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Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria

Abstract

Gram-negative bacteria express structurally diverse lipoproteins in their cell envelope. Here, we find that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. As we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system, whereby linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer-membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function.

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Fig. 1: Structural analysis of lipoproteins reveals that half of outer-membrane lipoproteins display an intrinsically disordered linker at the N terminus.
Fig. 2: The N-terminal linker displayed by lipoproteins is important for outer-membrane targeting.
Fig. 3: The length and the disordered character of the RcsF linker play key roles in RcsF targeting to the outer membrane.
Fig. 4: N-terminal disordered linkers interact with the Lol system to target lipoproteins to the outer membrane.

Data availability

Source data are provided with this paper. All other data generated or analyzed during this study are included in this paper and its Supplementary Information file.

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Acknowledgements

We thank A. Boujtat for technical help. We are indebted to the members of the Collet laboratory and to Nassos Typas (EMBL, Heidelberg) for helpful suggestions and discussions and to T. Silhavy (Princeton) and N. Buddelmeijer (Pasteur) for providing bacterial strains. J.S. was a research fellow of FRIA and J.-F.C. is an Investigator of FRFS-WELBIO. This work was funded by WELBIO (grant no. WELBIO-CR-20190-03), by grants from F.R.S.-FNRS, from Fédération Wallonie-Bruxelles (ARC 17/22-087), from the European Commission via the International Training Network Train2Target (721484) and from the EOS Excellence in Research Program of FWO and F.R.S.-FNRS (G0G0818N).

Author information

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Authors

Contributions

J.-F.C., J.E.R., J.S. and S.-H.C. designed and performed the experiments. J.E.R., J.S. and S.-H.C. constructed the strains and cloned the constructs. J.-F.C., J.E.R., J.S., S.-H.C. and A.M. analyzed and interpreted the data. B.I.I. performed the structural analysis, M.D the microscopy analysis and N.C. the dot-blot analysis. J.-F.C., J.E.R. and J.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jean-François Collet.

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The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Vladimir Uversky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structural analysis of lipoproteins reveals that half of outer membrane lipoproteins display an intrinsically disordered linker at the N-terminus.

Structures were generated via comparative modeling. X-ray and cryo-EM structures are green, NMR structures are cyan, and structures built via comparative modeling from the closest analog in the same PFAM group are orange. In all cases, the N-terminal linker is magenta. Lipoproteins targeting the outer membrane: OsmE, NlpC, MltB, NlpE, YajI, YcfL, NlpI, HslJ, YbaY, RlpA, AmiD, BamB, MltC, YcaL, MltA, Blc, YghG, EmtA, BamD, LolB, MlaA, LoiP, YraP, MliC, LpoB, CsgG, YddW, YbhC, YedD, YgeR, YfiB, YbjP, YiaD, GfcE, PqiC, YfeY, LptE, LpoA, BamE. Lipoproteins targeting the IM: DcrB, MetQ, NlpA, ApbE,YcjN, YehR. Synthetic constructs: RcsFGS, RcsFGS2, RcsFGS3, RcsF∆19–47, RcsFFkpA, RcsFcol,Pal∆26–56, BamC Δ28–98,NlpD∆29–64.

Extended Data Fig. 2 The N-terminal linker of lipoproteins is important for outer membrane targeting.

The outer membrane (OM) and inner membrane (IM) were separated via centrifugation in a three-step sucrose density gradient (Methods). While (a) NlpDWT, and (b) PalWT were found predominantly in the OM, NlpD∆29–64, and Pal∆26–56 were substantially retained in the IM. Data are presented as the ratio of signal intensity in a single fraction to the total intensity in all fractions. All variants were expressed from plasmids (Supplementary Table 4). DsbD and Lpp were used as controls for the OM and IM, respectively. Images in a, and b are representative of biological triplicates. Graphs in a and b were created by spline analysis of curves representing a mean of three independent experiments.

Source data

Extended Data Fig. 3 RcsFCDD is retained in the inner membrane.

a, Expression levels of RcsFWT and RcsFCDD. Cells expressing chromosomal RcsFWT or RcsFCDD were grown in LB until OD600 = 0.7 and precipitated with trichloroacetic acid (Methods). Immunoblots were performed using α-RcsF. b, The outer (OM) and inner (IM) membranes of cells expressing chromosomal RcsFWT or RcsFCDD were separated by sucrose density gradient (Methods). While RcsFWT was found predominantly in the OM, RcsFCDD was retained in the IM. Images in a and b are representative of three independent experiments.

Source data

Extended Data Fig. 4 Expression levels of RcsF∆19–47, Pal∆26–56, and NlpD∆29–64.

Cells were grown at 37 °C in LB until OD600 = 0.5 and precipitated with trichloroacetic acid (Methods). Immunoblots were performed with α-RcsF, α-NlpD, and α-Pal antibodies. All images are representative of three independent experiments.

Extended Data Fig. 5 Schematic of RcsF variants used in this study and their distributions in the outer membrane (OM) and inner membrane (IM).

RcsFGS, RcsFGS2, and RcsFGS3 have linkers that are disordered and mostly consist of GS repeats. The linker of RcsFGS is the same length as the linker of RcsFWT. RcsFGS2 and RcsFGS3 are shorter than RcsFWT. Regions of RcsFFkpA and RcsFcol fold into alpha helices borrowed from the sequences of FkpA and colicin Ia, respectively.

Extended Data Fig. 6 Complexes between LolA and RcsFWT or RcsF∆19–47 can be purified.

Both RcsFWT (a) and RcsF∆19–47 (b) were eluted in complex with LolA-His via affinity chromatography followed by size exclusion chromatography. Elution fractions were analyzed via SDS–PAGE and proteins stained with Coomassie Brilliant Blue (Methods). Chromosomally-encoded RcsF variants were detected by immunoblotting fractions with α-RcsF antibodies. Images are representative of three independent experiments.

Extended Data Fig. 7 Overexpression of LolCDE does not restore targeting of RcsF∆19–47.

a, Expression level of LolCDE-His. Cells were grown in LB plus 0.2% arabinose at 37 °C until OD600 = 0.7 (Methods). Membrane and soluble fractions were separated with a sucrose density gradient (Methods). LolE-His was detected in the inner membrane fraction by immunoblotting with α-His (Methods). Images are representative of three independent experiments. b, The outer membrane (OM) and inner membrane (IM) were separated with a sucrose density gradient. Expression of LolCDE did not rescue OM targeting of RcsF∆19–47. Images are representative of experiments performed in biological triplicate.

Source data

Extended Data Fig. 8 Overexpressing Lgt, LspA, and Lnt does not rescue the targeting of RcsF∆19–47 to the outer membrane.

a, Expression levels of Lgt, LspA, and Lnt. Cells were grown in LB (plus 25 µM IPTG for cells expressing LspA) at 37 °C until OD600 = 0.7 (Methods). Outer membrane (OM) and inner membrane (IM) were separated with a sucrose density gradient (Methods). Lgt-Myc and Lnt-Myc were detected in the IM via immunoblotting with α-Myc. LspA-Flag was detected in the IM with α-Flag. b, Cells overexpressing Lgt, LspA, or Lnt were exposed to a sucrose density gradient (Methods). RcsF∆19–47 was retained in the IM in all conditions. Images in a and b are representative of three independent experiments.

Source data

Extended Data Fig. 9 RcsF∆19–47 is processed by Lgt and Lnt.

a, Lgt replete (+) or deplete samples were obtained by growing cells in the presence or absence of L-arabinose. Overnight cultures were grown at 37 °C in LB medium with 0.2 % arabinose; cells were then diluted in fresh LB medium and grown at 37 °C in the absence of L-arabinose for the indicated times to deplete Lgt. b, Lnt replete (+) or deplete samples were obtained from cultures supplemented with L-arabinose or fucose, respectively. Overnight cultures were grown at 37 °C in LB medium in the presence of 0.2 % arabinose; cells were then washed with fresh LB medium, diluted in LB supplemented with 0.2 % fucose and grown at 37 °C for the indicated times to deplete Lnt. Protein samples were subjected to SDS–PAGE tricine or Tris-glycine 16% gel and probed for Lpp and RcsF by immunoblotting. PreproLpp (in the case of Lgt depletion) and apoLpp (in the case of Lnt depletion) are noted as *. The same annotation is used for RcsF variants. The prepro and apo forms of Lpp, RcsFWT and RcsF∆19-47 are detected only when Lgt and Lnt are depleted. This indicates that RcsF∆19–47 and, as expected RcsF and Lpp, are processed by Lgt and Lnt. Images in a and b are representative of biological triplicates.

Extended Data Fig. 10 The disordered linker of BamC and NlpD, two proteins that can be detected on the cell surface, is important for targeting to the outer membrane (OM).

a, NlpD is exposed on the cell surface. Fixed cells were spotted (with or without permeabilization with lysozyme and EDTA) onto nitrocellulose membranes and probed with anti-NlpD antibodies. NlpD is detected on non-permeabilized cells, indicating that the protein is on the cell surface. An HA-tagged version of the periplasmic, soluble protein FkpA, expressed from the chromosome, was used as a permeabilization control. HA-tagged FkpA can only be detected with anti-HA antibodies following permeabilization. b,c, The disordered linker of BamC is important for its targeting to the outer membrane (OM). b, Expression levels of BamCWT-Flag and BamC Δ28–98-Flag. The BamC variants were expressed from a plasmid with a C-terminal Flag-tag. Cells were grown in LB plus IPTG; when OD600 reached 0.6, cells were precipitated with trichloroacetic acid (Methods). Samples were subjected to SDS-PAGE and probed with α-Flag-tag antibodies. c. Cells expressing BamCWT-Flag and BamCΔ28–98-Flag were grown in LB plus IPTG until OD600 reached 0.7. Cells were then subjected to fractionation; the outer (OM) and inner (IM) membranes were separated by sucrose density gradient (Methods). Deletion of the linker of BamC alters the targeting of this lipoprotein to the OM: more BamCΔ28–98 is detected in the IM fractions than BamCWT. Images in a, b,c are representative of biological triplicate.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 2–5.

Reporting Summary

Supplementary Data 1

Rayes_Szewczyk_Supplementary Table 1.

Source data

Source Data Fig. 2

Uncropped gels of sucrose density gradient shown in Fig. 2a. a,b, Uncropped images of the sucrose density gradient of RcsFWT (a) and RcsF∆19–47 (b) showing the anti-DsbD, anti-RcsF and anti-Lpp western blots. The black box outlines the final cropped image.

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 4

Uncropped gels of pulldown shown in Fig. 4b a,b, Uncropped images of Coomassie blue gels and western blots of pulldown of RcsFWT (a) and RcsF∆19–47 (b) showing the anti- anti-RcsF western blot. The black box outlines the final cropped image.

Source Data Fig. 4

Statistical source data for Fig. 4a.

Source Data Extended Data Fig. 2

Uncropped gels of the sucrose density gradient shown in Extended Data Fig. 2. a,b, Uncropped images of the sucrose density gradient of NlpD (a) and Pal (b) showing the anti-DsbD, anti-RcsF and anti-Lpp western blots. The black box outlines the final cropped image.

Source Data Extended Data Fig. 2

Statistical source data for Extended Data Fig. 2.

Source Data Extended Data Fig. 3

Uncropped gels of the sucrose density gradient shown in Extended Data Fig. 3b. Uncropped images of the sucrose density gradient of RcsFWT and RcsFCDD showing the anti-DsbD, anti-RcsF and anti-Lpp western blots. The black box outlines the final cropped image.

Source Data Extended Data Fig. 7

Uncropped gels of the sucrose density gradient shown in Extended Data Fig. 7b. Uncropped images of the sucrose density gradient of RcsF∆19–47, when LolCDE complex is overexpressed or not, showing the anti-DsbD, anti-RcsF and anti-Lpp western blots. The black box outlines the final cropped image.

Source Data Extended Data Fig. 8

Uncropped gels of the sucrose density gradient shown in Extended Data Fig. 8b. Uncropped images of the sucrose density gradient of RcsF∆19–47, when one of the maturation enzymes is overexpressed or not, showing the anti-DsbD, anti-RcsF and anti-Lpp western blots. The black box outlines the final cropped image.

Source Data Extended Data Fig. 10

Uncropped gels of the sucrose density gradient shown in Extended Data Fig. 10c. Uncropped images of the sucrose density gradient of BamCWT-Flag and BamC∆28–98-Flag showing the anti-DsbD, anti-Flag and anti-Lpp western blots. The black box outlines the final cropped image.

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El Rayes, J., Szewczyk, J., Deghelt, M. et al. Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria. Nat Chem Biol 17, 1093–1100 (2021). https://doi.org/10.1038/s41589-021-00845-z

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