Charting the sequence-activity landscape of peptide inhibitors of translation termination
Charting the sequence-activity landscape of peptide inhibitors of translation termination
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DOI：
10.1073/pnas.2026465118
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发表时间：
2021-03-09
影响因子：
11.1
通讯作者：
Mankin, Alexander S.
中科院分区：
文献类型：
--
作者：
Baliga, Chetana;Brown, Tyler J.;Mankin, Alexander S.
Apidaecin（API）是一种由蜜蜂产生的富含脯氨酸的抗菌肽，是一种特异性的翻译终止抑制剂。它侵入释放后核糖体的新生肽出口隧道并捕获释放因子，阻止其回收。API在出口通道中以延伸构象结合，该延伸构象与新生多肽的位置相匹配，并与核糖体RNA（rRNA）和核糖体蛋白建立多个接触。这些相互作用中哪些对API的活动至关重要尚不清楚。我们通过分析细菌细胞中合成的API变体的所有可能的单氨基酸取代的活性来解决这个问题。通过有条件地表达工程改造的API基因，我们直接在细菌胞质溶胶中产生了API，从而绕过了从培养基中输入肽的需要。内源性表达的API，以及其N-末端截短的突变体，保留了天然肽的抗菌特性和作用机制。利用API表达系统和下一代测序，我们在一个实验中绘制了保留或减轻API突变体的靶向活性的所有单个氨基酸取代。失活突变的分析使得有可能确定与核糖体，转移RNA（tRNA）和释放因子的关键相互作用的API的药效团。我们还确定了API片段，耐受各种氨基酸取代;在这一段的改变可用于改善抗菌肽的药理学特性。
Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.