Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Departments of Pharmacology and Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
J Biol Chem. 2020 May 22;295(21):7529-7543. doi: 10.1074/jbc.RA120.012617. Epub 2020 Apr 6.
The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of 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 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 .
由于对扩展谱头孢菌素（ESCs）敏感性降低的菌株的传播，预计性传播疾病淋病的全球发病率将会上升。ESC 耐药性是由青霉素结合蛋白 2（PBP2）的镶嵌变体赋予的，这些变体与头孢菌素形成酰化加合物的能力降低。为了阐明 ESC 耐药性的分子机制，我们对来自低敏感性菌株 35/02 和 ESC 耐药菌株 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，突变会降低 PBP2 对头孢曲松的亲和力，并限制与酰化伴随的构象变化。具体来说，我们观察到 G545S 取代会阻碍形成酰化所需的氧阴离子孔的β3 链的旋转，并且还将头孢曲松固定在非典型构象中。此外，F504L 和 N512Y 取代似乎阻止了β3-β4 环的弯曲，该弯曲是在活性部位与头孢曲松的 R1 基团接触所必需的。其他突变似乎也通过降低蛋白质的灵活性起作用。总体而言，我们的发现表明，PBP2 中蛋白质动力学的限制是 ESC 耐药性的基础。
