Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis^1,2,3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets^4,5. The natural compound darobactin targets the bacterial insertase BamA⁶—the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins^{7,8,9,10,11,12,13}. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.

需要以新方式针对革兰氏阴性菌的抗生素来解决抗菌素耐药性危机^1,2,3。革兰氏阴性菌受到额外的外膜保护，使细胞表面的蛋白质成为有吸引力的药物靶标^4,5。天然化合物 darobactin 靶向细菌插入酶 BamA 6——^基本BAM 复合体的中心单元，它促进外膜蛋白^{7,8,9,10,11,12,13的折叠和插入}. BamA 缺乏一个典型的催化中心，并且像 darobactin 这样的小分子如何抑制它的功能并不明显。在这里，我们结合使用低温电子显微镜、X 射线晶体学、天然质谱、体内实验和分子动力学模拟，在原子水平上解决了 darobactin 的作用模式。两次环化以刚性 β 链构象预组织 darobactin 肽。这创建了一个模拟原生底物的识别信号，具有与 BamA 侧门结合的卓越能力。结合后，darobactin 取代了侧门的脂质分子，将膜环境用作扩展的结合袋。因为 darobactin 和 BamA 之间的相互作用主要由骨干接触介导，它对潜在的抗性突变特别有效。我们的结果将侧门确定为 BamA 中的功能热点，并将允许合理设计针对这种细菌致命弱点的抗生素。