Article | Published:

Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ

Nature Chemistry volume 10, pages 363371 (2018) | Download Citation

Abstract

Translocation of lipid II across the cytoplasmic membrane is essential in peptidoglycan biogenesis. Although most steps are understood, identifying the lipid II flippase has yielded conflicting results, and the lipid II binding properties of two candidate flippases—MurJ and FtsW—remain largely unknown. Here we apply native mass spectrometry to both proteins and characterize lipid II binding. We observed lower levels of lipid II binding to FtsW compared to MurJ, consistent with MurJ having a higher affinity. Site-directed mutagenesis of MurJ suggests that mutations at A29 and D269 attenuate lipid II binding to MurJ, whereas chemical modification of A29 eliminates binding. The antibiotic ramoplanin dissociates lipid II from MurJ, whereas vancomycin binds to form a stable complex with MurJ:lipid II. Furthermore, we reveal cardiolipins associate with MurJ but not FtsW, and exogenous cardiolipins reduce lipid II binding to MurJ. These observations provide insights into determinants of lipid II binding to MurJ and suggest roles for endogenous lipids in regulating substrate binding.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Protein Data Bank

References

  1. 1.

    , & The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010).

  2. 2.

    , & Lipid II: a central component in bacterial cell wall synthesis and a target for antibiotics. Prostaglandins Leukot. Essent. Fatty Acids 79, 117–121 (2008).

  3. 3.

    Lipid intermediates in the biosynthesis of bacterial peptidoglycan. Microbiol. Mol. Biol. Rev. 71, 620–635 (2007).

  4. 4.

    & An oldie but a goodie—cell wall biosynthesis as antibiotic target pathway. Int. J. Med. Microbiol. 300, 161–169 (2010).

  5. 5.

    , & Prospects for novel inhibitors of peptidoglycan transglycosylases. Bioorg. Chem. 55, 16–26 (2014).

  6. 6.

    & Structural variations of the cell wall precursor lipid II in Gram-positive bacteria—impact on binding and efficacy of antimicrobial peptides. Biochim. Biophys. Acta 1848, 3062–3071 (2015).

  7. 7.

    , , & Lantibiotic resistance. Microbiol. Mol. Biol. Rev. 79, 171–191 (2015).

  8. 8.

    et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).

  9. 9.

    & LipidII: just another brick in the wall? PLoS Pathog. 11, e1005213 (2015).

  10. 10.

    Lipid flippases for bacterial peptidoglycan biosynthesis. Lipid Insights 8, 21–31 (2016).

  11. 11.

    Filling holes in peptidoglycan biogenesis of Escherichia coli. Curr. Opin. Microbiol. 34, 1–6 (2016).

  12. 12.

    et al. Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane. EMBO J. 30, 1425–1432 (2011).

  13. 13.

    et al. Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis. Science 345, 220–222 (2014).

  14. 14.

    et al. Specificity of the transport of lipid II by FtsW in Escherichia coli. J. Biol. Chem. 289, 14707–14718 (2014).

  15. 15.

    et al. Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli. J. Bacteriol. 190, 7298–7301 (2008).

  16. 16.

    Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase in Escherichia coli. Proc. Natl Acad. Sci. USA 105, 15553–15557 (2008).

  17. 17.

    , & Crystal structure of the MOP flippase MurJ in an inward-facing conformation. Nat. Struct. Mol. Biol. 24, 171–176 (2017).

  18. 18.

    et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc. Natl Acad. Sci. USA 112, 6437–6442 (2015).

  19. 19.

    et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016).

  20. 20.

    et al. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nat. Microbiol. 2, 16253 (2017).

  21. 21.

    et al. Interplay between penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis. Sci. Rep. 7, 43306 (2017).

  22. 22.

    et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).

  23. 23.

    et al. Quantifying the stabilizing effects of protein–ligand interactions in the gas phase. Nat. Commun. 6, 8551 (2015).

  24. 24.

    et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016).

  25. 25.

    et al. Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. Nat. Chem. 8, 1152–1158 (2016).

  26. 26.

    et al. A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nat. Chem. 7, 255–262 (2015).

  27. 27.

    et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).

  28. 28.

    et al. The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase. Angew. Chem. Int. Ed. 54, 4577–4581 (2015).

  29. 29.

    et al. The effect of detergent, temperature, and lipid on the oligomeric state of MscL constructs: insights from mass spectrometry. Chem. Biol. 22, 593–603 (2015).

  30. 30.

    , , , & Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J. Bacteriol. 174, 3549–3557 (1992).

  31. 31.

    et al. Structure of a cation-bound multidrug and toxic compound extrusion transporter. Nature 467, 991–994 (2010).

  32. 32.

    , , , & Structural insights into H+-coupled multidrug extrusion by a MATE transporter. Nat. Struct. Mol. Biol. 20, 1310–1317 (2013).

  33. 33.

    et al. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter. Proc. Natl Acad. Sci. USA 110, 2099–2104 (2013).

  34. 34.

    et al. Structural basis for the drug extrusion mechanism by a MATE multidrug transporter. Nature 496, 247–251 (2013).

  35. 35.

    , & Multidrug transport protein NorM from Vibrio cholerae simultaneously couples to sodium- and proton-motive force. J. Biol. Chem. 289, 14624–14632 (2014).

  36. 36.

    , , & Escherichia coli intracellular pH, membrane potential, and cell growth. J. Bacteriol. 158, 246–252 (1984).

  37. 37.

    et al. Chemistry and biology of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity. Chem. Rev. 105, 449–476 (2005).

  38. 38.

    & Lipid II as a target for antibiotics. Nat. Rev. Drug Discov. 5, 321–332 (2006).

  39. 39.

    et al. A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential lipid II docking interface. Proc. Natl Acad. Sci. USA 106, 13759–13764 (2009).

  40. 40.

    , & Quantification of cardiolipin molecular species in Escherichia coli lipid extracts using liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass. Spectrom. 26, 2267–2274 (2012).

  41. 41.

    & Cardiolipin membrane domains in prokaryotes and eukaryotes. Biochim. Biophys. Acta 1788, 2084–2091 (2009).

  42. 42.

    et al. NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of deploarized mitochondria by mitophagy. Cell Death Diff. 23, 1140–1151 (2016).

  43. 43.

    et al. In vitro susceptibility of Staphyococcus aureus to thrombin-induced platelet microbicidal protein-1 (tPMP-1) is influenced by cell membrane phospholipid composition and asymmetry. Microbiology 153, 1187–1197 (2007).

  44. 44.

    , , & Impact of the β-lactam resistance modifier (−)-epicatechin gallate on the non-random distribution of phospholipids across the cytoplasmic membrane of Staphylococcus aureus. Int. J. Mol. Sci. 16, 16710–16727 (2015).

  45. 45.

    & Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat. Protoc. 2, 715–726 (2007).

  46. 46.

    , , , & Lipidomics profiling by high-resolution LC-MS and high-energy collisional dissociation fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal. Chem. 83, 940–949 (2011).

Download references

Acknowledgements

The authors acknowledge funding from an MRC programme grant (MR/N020413/1), an ERC Advanced Grant ENABLE (641317) and a Wellcome Trust Investigator Award (104633/Z/14/Z). The authors thank W. Vollmer for providing C55-P and H.-Y. Yen, J. Gault and M. Agasid for useful discussions.

Author information

Affiliations

  1. Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK

    • Jani Reddy Bolla
    • , Joshua B. Sauer
    • , Di Wu
    • , Shahid Mehmood
    • , Timothy M. Allison
    •  & Carol V. Robinson

Authors

  1. Search for Jani Reddy Bolla in:

  2. Search for Joshua B. Sauer in:

  3. Search for Di Wu in:

  4. Search for Shahid Mehmood in:

  5. Search for Timothy M. Allison in:

  6. Search for Carol V. Robinson in:

Contributions

J.R.B. and C.V.R. conceived and designed the experiments. J.R.B designed primers and generated the constructs for protein expression. J.R.B and J.B.S. expressed and purified the proteins. J.R.B. optimized the MS conditions and obtained all MS measurements. J.R.B. performed lipidomics analysis with the help of D.W.S.M. and T.M.A. assisted with data analysis. J.R.B. and C.V.R wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Carol V. Robinson.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchem.2919