| Izvleček: | Translocation of lipid II across cytoplasmic membrane is essential in peptidoglycan biogenesis. While 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 characterise 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, and chemical modification of A29 eliminate, lipid II binding to MurJ. 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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