Randazzo P A, Terui T, Sturch S, Fales H M, Ferrige A G, Kahn R A
Laboratory of Biological Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Jun 16;270(24):14809-15. doi: 10.1074/jbc.270.24.14809.
ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.
ADP核糖基化因子1（Arf1）是一种必需的N-肉豆蔻酰化的21 kDa GTP结合蛋白，其活性包括调节膜运输和磷脂酶D活性。该蛋白的N端以及与甘氨酸2结合的N-肉豆蔻酸酯先前已被证明对Arf在细胞中的功能至关重要。我们发现结合的核苷酸会影响Arf1的N端或与N端直接接触的残基的构象。通过研究该N端结构域中的突变对鸟苷5'-O-（3-硫代）三磷酸（GTPγS）和GDP结合及解离动力学的影响得以证明。与野生型蛋白相比，从N端缺失13或17个残基或在残基3-7处发生突变的Arf1突变体对GTPγS的亲和力更高，对GDP的亲和力更低。由于与靶蛋白相互作用需要N端，我们得出结论，Arf1的N端是一个GTP敏感的效应结构域。当Arf1被酰化时，GTP依赖性构象变化与添加的磷脂相互依赖。在没有磷脂的情况下，肉豆蔻酰化的Arf1对GTPγS的亲和力低于对GDP的亲和力，而在有磷脂的情况下，肉豆蔻酰化的蛋白对GTPγS的亲和力高于对GDP的亲和力。因此，N-肉豆蔻酰化是构建这种磷脂和GTP依赖性开关的关键组成部分。
