Schedlbauer Andreas, Han Xu, van Bakel Wouter, Kaminishi Tatsuya, Ochoa-Lizarralde Borja, Iturrioz Idoia, Çapuni Retina, Parry Ransford, Zegarra Ronny, Gil-Carton David, López-Alonso Jorge P, Barragan Sanz Kristina, Brandi Letizia, Gualerzi Claudio O, Fucini Paola, Connell Sean R
Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160 Derio, Spain.
Structural Biology of Cellular Machines Laboratory, Biobizkaia Health Research Institute, Cruces University Hospital, 48903 Barakaldo, Bizkaia, Spain.
bioRxiv. 2024 Sep 26:2024.09.26.614503. doi: 10.1101/2024.09.26.614503.
The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an 30S pre/C state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.
起始阶段是蛋白质合成（翻译）的限速步骤，受到精细调控，使其成为一个重要的药物靶点。在细菌中，起始过程由三种起始因子引导，涉及将起始位点定位在小核糖体亚基（30S）的P位点内的信使RNA上，在此处由起始tRNA进行解码。这一过程可被GE81112有效抑制，GE81112是一种天然的亲水性、非环状、非核糖体四肽。它在自然界中以三种结构变体（A、B和B1，分子量为643 - 658 Da）存在。先前对GE81112的生化和结构表征表明，这种抗生素的主要作用机制是：（1）阻止起始tRNA正确结合到P位点；（2）阻止起始因子IF3的构象重排，从而导致30S前/预起始状态。在本研究中，我们使用冷冻电镜确定了GE81112在起始复合物（3.2 - 3.7Å）和空载核糖体（2.09 Å）中的结合位点。该结合位点位于mRNA通道（E位点）内，但远离起始因子和起始tRNA的结合位点。这表明它通过变构作用阻止起始tRNA锁定到位。这种结合模式与先前的生化研究以及最近确定GE81112关键药效基团的工作一致。
