molecular dynamics (MD) stimulations of the crosslinked (green line) and unlinked wildtype (purple line) BAM-EspP complex in lipidic nanodisc (e,f) or unlinked wildtype BAM-EspP complex in nanodisc/PL bilayer (red line) and LPS and PL membrane (blue line) (g,h). For blot source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Flow charts for cryo-EM structure determination of BAM-pair1+4-EspP and BAM-pair1-EspP complexes in nanodisc.
a,f, Representitive cryo-EM microscope images of the BAM-pair1+4-EspP (a) and BAM-pair1-EspP (f) complexes. b,g, Scheme of data collection, two- and three- dimensional classifications, refinements and the local cryo-EM resolutions of the density maps for BAM-pair1+4-EspP (b) and BAM-pair1-EspP (g). c, Selected two-dimensional classification images of BAM-pair1+4-EspP. d,h, Gold-standard Fourier Shell Correction (FSC) curves for structure resolution determination for BAM-pair1+4-EspP (d) and BAM-pair1-EspP (h). e,i, Cryo-EM maps of the selected BamA and EspP β-strands of BAM-pair1+4-EspP (e) and BAM-pair1-EspP (i).
Extended Data Fig. 5 Flow charts for cryo-EM structure determination of BAM-pair2-EspP and BAM-pair3-EspP complexes in nanodisc.
a,f, Representative cryo-EM microscope image of BAM-pair2-EspP (a) and BAM-pair3-EspP (f) complex. b,g, Cryo-EM image collection, reconstitution, two-dimensional and three-dimensional classifications, refinements and local resolutions of the density map for BAM-pair2-EspP (b) and BAM-pair3-EspP (g). c, Selected two-dimensional image of BAM-pair2-EspP complex. d,h, FSC curves for the structure resolution determination for BAM-pair2-EspP (d) and BAM-pair3-EspP (h). e,i, Cryo-EM maps of the selected BamA and EspP β-strands of BAM-pair2-EspP (e) and BAM-pair3-EspP (i).
Extended Data Fig. 6 Superimposition of the BamA-EspP structures with other reported BAM structures.
a, Side views of hybrid barrels of BAM-pair1+4-EspP, showing the interface between the N-terminus of BamA and C-terminus of EspP (left) and the C-terminus of BamA and N-terminus of EspP with exposed hydrophobic side chains (right). The red circle indicates the position of the disulfide bond formed between BamA C1043 and EspP C781. b, Superimposition of the BAM-pair1+4-EspP and BAM-pair1-EspP structures from side view (left) and top view (right) c, Superimposition of the BamA barrel of the BAM-pair1-EspP structure (light green) with that of the closed crystal structure (PDB: 5D0O, salmon) (left) and open crystal structure (PDB: 5D0Q, pale yellow) (right). d, Superimposition of BAM-pair1+4-EspP complex (dark cyan) with BAM-BamA∆L1 complex (PDB: 6V05, dark purple) in the side (left) and top (mid) views of the hybrid barrels and the periplasmic ring (right). e, Superimposition of the BamA and EspP hybrid barrels of pair1 (light green) and pair1+4 (dark cyan) complexes from the top view. The side chains of R1297 of the pair1+4 (red) and pair1 (blue) structures, showing different orientations. f, In vivo folding assessment of BAM-EspP(R1297A) over 90min. MBP-EspP intermediate, mature EspP and BamA were detected by western blots under reducing conditions. The result shown is representative data of experiments n=3. For blot source data, see Supplementary Fig. 1.
Extended Data Fig. 7 In vitro assembly assays set-up.
a, Scheme of in vitro folding assay. Urea denatured EspP is mixed with BAM proteoliposomes in presence of SurA. b, The set-up of the in vitro EspP assembly assay by western blot. The folded EspP and BamA in liposomes are tested by heat denaturation. c, The orientation of BAM complex in liposomes was determined by proteinase K treatment. d, Unfolded EspP was degraded by proteinase K. e, Immunoprecipitation against SurA (Strep) from the in vitro assembly assay to pull out bounded EspP(Flag) variants to show normal presentation of SurA. f, Protein detection of EspP(∆L1) and EspP(∆L6) variants with or without alanine linker restoration following 30min in vivo assembly by western blot against EspP and BamA. Anti-RpoB was used as loading control. g, Cellular viability assays of BamA mutants in the presence of arabinose. The viability assays in the absence of arabinose as shown in Fig. 3h. h, SDS-PAGE of purified BAM complex of BamA mutants of g. i, Liposome reconstituted BamA mutants before and after heat denaturation. The results shown are representative data of experiments n=3. For source data, see Supplementary Fig. 1.
Extended Data Fig. 8 Conformational changes of the structures at different states.
a, Superimposition of the hybrid barrel of pair1+4 and pair2 structures (left), showing the closing lateral gate of the pair2 structure with the interactions on BamA ECL1 (right). b, Superimposition of the BamA barrels of the pair2 structure and the closed crystal structure (PDB: 5D0O, salmon). The zoomed section shows the interactions between BamA β1 and β16 of the closed crystal structure (PDB: 5D0O) (right panel) c, Superimposed BamA barrels of the four BAM-EspP structures, showing the conformational changes of the C-terminal end of β16. d, Superimpositions of the periplasmic ring of the pair1+4, pair2 and pair3 structures. e, Superimpositions of the BamA barrels and the periplasmic rings of the pair1 and pair2 structures, with the reference of the pair1+4 structure. The boxed section is showing curved BamA ECL1 in pair1 and pair2 structures with interactions from S436. f, g, Superimposed BamA barrel of pair1+4, pair2 and the lateral closed crystal (5D0O) structures, showing conformational changes in the BamA β1 periplasmic linker (f), POTRA domains (g, left) and POTRA3 loop (g, right). h–l, Superimposed four BAM-EspP structures with highlighted interactions between POTRA5, PT1 and PT4 (green) and between PT1 and BamD (orange). BAM-EspP pair1+4 in dark cyan, pair1 in light green, pair2 in medium purple and pair3 in royal blue.
Extended Data Fig. 9 EspP barrel closes in a zipper-like fashion.
a–e, EspP barrels from the pair1+4 (a), pair1 (b), pair2 (c), pair3 (d) and the crystal mature EspP (PDB: 2QOM, yellow orange) (e) structures (upper panels). The hydrogen bonds formed at the β-seam of the EspP barrel of the corresponding structures are shown in the lower panels. f–l, MD simulation of the EspP from our pair2 structure showing sequential formation of hydrogen bonds within the time monitored (f–k). The monitored hydrogen bonds are indicated in different colors. Structural comparison of our pair2 and the crystal folded EspP structures during the simulated barrel closure (j,l). m, The in vivo disulfide bond formation upon cysteine mutation of the observed interacted residues in the EspP of the pair2 structure. The result shown is representative data of experiments n = 3. For blot source data, see Supplementary Fig. 1.
Extended Data Fig. 10 Structural comparisons of BAM assembly intermediate with the reported crystal BAM structures.
Superimposition of pair1+4 (dark cyan), pair1 (light green), pair2 (medium purple), pair3 (purple) and crystal structures PDB: 5D0Q (pale yellow), 5EKQ (marine) and 5D0O (salmon). a–f, Conformational changes in the BamA barrel of the superimposed structures, showing state transition from the lateral opening to close. g–l, The overall conformational changes of BamA of the superimposed structures from the open state to close state. m–r, Top view of the POTRA1-5 of the superimposed structures from the open to the close states, showing the clockwise rotation of the POTRA domains.
Extended Data Fig. 11 Structural comparisons of the reported ligand bound BamA with our BAM-EspP structures.
a–c, Superimposition of the reported BamA-EspP PDB: 7TT5 (dirty violet) or 7TT7 (olive) with our pair1+4 (dark cyan) (a), pair1 (light green) (b) and pair2 (medium purple) (c) structures, showing the distinct intermediate conformations of BamA and EspP barrel. The lower panels are 90° rotation of the upper panels along x-axis, showing the top view of the hybrid barrels. d–g, Superimposition of BamA from the reported BAM-EspPβ9-12 (PDB: 7RI4) (light pink), closed crystal PDB: 5D0O (light orange) (d) and pair1+4 (dark cyan) structures (e-g), showing the distinct conformations of BamA POTRA domains (d,e) and barrel lateral opening (f) of the BAM-EspPβ9-12 structure. The EspPβ9-12 (medium orchid) is sandwiched between β1 and β16 of BamA (g).
Supplementary information
Supplementary Fig. 1
Uncropped gels and immunoblots. Cropped regions shown in the main and Extended Data Figures are indicated with red dashed lines.
Supplementary Video 1
Dynamics of BAM-EspP for the view of BAM. State transitions of the BAM-EspP complex, showing conformational changes of BAM and EspP for the view of the lateral gate of the BamA β-barrel.
Supplementary Video 2
Dynamics of BAM-EspP for the view of EspP. State transitions of the BAM-EspP complex, showing conformational changes of BAM and EspP for the view of the EspP β-barrel.
Supplementary Video 3
Dynamics of BAM-EspP from the front and top views. State transitions of the BAM-EspP complex, showing conformational changes of BAM and EspP from the front and top views.
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Shen, C., Chang, S., Luo, Q. et al. Structural basis of BAM-mediated outer membrane β-barrel protein assembly. Nature 617, 185–193 (2023). https://doi.org/10.1038/s41586-023-05988-8
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DOI: https://doi.org/10.1038/s41586-023-05988-8
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