Structural and functional insights into the mode of action of a universally conserved Obg GTPase.
Structural and functional insights into the mode of action of a universally conserved Obg GTPase.
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对普遍保守的 Obg GTPase 作用模式的结构和功能见解
DOI：
10.1371/journal.pbio.1001866
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发表时间：
2014-05
期刊：
影响因子：
9.8
通讯作者：
Gao N
中科院分区：
文献类型：
--
作者：
Feng B;Mandava CS;Guo Q;Wang J;Cao W;Li N;Zhang Y;Zhang Y;Wang Z;Wu J;Sanyal S;Lei J;Gao N
动力学和低温电子显微镜数据提供了对GTP酶ObgE？™S作为核糖体反结合因子的作用的见解，该因子受营养物质供应、生长控制与核糖体生物合成和蛋白质翻译的调节。OBG蛋白是一个P-loop GTP酶家族，从细菌到人类都是保守的。大肠杆菌中的Obg蛋白(ObgE)参与了多种不同的细胞功能，在核糖体组装和严格反应两个全球过程中发挥了分子作用。在这里，使用稳态前的快速动力学，我们证明了ObgE是一个反结合因子，它通过与50S亚基结合来阻止核糖体亚基的结合和下游的翻译步骤。ObgE是一种核糖体依赖的GTPase；然而，当ObgE与严格反应的全球调节因子四磷酸鸟苷(PpGpp)结合后，ObgE与50S亚基的相互作用增强，导致70S核糖体平衡解离成亚基。此外，我们对50s·ObgE·GMPPNP复合体的冷冻电子显微镜(Cryo-EM)结构表明，ObgE在进化上保守的N-末端结构域(NTD)是一个tRNA结构模拟物，与肽基转移酶中心具有特异的相互作用，显示出与I类释放因子的显著相似之处。这些结构数据可能将ObgE定义为与应激反应相关的专门翻译因子，并为未来阐明ObgE与核糖体相关(P)ppGpp调节因子之间的功能相互作用提供了一个框架。结合已发表的数据，我们的结果表明，在正常生长条件下，ObgE可能在50年代亚基组装的最后阶段发挥检查点的作用。更重要的是，ObgE作为一个(P)ppGpp效应子，在一定的应激条件下也可能对50s亚基的产生及其参与翻译起到调节作用。因此，我们的发现可能揭示了环境线索对翻译控制的一种未被充分认识的机制。GTP酶在生物系统中通常起分子开关的作用。通过在两种构象状态之间振荡，取决于鸟嘌呤核苷酸结合的类型(GTP或GDP)，GTP酶是细胞生物学许多方面的重要调节因子。通过合成针对GTP酶的其他鸟嘌呤核苷酸衍生物，可以获得更多的调控水平；例如，当营养物质有限时，细菌细胞产生四磷酸鸟苷/五磷酸鸟苷--(P)ppGpp--作为调节生长和生存之间平衡的“严格反应”的一部分。ObgE是一种GTPase，具有许多已报道的细胞功能，包括核糖体的生物发生，但其功能在分子水平上尚不清楚。在这里，我们从生化和结构上表征了ObgE与其细胞伙伴50s核糖体亚单位的结合。我们的结果表明，ObgE是一种抗结合因子，它结合到50s亚基上，阻止70s核糖体的形成，从而抑制翻译的启动。此外，ObgE的结合和抗结合活性受鸟嘌呤核苷酸和(P)ppGpp的调节。因此，我们认为ObgE是50s亚单位组装中的一个检查点蛋白，它通过(P)ppGpp水平感知细胞能量应激，并将核糖体组装与其他全球生长控制途径联系起来。
Kinetics and cryo-electronmicroscopy data provide insights into GTPase ObgE’s role as a ribosome anti-association factor that is modulated by nutrient availability, coupling growth control to ribosome biosynthesis and protein translation. Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues. GTPases commonly act as molecular switches in biological systems. By oscillating between two conformational states, depending on the type of guanine nucleotide bound (GTP or GDP), GTPases are essential regulators of many aspects of cell biology. Additional levels of regulation can be acquired through the synthesis of other guanine nucleotide derivatives that target GTPases; for instance, when nutrients are limited, bacterial cells produce guanine tetraphosphate/pentaphosphate—(p)ppGpp—as part of the “stringent response” to adjust the balance between growth and survival. ObgE is a GTPase with many reported cellular functions that include ribosome biogenesis, but none of its functions is understood at the molecular level. Here we characterize, both biochemically and structurally, the binding of ObgE to its cellular partner, the 50S ribosomal subunit. Our results show that ObgE is an anti-association factor, which binds to the 50S subunit to block the formation of the 70S ribosome, thereby inhibiting the initiation of translation. Furthermore, the binding and anti-association activities of ObgE are regulated by guanine nucleotides, as well as by (p)ppGpp. We thus propose that ObgE is a checkpoint protein in the assembly of the 50S subunit, which senses the cellular energy stress via levels of (p)ppGpp and links ribosome assembly to other global growth control pathways.
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影响因子：
3.7
作者：
Atkinson GC;Tenson T;Hauryliuk V
通讯作者：
Hauryliuk V
DOI：
10.1126/science.1179709
发表时间：
2009-10-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gao YG;Selmer M;Dunham CM;Weixlbaumer A;Kelley AC;Ramakrishnan V
通讯作者：
Ramakrishnan V
影响因子：
3
作者：
Heymann, J. Bernard;Belnap, David M.
通讯作者：
Belnap, David M.
影响因子：
16
作者：
Ali, Iraj K.;Lancaster, Laura;Noller, Harry F.
通讯作者：
Noller, Harry F.
影响因子：
64.5
作者：
Diaconu, M;Kothe, U;Wahl, MC
通讯作者：
Wahl, MC