Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics
Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics
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DOI：
10.1074/jbc.m302109200
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发表时间：
2003-06-13
影响因子：
4.8
通讯作者：
Mankin, AS
中科院分区：
文献类型：
--
作者：
Colca, JR;McDonald, WG;Mankin, AS
恶唑烷酮类抗生素是一类重要的新型合成抗菌药物，通过干扰核糖体功能抑制蛋白质合成。恶唑烷酮作用的确切位点和机制尚未阐明。尽管遗传数据指出核糖体肽基转移酶是药物作用的主要位点，但一些体外生物化学研究表明与核糖体不同区域的相互作用。这些在体内和体外获得的不一致的观察结果使恶唑烷酮作用的理解变得复杂。为了定位恶唑烷酮在活细胞中的作用位点，我们将光敏药物类似物与其在完整的、活跃生长的金黄色葡萄球菌中的靶标交联。恶唑烷酮特异性交联23 S rRNA、tRNA和两种多肽。与23 S rRNA交联的位点定位于普遍保守的A-2602。交联的多肽是核糖体蛋白L27，其N末端可以到达肽基转移酶中心，和LepA，一种与翻译因子同源的蛋白质。只有核糖体相关的LepA，而不是游离蛋白，交联，表明LepA交联的核糖体结合的抗生素。证据表明，一个特定的恶唑烷酮结合位点是在翻译核糖体中形成的肽基转移酶中心附近。
Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.