Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
J Biol Chem. 2019 Sep 20;294(38):14020-14032. doi: 10.1074/jbc.RA119.009942. Epub 2019 Jul 30.
Resistance of to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which becomes resistant to ESCs is by acquiring a mosaic allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the β3 strand to form the oxyanion hole, and rolling of the β3-β4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of β3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.
对扩展谱头孢菌素 (ESC) 的耐药性已成为人类健康的主要威胁。 导致 对 ESC 产生耐药性的主要机制是获得镶嵌 等位基因，该基因编码青霉素结合蛋白 2 (PBP2) 变体，与 WT 相比含有多达 62 个突变，其中一部分有助于耐药性。为了解释头孢菌素耐药性的分子机制，有必要了解 ESC 如何酰化 PBP2。在这里，我们报告了 WT PBP2 的转肽酶结构域与头孢克肟和头孢曲松复合物的晶体结构，以及 PBP2 的 形式和在活性位点结合磷酸离子的结构，分辨率分别为 1.9 Å 和 1.7-1.9 Å。这些结构表明，ESC 对 PBP2 的酰化伴随着 KTG 基序中 Thr-498 侧链的旋转以与头孢菌素羧酸接触，β3 链的扭曲以形成氧阴离子穴，以及β3-β4 环向活性位点滚动。对头孢菌素羧酸的识别似乎是形成 PBP2 酰化能力状态的关键触发因素。这些结构还开始解释与 ESC 耐药性相关的突变的影响。特别是，G545S 突变可能会阻碍β3 的扭曲，因为其侧链羟基与 Thr-498 形成氢键。总体而言，我们的数据表明，酰化是由 ESC 结合引发或捕获的构象变化引发的，这些运动受到与 ESC 耐药性相关的突变的限制。
