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.

脂质 II 跨细胞质膜的易位在肽聚糖的生物发生中是必不可少的。尽管大多数步骤都已被理解，但识别脂质 II 翻转酶产生了相互矛盾的结果，而两种候选翻转酶——MurJ 和 FtsW——的脂质 II 结合特性在很大程度上仍然未知。在这里，我们将天然质谱应用于两种蛋白质并表征脂质 II 结合。与 MurJ 相比，我们观察到与 FtsW 结合的脂质 II 水平较低，这与 MurJ 具有更高的亲和力一致。MurJ 的定点诱变表明 A29 和 D269 的突变减弱了脂质 II 与 MurJ 的结合，而 A29 的化学修饰消除了结合。抗生素 ramoplanin 将脂质 II 从 MurJ 中解离出来，而万古霉素与 MurJ:lipid II 结合形成稳定的复合物。此外，我们揭示了与 MurJ 相关的心磷脂，但与 FtsW 无关，并且外源性心磷脂降低了脂质 II 与 MurJ 的结合。这些观察结果提供了对脂质 II 与 MurJ 结合的决定因素的见解，并提出了内源性脂质在调节底物结合中的作用。