From the Department of Chemistry and
the Synmikro Center for Synthetic Microbiology, 35043 Marburg, Germany, and.
J Biol Chem. 2018 Dec 21;293(51):19699-19709. doi: 10.1074/jbc.RA118.003070. Epub 2018 Oct 26.
Efficient adaptation to environmental changes is pivotal for all bacterial cells. Almost all bacterial species depend on the conserved stringent response system to prompt timely transcriptional and metabolic responses according to stress conditions and nutrient depletion. The stringent response relies on the stress-dependent synthesis of the second messenger nucleotides and alarmones (p)ppGpp, which pleiotropically target and reprogram processes that consume cellular resources, such as ribosome biogenesis. Here we show that (p)ppGpp acts on the ribosome biogenesis GTPase A (RbgA) of Gram-positive bacteria. Using X-ray crystallography, hydrogen-deuterium exchange MS (HDX-MS) and kinetic analysis, we demonstrate that the alarmones (p)ppGpp bind to RbgA in a manner similar to that of binding by GDP and GTP and thereby act as competitive inhibitors. Our structural analysis of RbgA bound to ppGpp and pppGpp at 1.8 and 1.65 Å resolution, respectively, suggested that the alarmones (p)ppGpp prevent the active GTPase conformation of RbgA by sterically blocking the association of its G2 motif via their 3'-pyrophosphate moieties. Taken together, our structural and biochemical characterization of RbgA in the context of the alarmone-mediated stringent response reveals how (p)ppGpp affects the function of RbgA and reprograms this GTPase to arrest the ribosomal large subunit.
有效适应环境变化对所有细菌细胞都至关重要。几乎所有的细菌物种都依赖于保守的严谨反应系统，根据应激条件和营养枯竭来及时触发转录和代谢反应。严谨反应依赖于第二信使核苷酸和警报素（p）ppGpp的应激依赖性合成，它们多效性地靶向和重新编程消耗细胞资源的过程，如核糖体生物发生。在这里，我们表明（p）ppGpp 作用于革兰氏阳性菌的核糖体生物发生 GTP 酶 A（RbgA）。通过 X 射线晶体学、氢氘交换 MS（HDX-MS）和动力学分析，我们证明警报素（p）ppGpp 以类似于 GDP 和 GTP 结合的方式结合 RbgA，从而作为竞争性抑制剂。我们对分别以 1.8 和 1.65 Å分辨率结合 ppGpp 和 pppGpp 的 RbgA 的结构分析表明，警报素（p）ppGpp 通过其 3'-焦磷酸部分阻止其 G2 基序的缔合，从而阻止 RbgA 的活性 GTP 酶构象。综上所述，我们在警报素介导的严谨反应背景下对 RbgA 的结构和生化特性的表征揭示了（p）ppGpp 如何影响 RbgA 的功能并重新编程该 GTP 酶以阻止核糖体大亚基的形成。
