Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3-β4 loop conformation and a high-affinity state with an inward β3-β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3-β4 loop conformation or stabilizing the extended β3-β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

中文翻译：

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来自头孢曲松耐药性淋病奈瑟菌的 PBP2 突变改变了 β3-β4 环的动力学，有利于低亲和力药物结合状态。

致病菌淋病奈瑟菌对超广谱头孢菌素头孢曲松的耐药性是由青霉素结合蛋白 2 (PBP2)（抗生素的致死靶点）的突变赋予的，但这些突变如何在分子水平上发挥作用尚不清楚。使用溶液核磁共振、X 射线晶体学和等温滴定量热法，我们报告说 WT PBP2 在具有扩展 β3-β4 环构象的低亲和力状态和具有向内 β3-β4 环构象的高亲和力状态之间动态交换。Histidine-514 位于 β4 链的边界，在这两种构象状态之间的交换过程中起着重要作用。我们还发现 H041 的 PBP2 中存在突变，H041 是一种耐头孢曲松的淋病奈瑟菌菌株，通过破坏内向的 β3-β4 环构象或稳定延伸的 β3-β4 环构象以有利于低亲和力药物结合状态来增加对头孢曲松的抗性。这些观察结果揭示了头孢曲松耐药的独特机制，即 PBP2 的突变降低了处于高亲和力药物结合状态的靶分子的比例，从而降低了在较低药物浓度下的抑制作用。