A Coordinated Proteomic Approach for Identifying Proteins that Interact with the <i>E. coli</i> Ribosomal Protein S12

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions (Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.细菌核糖体蛋白S12（ribosomal protein S12）含有一个普遍保守的D88残基，位于一段环区之中。该环区因靠近30S亚基的A位点，被认为在翻译过程中发挥关键作用。尽管D88突变体具有致死性，但该残基已被证实可被翻译后修饰为β-甲基硫代天冬氨酸——这是一种在多种系统发育不同的细菌的S12直系同源物中鉴定得到的翻译后修饰（post-translational modification, PTM）。在一项针对该翻译后修饰的表征研究中，我们的实验结果提供了证据，表明这一保守环区可能参与形成多种蛋白质-蛋白质相互作用（Strader, M. B.; Costantino, N.; Elkins, C. A.; Chen, C. Y.; Patel, I.; Makusky, A. J.; Choy, J. S.; Court, D. L.; Markey, S. P.; Kowalak, J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199）。 为跟进该项研究，我们针对含有D88的环区开展探针实验，采用两步互补亲和纯化策略以鉴定候选结合蛋白。第一步为利用内源表达的带有C端标签的S12蛋白，捕获S12的结合伙伴；第二步则使用合成的生物素标记肽段（对应D88保守环区），捕获与S12环区相互作用的伴侣蛋白。两种方法中捕获得到的蛋白均通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与一维液相色谱-串联质谱（one-dimensional liquid chromatography–tandem mass spectrometry）进行检测。 本报告呈现的实验结果鉴定出了可与30S亚基直接相互作用的蛋白，并阐明了哪些蛋白可能与S12发生相互作用。此外，我们提供的实验证据表明，两种在胁迫条件下参与调控核糖体和/或mRNA转录水平的蛋白——核糖核酸酶R（RNase R）与Hfq蛋白，可与S12保守环区形成直接相互作用，提示该环区极有可能是蛋白质结合界面的组成部分。