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首页> 外文期刊>Biochemistry >Conformational States of ADP Ribosylation Factor 1 Complexed with Different Guanosine Triphosphates As Studied by 31P NMR Spectroscopy
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Conformational States of ADP Ribosylation Factor 1 Complexed with Different Guanosine Triphosphates As Studied by 31P NMR Spectroscopy

机译：31 P NMR光谱研究的ADP核糖化因子1与不同的鸟苷三磷酸络合的构象态

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Guanine nucleotide binding proteins (GNBproteins)nplay an essential role in cellular signaling, acting asnmolecular switches, cycling between the inactive, GDP-boundnform and the active, GTP-bound form. It has been shown thatnconformational equilibria also exist within the active form ofnGNB-proteins between conformational states with differentnfunctional properties. Here we present 31P NMR data onnADP ribosylation factor 1 (Arf1), a GNB-protein involved innGolgi traffic, promoting the coating of secretory vesicles. Toninvestigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety ofncommonly used GTP analogues, namely, GppCH2p, GppNHp, and GTPγS, were analyzed. To gain deeper insight into thenconformational state of active Arf1, we titrated with Cu2+-cyclen and GdmCl and formed the complex with the Sec7 domain ofnnucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, fromn31P NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This statenseems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effectorndomain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward anconformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchangenfactors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effectorninteracting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuningnmechanism of regulation and thereby an interesting target for the modulation of protein activity.
机译：鸟嘌呤核苷酸结合蛋白（GNB蛋白）在细胞信号传导，非分子开关，非活性GDP结合形式和活性​​GTP结合形式之间循环中起重要作用。已经显示，构象平衡也存在于具有不同功能性质的构象状态之间的nGNB蛋白的活性形式中。在这里，我们介绍nADP核糖基化因子1（Arf1）的31P NMR数据，该蛋白是参与nnGolgi转运的GNB蛋白，可促进分泌性囊泡的覆盖。分析活性Arf1，与GTP复合的野生型和switch I突变体以及多种常用的GTP类似物（即GppCH2p，GppNHp和GTPγS）的构象平衡。为了更深入地了解活性Arf1的构象状态，我们用Cu2 + -cycln和GdmCl进行了滴定，并形成了具有核苷酸交换因子ARNO的Sec7结构域和效应GAT结构域的复合物。与相关蛋白Ras，Ral，Cdc42和Ran相比，从n31P NMR光谱图看，Arf1主要以单一构象存在，而与所用的GTP类似物无关。根据与命名效应域相互作用的Ras命名法，该状态似乎对应于所谓的状态2（T）构象。第48位的高度保守的苏氨酸与丙氨酸的交换导致平衡向构象态转移，该构象态具有Ras中状态1（T）的典型特性，例如与鸟嘌呤核苷酸交换因子的相互作用，对核苷三磷酸的亲和力较低，对离液剂的敏感性更高。在活性Arf1（wt）中，强烈推荐相互作用的构象。活性GNB蛋白的这些固有构象平衡可能是调节的微调机制，从而成为调节蛋白活性的有趣靶标。

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  • 来源
    《Biochemistry》 |2011年第29期|p.6316-6327|共12页
  • 作者

    Tanja Meierhofer Maren Eberhardt and Michael Spoerner;

  • 作者单位

    University of Regensburg, Institute of Biophysics and Physical Biochemistry, Universit€atsstrasse 31, D-93053 Regensburg, Germany;

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