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SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains

Nature volume 626, pages 617–625 (2024)Cite this article

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Abstract

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid—lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2,3,4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic β-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate β-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

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Fig. 1: slyB is essential under PhoP/Q-inducing OM-stress conditions.
Fig. 2: SlyB acts as an OM guard protein under LPS-stress conditions.
Fig. 3: SlyB binds to the OM proteome under stress.
Fig. 4: SlyB forms lipid nanodomains.
Fig. 5: The role of SlyB during loss of lipid asymmetry.

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Data availability

Cryo-EM potential maps and 3D coordinates of the reported complexes have been deposited at the EMDB and PDB under the following accession codes: SlyB10 (EMD-12945), SlyB11 (EMD-12950; 7OJG), SlyB12 (EMD-12946), SlyB13 (EMD-12947), SlyB13–BamA (EMD-12949; 7OJF) and SlyB13–BamA (EMD-12948). The MS proteomics data have been deposited at the ProteomeXchange Consortium through the PRIDE partner repository under dataset identifier PXD041008. Uncropped gels and western blots are available in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

We thank M. Fislage and A. Schröfel for assistance with data collection at the VIB-VUB facility for Bio Electron Cryogenic Microscopy (BECM); and E. Parthoens at VIB Ghent Imaging Core for assistance in confocal imaging. A.J. and V.S.N. are recipients of a PhD and senior post-doctoral fellowship of the Flanders Research Foundation (FWO; FWOTM903 and 12ZM421N, respectively). We acknowledge financial support from VIB, FWO EOS grant G0G0818N and FWO research infrastructure grant G0H5916N. A.J.C. and A.J.P. acknowledge support from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under grant R21AI168838.

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Author notes

  1. These authors contributed equally: Arne Janssens, Van Son Nguyen

Authors and Affiliations

  1. Structural and Molecular Microbiology, VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium

    Arne Janssens, Van Son Nguyen, Sander E. Van der Verren & Han Remaut

  2. Department of Bioengineering Sciences, Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Arne Janssens, Van Son Nguyen, Sander E. Van der Verren & Han Remaut

  3. Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, USA

    Adam J. Cecil & Alexander J. Pak

  4. VIB Proteomics Core, Ghent, Belgium

    Evy Timmerman & Francis Impens

  5. VIB-UGent Center for Medical Biotechnology, Ghent, Belgium

    Evy Timmerman & Francis Impens

  6. Faculty of Medicine and Health Sciences, Department of Biochemistry, Ghent University, Ghent, Belgium

    Evy Timmerman & Francis Impens

  7. Walloon Excellence in Life Sciences and Biotechnology, WELBIO, Brussels, Belgium

    Michaël Deghelt & Jean-François Collet

  8. De Duve Institute, Université Catholique de Louvain, Brussels, Belgium

    Michaël Deghelt & Jean-François Collet

  9. Quantitative Biosciences and Engineering, Colorado School of Mines, Golden, CO, USA

    Alexander J. Pak

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Contributions

V.S.N. produced expression constructs and performed biochemical and structural studies of SlyB complexes. V.S.N. and H.R. collected cryo-EM data. A.J. produced mutant strains and performed biochemical and cell-based experiments and microscopy. S.E.V.d.V. assisted with cryo-EM grid preparation and data analysis. A.J.C. and A.J.P. performed and supervised the molecular-dynamics simulations. M.D. and J.-F.C. contributed materials and performed microscopy. E.T. and F.I. performed and coordinated LC–MS/MS proteomics. H.R. supervised and conceptualized the study. V.S.N., A.J. and H.R. analysed data and wrote the paper, with contributions from all of the authors.

Corresponding author

Correspondence to Han Remaut.

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Competing interests

A.J., V.S.N. and H.R. are named as inventors on a patent application describing the use of SlyB nanodomains as a vaccination platform.

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Extended data figures and tables

Extended Data Fig. 1 Identification of the SlyB:BamA and SlyB:YnfB complexes.

(a,b) SEC profiles (a) and SDS-PAGE analysis (b) of recBamA purified from E. coli BL21AI pBamA (blue; left y-axis) showed the presence of BamA (B) as well as a contaminant shoulder at higher molecular weight (A). The latter fraction contained BamA as well as a ~14 kDa protein, identified as SlyB by MS peptide fingerprinting (red arrow). Shown in green is the SEC profile of SlyB:BamA (labled 3; right y-axis) purified from E. coli BL21AI pSlyB_BamA by 2-step affinity chromatography. (c) SDS-PAGE of purified SlyB (lane 1), BamA (lane 2), or the SlyB:BamA complex purified by 2-step affinity chromatography (i.e. lane 3). (d) α-BamA and α-SlyB stained western blot of the fraction containing the SlyB:BamA complex (lane 3). (e) Representative cryoEM micrograph of SlyB:BamA complex at 60K magnification. Top and side views are indicated by red and blue circles, resp. (f) Selected 2D cryoEM classes showing side (upper) and top (lower) views of SlyB:BamA complex, as well as the schematic presentation of the complex in both views. Green: BamA, blue: SlyB and orange: membrane/detergent (M). The topview reveals a SlyB oligomer comprising 13 or 14 SlyB copies that enclose a single BamA copy (SlyB13-BamA and SlyB14-BamA, resp.). (M: detergent micelle). (g) WB analysis of whole cell extract of BW25113 pslyBHis-ynfBStrep cells grown on LB, affinity purified over Ni-IMAC (1; ‘His-pulldown’) or streptavidin (2; ‘Strep-pulldown) and stained with α-His (left) or α-Strep (right) primary antibody. Experiments shows the dual affinity pulldown of a SlyB:YnfB complex. The strep-pulldown shows the presence of a N-terminal breakdown product of YnfB (*) not found in the his-pulldown, and thus unable to form a SlyB complex. (h) Superose 6 size exclusion chromatogram and coomassie stained SDS PAGE (inset) of a 1% DDM extract and Strep pulldown of BW25113 pslyBHis-ynfBStrep shows the presence of a high (i) and low (ii) molecular mass fraction, corresponding to SlyB:YnfB and monomer or low oligomeric YnfB, resp. (i) cryoEM electron micrograph and selected 2D class averages of the BW25113 pslyBHis-ynfBStrep strep-pulldown (ie. sample (2)) shows YnfB binds the periplasmic side of a SlyB OMD. (j) Growth curves of BW25113 (WT) and its isogenic ΔslyB or ΔynfB mutants, grown on LB or LB supplemented with 1mM EDTA. Mean ± s.d. (n = 3 biological replicates). Loss of YnfB does not result in the conditional lethality seen for the ΔslyB mutant, indicating the YnfB complex is not implicated in the stabilizing activity of SlyB during EDTA-induced OM destabilization.

Source Data

Extended Data Fig. 2 SlyB is required for growth under OM stress conditions.

(a) Close-up of early exponential phase growth of E. coli BW25113 and derivative strains ΔslyB, ΔphoP and ΔslyB::slyB grown on LB and LB + 1mM EDTA. Calculated generation times for the respective strains and growth media. (n = 3 biological replicates). (b) Phase contrast imaging of BW25113 ΔslyB grown on LB agar + 5mM EDTA. A single colony is shown at indicated time-points from innoculation, corresponding to generation 5 to 9 of a single parental cell. Under EDTA stress, ΔslyB stop dividing and abruptly lyse after ~4-6 generations. Images representative of >20 single colonies tracked, from at least 3 biological replicates. Scale bar = 5 μm. (c) OD600 growth curves of E. coli BW25113 and derivative strains ΔslyB, ΔphoP and ΔslyB::slyB in N minimal medium with defined concentrations of Mg2+. Mean ± s.d. (n = 3 biological replicates). (d) OD600 growth curves of E. coli BW25113 (WT) and BW25113 ΔslyB, transformed with ppmrAR81S. All cells grown in LB, with (I) or without (NI) 0.1% l-arabinose for pmrAR81S induction. Mean ± s.d. (n = 3 biological replicates). (e) OD600 growth curves of S. enterica Sv. Thyphimurium strains LT2, and adherent invasive E. coli strain LF82, grown in LB or LB supplemented with 1 mM EDTA. Mean ± s.d. (n = 3 biological replicates). (f) OD600 growth curves of E. coli BW25113 (WT), BW25113 ΔslyB, BW25113 ΔslyB attTn7::SlyB and BW25113 ΔslyB attTn7::SlyBTEVHIS grown on LB and LB + EDTA (1 mM). Mean ± s.d. (n = 3 biological replicates).

Source Data

Extended Data Fig. 3 OMP and OM stabilizing activity of SlyB OMDs.

(a, b) Timelapse epifluorescence imaging of BW25113 (WT) or BW25113 ΔslyB cells transformed with pTHV037-ssdsbA-sfTq2C70V-mcherry for labelling of periplasm and cytoplasm with sfTq2OX and mCherry, resp. Cells were grown on LB, stressed with 1mM EDTA (a) or 100 μg.mL−1 Bac2A (b) and imaged at indicated time post stress. EDTA stressed cells show a polar accumulation of the sfTq2OX signal by plasmolysis. In ΔslyB cells, the heightened periplasm at the poles is shed in the form of OMVs. Scale bars = 1 μm. (c, d) RNAseq transcriptome data of BW25113 (WT) and BW25113 ΔslyB cells grown on LB and exposed to 1mM EDTA for the indicated time (SI Table 3). Shown are volcano plots comparing WT transcriptome prior to and 60 min after addition of EDTA (c) or comparing BW25113 (WT) and BW25113 ΔslyB transcripts at 30 and 60 min after addition of EDTA (log2 fold change and Wald test p values; n = 6 biological replicates). Transcripts are coloured according subcellular location: OMP (red), OML (blue), periplasmic (orange), IMP (black) and cytoplasmic (grey), Secreted (pink). In d, transcripts for cytoplasmic proteins are omitted for clarity. EDTA stress results in a large-scale shift in gene expression profiles, with some notable high significance, high fold change cell-envelope associated transcripts annotated: periplasmic metal (Zn2+) import cofactors znuA and zinT, lysozyme inhibitor pliG, and periplasmic or outer membrane lipoproteins of unknown function yahO, ydcL, ybjP and blc. With few exceptions (iron import pores fhuE and fepA, and high osmolarity porin ompC), EDTA stress does not result in a significant up- or downregulation of OMPs. No significant differences in OMP expression are found between WT or ΔslyB cells. (e, f) Quantitative Western analysis of cell-associated or shed BamA (i.e. in the cell pellet (e) or ultracentrifugation of culture supernatant (f)) of BW25113 and BW25113 ΔslyB grown on LB and stressed for 30 min with (buffer; ‘LB’), 1mM EDTA, 100 μg.mL−1 Bac2A or 0.1 μg.mL−1 PF-04753299 (LpxCINH). Cell associated [BamA] levels (e) are normalized to WT buffer control levels. Mean ± s.d. N = 3 biological replicates. Supernatant and OMV-associated BamA shown as absorption units. Null hypothesis was analysed by unpaired two-sided t-test where in (e) *: p = 0.02, **: p = 0.004 and ns: non-significant and in (f) *: p = 0.01.

Source Data

Extended Data Fig. 4 SlyB expression and interactome under stressed and non-stressed conditions.

(a) WB of whole cell SDS PAGE of BW25113 (WT), the complemented knockout strain BW25113 ΔslyB attTn7::slyB (‘C’) and BW25113 ΔphoP (right) grown on LB supplemented with 200 mM Mg2+ (1, LB+), on LB (2), or on LB supplemented with 1mM EDTA (3) or 100 μg.mL−1 Bac2A (4). Experiment shows the proportion of PhoP-independent SlyB expression and the PhoP-dependent induction of SlyB during LPS destabilizing conditions. (b) α-SlyB Western analysis of snPAGE of BW25113 ΔphoP whole cell lysates. In absence of phoP, little to no SlyB OMDs (SlyBC) are formed. (c) α-SlyB stained snPAGE of BW25113 cells grown up to OD600 of 0.1 on N minimal medium with indicated Mg2+ concentrations. (d, e) SEC profile and fractionated coomassie-stained (d) snPAGE and its α-SlyB Western blot (e) of Ni-NTA pulldowns from BW25113 ΔslyB::slyBTEV_His grown on LB-EDTA (5mM) medium. Elution volumes of MW standards indicated by vertical lines. SEC elution fractions are indicated above lanes. Bands corresponding to monomeric SlyB or high molecular weight SlyB complexes are labelled SlyBM and SlyBC, resp. (f, g) Western analysis of the snPAGE (f) and denaturing SDS-PAGE (g) of the SEC fractionated Ni-NTA pulldowns as in panel a. Western blot analysis use α-BamA, α-LptD, α-OmpA, α-OmpC or α-SlyB as primary immune serum; as indicated. MW: standards with molecular weight indicated in KDa. (h, i) Representative 2D cryoEM classes from Ni-NTA pulldowns of BW25113 ΔslyB::slyBTEV_His grown in LB (h) or LB + 5mM EDTA medium (i), generated by cryoSPARC52. Both datasets contain a series of homogeneous classes corresponding to a SlyB:YnfB complex (see Extended Data Fig. 1g, h, j) corresponding to 6 and 14 % of the aligned particles in the LB and LB + EDTA datasets, resp. (top row in h and i). The YnfB protein was identified as the dominant tryptic peptide in the MS peptide fingerprint and the sole significant periplasmic SlyB binding partner in the LB dataset (see SI Table 3). The remaining particles in the LB correspond to SlyB oligomers (SlyBO; middle five rows) of variable diameter and protomer number (as judged by top views), as well as a smaller fraction of particles corresponding to micelles with a low molecular weight SlyB complex (a single to a few SlyB protomers only; bottom row). In the LB + EDTA dataset, the majority of particles corresponds to SlyB oligomers as well as different SlyB:OMP complexes (bottom five rows, and Fig. 3f). For some classes, the molecular identity of the encapsulated OMP can be decerned by its structural properties (See Fig. 3f). Box size = 275 Å.

Source Data

Extended Data Fig. 5 Structure determination of SlyB oligomers.

(a) SEC profile (Superdex S200 16/600 column) of affinity purified SlyB. SlyB was over-expressed in E. coli BW25113 carrying pSlyB following by a 2-step Ni-IMAC purification on SlyB. (b) Blue-native PAGE of the elution fractions from SEC run shown in (a). Red arrow indicates the fraction that was used for cryo-EM single particle analysis. Bands corresponding to SlyB complexes or SlyB oligomers are labelled SlyBC and SlyBO, resp. (c) 2D classification analysis of SlyBO single particles corresponding to the fraction indicated with the red arrow in panel a and b. Panel shows representative 2D classes generated by Cryosparc52. (d) Selected 2D classes showing top views of different SlyB oligomerization states (labelled SlyBX, with X = the number of protomers in the particle) present in the analysed fraction. Relative abundance of the oligomeric states indicated in parentheses. (e) Summary of the data processing strategies for the different SlyB oligomer reconstructions described in this paper. See methods section for full description of processing procedure. Steps performed using Relion51 or Cryosparc52 are coloured green or red, resp. Mask corrected FSC curves are shown in Extended Data Figure 6b. Angular distribution plot for particles used for reconstruction of the highest resolution model, SlyB11, is shown in SI Figure 2. Data and model statistics for SlyB11 are found in Extanded Data Figure 10.

Source Data

Extended Data Fig. 6 Structure determination of the SlyB:BamA complex.

(a) Summary of the data processing strategies for the cryoEM 3D reconstruction of SlyB:BamA complexes (see Extended Data Fig. 1 for raw particles and representative 2D classes). Steps performed using Relion52 or cryoSPARC52 are coloured green or red, resp. Details of the processing procedures are described in the methods section. (b, c) Mask corrected Fourier Shell Correlation curves for the final 3D reconstructions of SlyB oligomer (b) and SlyB:BamA (c) complexes. Average map resolutions mentioned throughout the manuscript are according to the FSC 0.143 criterion (shown as dotted line). Data and model statistics for SlyB11 (PDB entry 7OJG) and SlyB13:BamA (PDB entry 7OJF) are found in Extanded Data Figure 10.

Source Data

Extended Data Fig. 7 Structure determination of the SlyB:BtuB and SlyB:TSX complexes.

(a, b) Summary of the data processing strategies for the cryoEM 3D reconstruction of SlyB:BtuB (a) and SlyB:TSX (b) complexes. Steps performed using Relion52 or cryoSPARC52 are coloured green or red, resp. Details of the processing procedures are described in the methods section. FSC curves represent mask corrected FSC of the final 3D reconstructions of the indicated models.

Extended Data Fig. 8 Outer leaflet phospholipid triggers SlyB oligomerization.

(a) cryoEM map of SlyB12 shown in mesh representation carved (3 Å) around a SlyB monomer, viewed from the luminal side, and with map coloured: orange (LPS), magenta (PL), light blue (SlyB) and blue (N-terminal lipid anchor, i.e palmitic acid amide PLM and diacyl glycerol thioether, DAG). Selected residues are labelled: Cys18, Ile65, Val69 and Phe73. Inset: coomassie-stained and α-LPS stained Western blot of SlyBC complexes run on snPAGE with (+) or without (-) heating (5 min 95 °C) of sample. LPS remains bound to SlyBC under semi-native conditions. Map contoured at σ = 2, with highlighted sections carved at 3.0 Å around the corresponding model coordinates. (b) Solvent accessible surface representation of the SlyB12 model (coloured sand and with 1 SlyB protomer in rainbow from blue (N-terminus) to red (C-terminus)), shown in side view (left) and cross-sectional side view (right). The bound LPS, PL and the N-terminal lipid anchor are shown in stick representation, and with cryoEM map shown as mesh, coloured grey, blue and magenta, respectively. Map contoured at σ = 2, with highlighted sections carved at 3.0 Å around the corresponding model coordinates. (c) Density gradient ultracentrifugation (2.02 – 1.44 – 0.77 M sucrose) of total membranes isolated from BW25113 ΔslyB with chromosomal complementation with slyBTEV_His (left) or slyBPL (right), a mutant (I65A, V69A, F73A) disrupting the outer leaflet PL binding site. Fractions (high to low density from left to right) are run on denaturing SDS-PAGE followed by Western analysis with α-SlyB or α-DsbD and α-OmpC as representative inner and outer membrane proteins. (d) Time-series profiles from coarse-grained (CG) MD simulations. Top row: 11-fold symmetry order parameter (\({\psi }_{11}^{2D}\)) of the transmembrane domain of SlyB over simulation time for 3 replicas each with symmetrical luminal PL:PL bilayer (left), asymmetrical LPS:PL bilayer (middle), or empty pore (right). Bottom row: RMSD of the transmembrane domain of SlyB with respect to the CG-mapped atomic model over simulation time. All time-series plots are calculated from the NPT production runs. (e) Volcano plot of the SlyBPL interactome identified by peptide mass fingerprint of Ni-NTA pulldowns from BW25113 ΔslyB::slyBPL-TEV_His in LB+EDTA showing log2 fold change and t-test p values (n = 3 biological replicates) (SI Table 5). Proteins are coloured according to subcellular localization: OMP (red), OML (blue), periplasmic (orange), IMP (black) and cytoplasmic (purple). N = 3 biological triplicates. (f) Thermal unfolding plot of purified BamA (blue) and SlyB:BamA complexes (red), showing % denatured BamA as monitored by snPAGE (inset panels; C: high MW SlyB:BamA complexes, U: unfolded BamA and F: folded BamA). Mean ± s.d. (n = 3). Tm: melting temperature (T at 50% denaturation). Mean ± s.d. (n = 3). (g) Cellular Thermal Shift Assay (CETSA) following folded and unfolded BamA in BW25113 (WT) and BW25113 ΔslyB cells grown on LB + EDTA for 1 h, and subjected to 5 min incubations to increasing temperatures prior to whole cell snPAGE and α-BamA Western analysis. (h) Quantitative Western analysis of the fraction of free BamA and OmpC (i.e. flowthrough on Ni IMAC, labelled FT) versus BamA and OmpC found in the form of SlyB nanodomain complexes (i.e. retained on Ni-IMAC as result of interaction with SlyBTEV_His) using AJ1 attTn7::slyBTEV_His cells grown on LB (-), or cells grown on LB and stressed for 30’ with 1mM EDTA.

Source Data

Extended Data Fig. 9 Structure properties of the 2TM glycine zipper domain.

(a) helical wheel representation of the SlyB α1 and α2 helices that together form a conserved 2TM glycine zipper domain, and multiple sequence alignment of the corresponding regions of the six different 2TM Gly zipper domain containing proteins commonly identified in the E. coli genome (sequences show Uniprot ID and protein name, as well as MW of the full length proteins; numbering according to SlyB sequence). Hydrophobic and polar side chains are coloured orange sand and white, resp. the conserved Gly or Ala residues in the GXXXG motifs forming the Gly zipper are coloured magenta. Dotted lines connect opposing residues in the α1 - α2 helical packing. (b) Ribbon representation of the 2TM Gly zipper domain as found in the SlyB structures reported in this study. Side chains are shown in stick representation, with N atoms coloured blue, O atoms coloured red and C atoms colour coded according to (a). Shown as pruple, yellow and magenta stick representation are the N-terminal lipid anchor, and a bound LPS and PL molecule in the SlyB protomers. LPS binds the loop connecting α1 and α2 by means of three H-bonds (red dotted lines), two between LPS fatty acids and the G79 and G80 backbone amine, and one between the D-glycosamine-4-phosphate and T82 side chain hydroxyl. In the transmembrane region α1 and α2 pack by the GXXXG knobs-in-hole pattern, with an additional stabilization of three H-bonds connecting the N- and C-terminal region of α1 and α2 resp. (i.e. N60 carbonyl – Q100 side chain amide and the N60 side chain amide with the Q103 side chain amide and the S104 hydroxyl). OM: outer membrane. (c, d) Side and top view ribbon representation of two adjacent SlyB protomers in the SlyB11 oligomer structure (see Fig. 3), with the 2TM domains, lipid anchor, LPS and PL coloured as in Extended Data Fig. 5; and the periplasmic domains coloured blue to red from N- to C-terminus. Secondary structure elements are labelled α1 - α2 and β1 - β6 with A or B superscript for the left or right protomer, resp. Protomers interact by β-sheet augmentation in the periplasmic domain, i.e. strand β1 pairs with β4 of the adjacent SlyB protomer. In the transmembrane region, the protomers interact by non-specific hydrophobic contacts, so that SlyB does not form an obligate oligomer, and can also exist as a stable monomeric or non-circular, low MW oligomeric transmembrane protein. (e) Side-by-side comparison of side and top view cryoEM 2D classes of the OmpC trimer, LMW SlyB:OmpC complexes and SlyB18-OmpC complexes identified in a SlyB – OmpC double affinity purification. Top view classes of LMW SlyB:OmpC complexes did not show a well-resolved binding site for SlyB. (f) Schematic representation of SlyB:OMP nanodomains and side and top view cryoEM 2D classes of SlyB14:BtuB and SlyB12:TSX purified by double affinity purification. Top view classes show the presence of the BtuB or TSX β-barrels encapsulated by a SlyB oligomer of variable protomer number corresponding to the diameter of the enclosed OMP. (left) side view cross section and top view of the cryoEM reconstructions of the SlyB14:BtuB and SlyB12:TSX complexes. The N-terminal lipid anchor, PL, LPS and detergent micelle (M) are coloured slate, magenta, yellow and light blue, resp. β-barrels and SlyB oligomer are separated by a luminal PL bilayer, and SlyB oligomers are surrounded by a LPS ring. Side view classes show the inner core glycans protruding over the rim of the SlyB oligomer, as also seen for SlyB oligomers (Extended Data Fig. 4) and SlyB:BamA complexes (Extended Data Fig. 5). Maps contoured at σ = 3.4 (SlyB12:TSX) or σ = 3.6 (SlyB14:BtuB), with highlighted sections carved at 3.0 Å around the corresponding model coordinates. (g) Species wheel representation of the genomic distribution of the 2TM Gly zipper family (PFAM family PF05433; https://pfam.xfam.org/family/Rick_17kDa_Anti#tabview=tab7). At least 12 discrete protein architectures containing the 2TM Gly zipper domain can be discerned, encompassing at least 4133 sequences originating from 1832 annotated species. Individual genomes hold 1 to 12 2TM Gly zipper containing sequences. The primary occurrence of 2TM Gly domain containing sequences is in Gram-negative phylum proteobacteria, with a smaller fraction in Ascomycete fungi. SP: signal peptide; 2TM: 2TM Gly Zipper domain (green); OmpA: OmpA domain (blue; PFAM: PF00691; cell wall anchoring); Rick_17KDa: 17 kDa outer membrane surface antigen domain (fuchsia; PFAM: PF16998); CVNH: CyanoVirin-N Homology domain (yellow, PFAM: PF08881; glycan binding domain); LysM: LysM domain (magenta; PFAM: PF01476; peptidoglycan binding); β/γ cryst: bacterial β/γ crystallin domain (brown; PFAM: PF00030); RcnB: Nickel/cobalt transporter regulator domain (dark orange; PFAM: PF11776).

Extended Data Table 1 CryoEM data and model statistics
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Supplementary information

Supplementary Information

Supplementary Fig. 1: uncropped gel and western blot images. Supplementary Fig. 2: orientation distribution plots of Cryo-EM 3D reconstructions used in this study. Supplementary Table 1: strains, plasmids, primers and proteins, and additional references.

Reporting Summary

Supplementary Table 2

Quantitative LC–MS analysis of the proteome of WT and AJ1 cells with and without the addition of EDTA. The table contains the full MaxQuant proteomics data and comparisons between strains and conditions (Methods).

Supplementary Table 3

Transcriptomics data of the RNA-seq of WT and AJ1 cells at 0 min, 30 min and 60 min after the addition of 1 mM EDTA. The table contains the raw counts of the RNA-seq and comparisons between different strains or different times (Methods).

Supplementary Table 4

LC–MS analysis of the SlyB interactome. The table contains the full MaxQuant proteomics data of the SlyB-TEV-His pull-downs under LB and LB + EDTA conditions and comparison between conditions (Methods).

Supplementary Table 5

LC–MS analysis of the SlyBPL interactome. The table contains the full MaxQuant proteomics data of the SlyBPL-TEV-His pull-downs under LB and LB + EDTA conditions and a comparison between conditions (Methods).

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Janssens, A., Nguyen, V.S., Cecil, A.J. et al. SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains. Nature 626, 617–625 (2024). https://doi.org/10.1038/s41586-023-06925-5

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