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Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics.
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2020-05-22 , DOI: 10.1074/jbc.ra120.012617
Avinash Singh 1 , Jonathan M Turner 1 , Joshua Tomberg 2 , Alena Fedarovich 1 , Magnus Unemo 3 , Robert A Nicholas 2 , Christopher Davies 1
Affiliation  

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3-β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

中文翻译:

头孢菌素耐药淋病奈瑟菌青霉素结合蛋白 2 的突变通过限制蛋白质动力学来阻碍头孢曲松酰化。

由于对超广谱头孢菌素 (ESC) 敏感性降低的淋病奈瑟菌菌株的传播,性传播疾病淋病的全球发病率预计将上升。ESC 耐药性是由青霉素结合蛋白 2 (PBP2) 的嵌合变体赋予的,该变体与头孢菌素形成酰化加合物的能力减弱。为了阐明 ESC 抗性的分子机制,我们对源自易感性降低的淋病奈瑟菌菌株 35/02 和 ESC 抗性菌株 H041 的 PBP2 变体进行了生化和高分辨率结构分析。我们的数据表明,突变既降低了 PBP2 对头孢曲松的亲和力,又限制了通常伴随酰化的构象变化。具体来说,我们观察到 G545S 取代阻碍了形成酰化氧阴离子孔所需的 β3 链的旋转,并且还将头孢曲松捕获在非规范构型中。此外,F504L 和 N512Y 取代似乎可以防止 β3-β4 环弯曲,而β3-β4 环是接触活性位点头孢曲松 R1 基团所需的。其他突变似乎也通过降低蛋白质的灵活性来发挥作用。总体而言,我们的研究结果表明,PBP2 中蛋白质动力学的限制是淋病奈瑟菌的 ESC 抗性的基础。
更新日期:2020-05-22
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