Conformational States of ADP Ribosylation Factor 1 Complexed with Different Guanosine Triphosphates As Studied by 31P NMR Spectroscopy
Conformational States of ADP Ribosylation Factor 1 Complexed with Different Guanosine Triphosphates As Studied by 31P NMR Spectroscopy
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DOI：
10.1021/bi101573j
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发表时间：
2011-07-26
期刊：
影响因子：
2.9
通讯作者：
Spoerner, Michael
中科院分区：
文献类型：
--
作者：
Meierhofer, Tanja;Eberhardt, Maren;Spoerner, Michael
鸟嘌呤核苷酸结合蛋白（GNB-蛋白）在细胞信号传导中起重要作用，充当分子开关，在无活性的GDP结合形式和活性的GTP结合形式之间循环。已经表明，在具有不同功能特性的构象状态之间的活性形式的GNB-蛋白质内也存在构象平衡。在这里，我们提出的P-31 NMR数据ADP核糖基化因子1（Arf 1），GNB蛋白参与高尔基体交通，促进分泌囊泡的涂层。为了研究活性Arf 1的构象平衡，分析了与GTP和各种常用GTP类似物（即GppCH（2）p、GppNHp和GTP γ S）复合的野生型和开关I突变体。为了更深入地了解活性Arf 1的构象状态，我们用Cu 2 +-cyclen和GdmCI滴定，并与核苷酸交换因子ARNO的Sec 7结构域和效应物GAT结构域形成复合物。与相关蛋白Ras、Ral、Cdc 42和Ran相反，从P-31 NMR光谱的观点来看，Arf 1主要以独立于所使用的GTP类似物的单一构象存在。根据Ras命名法，这种状态似乎对应于所谓的状态2（T）构象，其与效应结构域相互作用。位置48处高度保守的苏氨酸与丙氨酸的交换导致平衡向具有Ras中状态1（T）所获得的典型性质的构象状态移动，例如与鸟嘌呤核苷酸交换因子的相互作用、对核苷三磷酸的较低亲和力以及对离液剂的更高敏感性。在活跃的Arf 1（野生型），效应相互作用的构象是非常有利的。这些固有的构象平衡的活性GNB-蛋白质可能是一个微调机制的调节，从而为蛋白质活性的调制一个有趣的目标。
Guanine nucleotide binding proteins (GNB-proteins) play an essential role in cellular signaling, acting as molecular switches, cycling between the inactive, GDP-bound form and the active, GTP-bound form. It has been shown that conformational equilibria also exist within the active form of GNB-proteins between conformational states with different functional properties. Here we present P-31 NMR data on ADP ribosylation factor 1 (Arf1), a GNB-protein involved in Golgi traffic, promoting the coating of secretory vesicles. To investigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety of commonly used GTP analogues, namely, GppCH(2)p, GppNHp, and GTP gamma S, were analyzed. To gain deeper insight into the conformational state of active Arf1, we titrated with Cu2+-cyclen and GdmCI and formed the complex with the Sec7 domain of nucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, from P-31 NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This state seems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effector domain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward a conformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchange factors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effector interacting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuning mechanism of regulation and thereby an interesting target for the modulation of protein activity.