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


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.

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Figure 1: Schematic representation of PG biosynthesis and assays used to measure the translocation of lipid II.
Figure 2: Mass spectra of MurJ and FtsW before and after addition of lipid II.
Figure 3: Determination of dissociation constants for lipid II binding to MurJ.
Figure 4: Structure of lipid II showing binding sites for antibiotics and mass spectra recorded after addition of antibiotics to MurJ and the Mur:lipid II complex.
Figure 5: Structures of MurJ with residues investigated through mutation, their effects on lipid II binding and spectra recorded for wild type and A29C after treating with MTSES in the presence of lipid II.
Figure 6: Mass spectra reveal the effect of increasing CDL concentration on lipid II binding to MurJ.
Figure 7: Schematic representation of the competition for lipid II binding between proteins, formation of a ternary complex with the antibiotic vancomycin, and interplay between CDL and lipid II binding to MurJ.

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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.

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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.

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Correspondence to Carol V. Robinson.

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Bolla, J., Sauer, J., Wu, D. et al. Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ. Nature Chem 10, 363–371 (2018).

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