Since their inception in antibacterial therapy, macrolide-based antibiotics have significantly shaped the evolutionary pathways of pathogenic bacteria, driving them to develop diverse antimicrobial resistance (AMR) mechanisms. Among these, macrolide esterase, commonly referred to as erythromycin esterase, emerged as a critical defense mechanism, enabling bacteria to detoxify macrolides by hydrolyzing the macrolactone ring within the bacterial cell. In this study, we delve into the intricate interactions and conformational dynamics of erythromycin esterase C (EreC), a key member of the Ere enzyme family. We have focused on three FDA-approved and widely prescribed macrolides─erythromycin, clarithromycin, and azithromycin─by employing classical molecular dynamics, absolute binding free energy calculations, and 2D well-tempered metadynamics simulations to explore their interactions with EreC. To estimate the absolute binding free energies, we have used the recently developed and robust "Streamlined Alchemical Free Energy Perturbation (SAFEP)" protocol. The results from our molecular dynamics simulations and advanced analyses portrayed the crucial role of hydrophobic interactions within the macrolide binding cleft of EreC, along with the significant influence of the minor lobe in facilitating overall structural fluctuation. In silico alanine scanning identified top three hydrophobic residues, i.e., PHE248, MET333, and PHE344, responsible for macrolide binding inside that cleft. According to the free energy calculations, azithromycin and clarithromycin showed greater binding affinities toward EreC than the parent macrolide erythromycin. Moreover, 2D metadynamics simulations along with graph theory-based eigenvector centrality analyses revealed a metastable "semiopen" state during the hypothesized "active loop closure" of the EreC protein triggered by subtle conformational changes of an important histidine residue, HIS289, upon macrolide capture, drawing a fascinating parallel to the renowned "Venus flytrap" mechanism.

中文翻译：

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深入研究红霉素酯酶 C 中的大环内酯结合亲和力和相关结构调节：深入了解捕蝇草机制。


自抗菌治疗开始以来，基于大环内酯类抗生素的抗生素显著塑造了病原菌的进化途径，促使它们发展出多种抗菌素耐药性 （AMR） 机制。其中，大环内酯酯酶，通常称为红霉素酯酶，成为一种关键的防御机制，使细菌能够通过水解细菌细胞内的大内酯环来解毒大环内酯类药物。在这项研究中，我们深入研究了红霉素酯酶 C （EreC） 的复杂相互作用和构象动力学，Ere 酶家族的关键成员。我们专注于三种 FDA 批准和广泛使用的大环内酯类药物——红霉素、克拉霉素和阿奇霉素——通过采用经典分子动力学、绝对结合自由能计算和 2D 调理的元动力学模拟来探索它们与 EreC 的相互作用。为了估计绝对结合自由能，我们使用了最近开发且强大的“简化炼金术自由能扰动 （SAFEP）”协议。我们的分子动力学模拟和高级分析的结果描述了 EreC 大环内酯结合裂隙内疏水相互作用的关键作用，以及小叶在促进整体结构波动中的显着影响。在计算机模拟中，丙氨酸扫描确定了前三个疏水残基，即 PHE248、MET333 和 PHE344，负责该裂隙内的大环内酯结合。根据自由能计算，阿奇霉素和克拉霉素对 EreC 的结合亲和力大于母体大环内酯类红霉素。 此外，二维元动力学模拟以及基于图论的特征向量中心性分析揭示了在大环内酯捕获后，重要组氨酸残基 HIS289 的细微构象变化触发的 EreC 蛋白的假设“主动环闭合”期间存在亚稳态“半开”状态，这与著名的“捕蝇草”机制有着迷人的相似之处。