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Structure of a nascent membrane protein as it folds on the BAM complex

Nature volume 583, pages 473–478 (2020)Cite this article

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Abstract

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure1,2. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3,4,5. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins5,6,7,8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10,11,12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

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Fig. 1: Deletion of extracellular loops causes BamAS to stall at different stages of folding on the BAM complex.
Fig. 2: The N-terminal β-strand of BamAM interacts with the C-terminal β-strand of BamAS by β-sheet augmentation.
Fig. 3: The hybrid barrel is asymmetric, as the C-terminal β-strand of BamAM and the N-terminal β-strand of BamAS are unpaired in the membrane.
Fig. 4: Release from BamAM requires structural features of the N and C termini of BamAS that allow stepwise hydrogen-bond exchange.

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Data availability

The electron microscopy maps have been deposited into the Electron Microscopy Data Bank (EMDB) under accession codes EMD-20969 (4.1 Å) and EMD-21313 (6.5 Å). The atomic model, built from the 4.1 Å map, has been deposited into the Protein Data Bank (PDB) under accession code 6V05. All data are available in the Article and its Supplementary Information.

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Acknowledgements

We thank S. Sterling, R. Walsh and M. Chambers for assistance with electron microscopy; R. Losick, S. Walker, A. Kruse, M. May, K. Pahil, R. Taylor and M. Mandler for providing feedback on the manuscript; and H. Bernstein (NIH) for the kind gift of the pJH114 plasmid. Cryo-EM data were collected at the Harvard Cryo-EM Center for Structural Biology at Harvard Medical School. This work was supported by NIH grants F31GM116210 (to J.L.), F32GM108258 (to J.S.W.), and R01GM066174 and R01AI081059 (to D.K.). S.C.H. is an Investigator in the Howard Hughes Medical Institute.

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Author notes

  1. James Lee

    Present address: Laboratory of Membrane Biophysics and Biology, The Rockefeller University, New York, NY, USA

  2. Joseph S. Wzorek

    Present address: Novartis Institutes for BioMedical Research, Cambridge, MA, USA

Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

    David Tomasek, James Lee & Daniel Kahne

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    David Tomasek, James Lee, Joseph S. Wzorek & Daniel Kahne

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Shaun Rawson, Stephen C. Harrison, Zongli Li & Daniel Kahne

  4. Howard Hughes Medical Institute, Boston, MA, USA

    Stephen C. Harrison & Zongli Li

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Contributions

D.T. designed and all performed biochemical experiments with assistance from J.L. and J.S.W. at early stages of the project. D.T. performed protein purification and screened samples using negative-stain electron microscopy. Z.L. collected cryo-EM data. Z.L. and S.R. processed cryo-EM data. D.T. and S.R. built the atomic models. D.K. and S.C.H. supervised the project. D.T. and D.K. wrote the manuscript and all authors contributed to editing.

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Correspondence to Stephen C. Harrison, Zongli Li or Daniel Kahne.

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The authors declare no competing interests.

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Peer review information Nature thanks Yihua Huang, Nikolaus Pfanner, Bert van den Berg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Capturing BamAS(ΔL1) on the BAM complex using disulfide-bond formation.

a, Topological map of the β-barrel domain of BamA with extracellular loops labelled. b, Residues near the C terminus of the β-barrel domain of BamAM that were substituted with cysteine (yellow sticks) shown on the structure of the β-barrel domain of BamA (green) (PDB code 5D0O). The N- and C-terminal β-strands are labelled and shown in salmon. c, As in b, but showing residues near the N terminus of the β-barrel domain of BamAS(ΔL1) (blue) that were substituted with cysteine. The positions of these cysteine substitutions were selected on the basis of reports that the C-terminal region of BamAM is in proximity to the N-terminal region of the substrate during folding10,11,12. d, Disulfide-bond formation between 6×His-tagged BamAM and 3×Flag-tagged BamAS(ΔP345/ΔL1) containing the cysteine substitutions shown in b and c. The presence of a BAM-complex–substrate disulfide bond is detected as a high-molecular-weight adduct on the anti-Flag (middle) immunoblot. The adducts generated via disulfide-bond formation were not present in an amount high enough for detection with the anti-His antibody but could be detected with the anti-Flag antibody. An anti-Flag immunoblot of total cell lysates (bottom) shows that the BamAS variants containing different cysteine substitutions are expressed at similar levels. In this experiment, no oxidizing agent (for example, copper(II) sulfate and 1,10-phenanthroline) was added. e, As in d, but without any cysteine substitutions introduced into BamAM. Disulfide-bond formation is not observed between BamAM and BamAS when no cysteine is introduced into BamAM. f, As in d, but with BamAS containing all five POTRA domains. Disulfide-bond formation between BamAM and BamAS containing all POTRA domains indicates that the stalling of BamAS in d is not due to the deletion of POTRA domains 3, 4 and 5. Data shown in d–f are representative of results from two biological replicates.

Extended Data Fig. 2 Expression and purification of substrate-bound BAM complex for cryo-EM.

a, Constructs used for protein expression. Mutations and affinity tags introduced into each protein are indicated. SP, signal peptide; P1 to P5, POTRA domains of BamA. The BamAS variants also contain deletion of POTRA domains 3, 4 and 5 (Δ172–421) to avoid the possibility of them forming BAM complexes if they complete folding. The deletion does not prevent folding of an otherwise wild-type BamA, as shown in b. b, Assessment of heat modifiability of BamAS(ΔL1) used for cryo-EM (ΔL1; right half of blot) that contains the T467C mutation, and BamAS that contains loop 1 and the T467C mutation (WT; left half of blot). The samples in the unboiled lanes can be used to calculate the fraction of each substrate that is folded in vivo (indicated below the blot). BamAS(ΔL1) accumulates on the BAM complex because of the deletion of loop 1. c, Representative size-exclusion chromatogram of the substrate-bound BAM complex in which disulfide-bond formation between the N terminus of BamAS(ΔL1) and the C terminus of BamAM was induced with the oxidizing agent copper sulfate and 1,10-phenanthroline (CuP). d, SDS–PAGE gels showing peak fractions from size-exclusion chromatography of the complex in c. The left gel shows the complex without addition of β-mercaptoethanol (βME), and the right gel shows the complex after addition of βME to break the disulfide bond. Data shown in b–d are representative of results from two biological replicates.

Extended Data Fig. 3 Cryo-EM data processing and analysis for the substrate-bound BAM complex.

a, Representative cryo-EM micrograph of the substrate-bound BAM complex embedded in vitreous ice. b, Selected two-dimensional class averages of cryo-EM particle images. c, Scheme of three-dimensional classification and refinement of cryo-EM particle images. d, Gold-standard Fourier shell correlation (FSC) curves calculated with different masks in cryoSPARC. The resolutions were determined at FSC = 0.143 (horizontal blue line). The final corrected mask gave an overall resolution of 4.1 Å. e, Distribution of orientations over azimuth and elevation angles for particles included in the calculation of the final map. f, Cryo-EM map coloured by local resolution as shown in Fig. 1g, but at a lower contour level.

Extended Data Fig. 4 Fit of the atomic model into the cryo-EM map.

a–h, The atomic model (in stick representation) shown with the corresponding portion of the cryo-EM map (shown in grey mesh) for selected regions in the BamAM β-barrel domain (a); the BamAM POTRA domains (b); BamB (c); BamC (d); BamD (e); BamE (f); BamAS (g); and the BamAM–BamAS interaction (h). Side-chain densities are visible, and individual β-strands can be resolved. Images were prepared in UCSF Chimera using a 2 Å carve radius.

Extended Data Fig. 5 Comparison of the architecture of the substrate-bound BAM complex to that of substrate-free BAM complexes.

a–c, Alignments of the substrate-bound BAM complex (all components shown in green) with published structures14,15 of the BAM complex (shown in different colours). Alignments are performed using the β-barrel domains of BamA.

Extended Data Fig. 6 Conformational differences between BAM-complex components from substrate-bound and substrate-free complexes.

a–c, Alignments of atomic models of BamAM (a), BamD (b) and BamE (c) from our substrate-bound complex (coloured as in Fig. 2) with the corresponding components from a substrate-free, laterally open BAM complex (light orange) (PDB code 5EKQ). d–f, As in a–c, but using a different substrate-free, laterally open BAM complex (blue) (PDB code 5D0Q). g–j, Alignments of atomic models of BamAM (g), BamB (h), BamD (i) and BamE (j) from our substrate-bound complex with the corresponding components from a substrate-free, laterally closed BAM complex (dark red) (PDB code 5D0O). Alignments of BamAM are performed using the β-barrel domains. Alignments of BamB are not shown for complexes in which BamB is absent. Alignments of BamC are not shown because BamC in our structure was obtained by docking BamC as a rigid body from PDB code 5D0Q. The root mean square deviation (RMSD) value for each alignment, obtained with PyMOL, is indicated.

Extended Data Fig. 7 BamAS(ΔL1) accumulates on the BAM complex in a largely folded state.

Alignment of the atomic model of the BamAS(ΔL1) substrate (blue) with that of folded wild-type BamA (cyan) (PDB code 4N75). The first two β-strands (β1 and β2, labelled on the structure of wild-type BamA) of BamAS(ΔL1) in our structure could not be resolved. The similarity between the trapped BamAS(ΔL1) and folded wild-type BamA suggests that the former represents an on-pathway folding intermediate.

Extended Data Fig. 8 Low-resolution cryo-EM structure of a substrate-bound BAM complex with an alternative cysteine crosslink.

a, Residue near the N terminus of the β-barrel domain of BamAM that was substituted with cysteine (yellow stick) shown on the structure of the β-barrel domain of BamA (green) (PDB code 5D0O). The N- and C-terminal β-strands are labelled and shown in salmon. b, As in a, but showing residues near the C terminus of the β-barrel domain of BamAS(ΔL1) (blue) that were substituted with cysteine. c, Cysteine crosslinking between 6×His-tagged BamAM and 3×Flag-tagged BamAS(ΔP345/ΔL1) containing the cysteine substitutions shown in a and b. The crosslinker 1,2-bis(maleimido)ethane (BMOE), which has an 8.0 Å spacer arm, was used. The presence of a BAM-complex–substrate crosslink is detected as a high-molecular-weight adduct on anti-His (top) and anti-Flag (middle) immunoblots. An anti-Flag immunoblot of total cell lysates (bottom) shows that BamAS containing different cysteine substitutions is expressed at similar levels. On the basis of these results, E800C was selected for use within BamAS(ΔL1) for cryo-EM and S439C was introduced into BamAM. d, Assessment of heat modifiability of BamAS(ΔL1) used for cryo-EM (ΔL1; right half of blot) that contains the E800C substitution, and BamAS that contains loop 1 and the E800C substitution (WT; left half of blot). The samples in the unboiled lanes can be used to calculate the fraction of each substrate that is folded in vivo (indicated below the blot). BamAS containing the E800C mutation retains the ability to fold in vivo. e, Representative size-exclusion chromatogram of the substrate-bound BAM complex in which cysteine-crosslink formation between the C terminus of BamAS(ΔL1) and the N terminus of BamAM was induced with BMOE. f, SDS–PAGE gels showing peak fractions from size-exclusion chromatography. The left gel shows the complex from e with addition of βME, which cannot break the crosslink formed by BMOE. The right gel shows the complex crosslinked with dithiobismaleimidoethane (DTME), which is similar to BMOE but has a 13.3 Å spacer arm and is cleavable in the presence of βME to visualize the individual components of the complex. g, Cryo-EM map obtained using the BamAM(S439C) and BamAS(ΔP345/ΔL1/E800C) cysteine pair crosslinked with BMOE. The two images show the same view at different contour levels. BamAM and BamAS are labelled. h, Gold-standard Fourier shell correlation (FSC) curves for the low-resolution cryo-EM structure calculated in Relion. The final corrected mask gave an overall resolution of 6.5 Å at FSC = 0.143 (horizontal blue line). i, Comparison of the 4.1 Å structure and the 6.5 Å structure that contains the alternative cysteine pair in BamAM and BamAS. Left, a top-down slice through the 4.1 Å cryo-EM map is shown (similar to the slice in Fig. 3c) and is overlaid with the atomic model generated from that map. Right, a slice, similar to that on the left, through the 6.5 Å cryo-EM map is shown and is overlaid with the atomic model generated from the 4.1 Å map after fitting of the atomic model into the 6.5 Å map as a rigid body. The dashed oval represents the approximate location of the cysteine tether introduced into each complex. The use of different cysteine pairs yielded similar cryo-EM structures, showing that the presence of the disulfide bond in the main (4.1 Å) structure did not introduce a nonnative conformation into the complex. Data shown in c–f are representative of results from two biological replicates.

Extended Data Fig. 9 In vivo photo-crosslinking with wild-type BamA and BamAS(ΔL1).

a, Residues in BamA substituted with the photo-crosslinkable amino acid pBPA (yellow sticks) shown on the structure of the β-barrel domain of BamA (blue) (PDB code 5D0O). These pBPA substitutions were used to observe crosslinks to LPS. The N and C termini of BamA are shown in salmon. All highlighted residues have side chains oriented outward, towards the membrane environment. b, In vivo photo-crosslinking of BamA to LPS. In a and b, the pBPA substitutions that were subsequently used to test crosslinking of stalled substrates to LPS (in Fig. 3d) are indicated in green. c, Residues in β1 and β2 of BamAS(ΔL1) substituted with pBPA shown on the structure of the β-barrel domain of BamA. Colours are as described in a. These positions were selected because β1 and β2 of the substrate are not visible in the cryo-EM structure (Extended Data Fig. 7). d, In vivo photo-crosslinking of Strep-tagged BamAS(ΔL1) containing pBPA at positions F428 or F440 and deletion of POTRA domains 3–5. Immunoblotting was performed using anti-His, anti-Strep and anti-Flag antibodies to detect BamD (loading control), BamAS(ΔL1) and BamAM, respectively. For anti-Flag immunoblot, a longer exposure (top) to detect crosslinks, and a shorter exposure (bottom) are shown. Although β1 and β2 of BamAS(ΔL1) are not visible in the cryo-EM structure, the in vivo photo-crosslinking results show that residues within these β-strands are in proximity to BamAM. The experiment in b was performed once, and the pBPA substitutions indicated in green were tested again in Fig. 3d. Data shown in d are representative of results from two biological replicates.

Extended Data Fig. 10 Proposed model of β-barrel assembly by the BAM complex.

a, The substrate is recruited to the BAM complex. BamAM is in a closed state before the substrate-induced opening of its lateral gate64. For simplicity, only BamAM, the substrate and BamD are shown. b–d, The C terminus of the substrate interacts with the exposed N-terminal edge of BamAM, and β-strands or β-hairpins of the substrate are added sequentially from the C terminus to the N terminus. Early folding may occur within the interior of the BamAM β-barrel12, and folded portions of the substrate may then be released outward. Full membrane integration could occur after a substantial amount of folding. The steps shown in b and c represent intermediate stages to that shown in d, which corresponds to the cryo-EM structure of the substrate-bound BAM complex. e, The substrate is released into the membrane environment once its N- and C-terminal ends are joined.

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This file contains Supplementary Figure 1, the uncropped gels, and Supplementary Tables 1-5.

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Tomasek, D., Rawson, S., Lee, J. et al. Structure of a nascent membrane protein as it folds on the BAM complex. Nature 583, 473–478 (2020). https://doi.org/10.1038/s41586-020-2370-1

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