GTP-BINDING PROTEIN STRUCTURE/FUNCTION STUDIES

adp -核糖基化因子（ARFs）是一种多功能、多结构域的蛋白质，可调节内质网和高尔基体（以及其他部位）的囊泡运输。ARF功能需要在gtp结合的活性形式和gdp结合的非活性形式之间进行调节。GTP的结合由鸟嘌呤核苷酸交换蛋白（GEPs）催化，GTP酶激活蛋白（gap）灭活GTP。目前已经发现了两种常见的GEPs，一种是被BFA（一种干扰蛋白质分泌并导致高尔基池可逆解体的药物）抑制的~200 kda的GEPs家族，另一种是BFA耐药的~55 kda的GEPs家族。它们都有大约200个氨基酸组成的所谓Sec7结构域，负责GEP活性及其BFA抑制。为了评估Sec7结构域外结构的影响，我们比较了细胞素-1（一个~55-kDa的GTP）及其Sec7结构域（C-1Sec7）对几种ARF和ARL蛋白的活性。ARL1于1991年首次被其他工作人员描述为一种与arf相关的蛋白，不会激活霍乱毒素，其氨基酸序列与ARF1相同56%。cytohesin-1和C-1Sec7都能激活ARF1，但只有后者能激活ARL1，即底物特异性受到催化Sec7结构域外结构的影响。几年前制备了由ARF1和ARL1序列组成的嵌合蛋白，以表明ARF1的不同区域负责霍乱毒素和磷脂酶D的激活，并确定了与arfaptin相互作用的关键区域。我们在最近的研究中使用了这些结构和其他结构来确定ARF1中参与与细胞分裂素-1功能相互作用的多个结构元件。例如，细胞增殖素-1作用所必需的ARF n端，对C-1Sec7来说并不是必需的。来自这些实验的数据意味着，基于ARNO Sec7结构域晶体结构的模型（与C-1Sec7有87%的相同）与缺乏前17个残基的ARF1相关，并不能完全或正确地反映arf -细胞分裂素复合物中的相互作用。- gtp结合蛋白，ARF， ARL，磷脂酶D，霍乱毒素

ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere). ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase-activating proteins (GAPs). Two general types of GEPs have been recognized, a family of ~200-kDa proteins that are inhibited by BFA (a drug that interferes with protein secretion and causes reversible disintegration of Golgi cisternae) and smaller ~55-kDa GEPs that are BFA-resistant. All have so-called Sec7 domains of ~200 amino acids that are responsible for GEP activity, as well as its BFA inhibition. To assess the effects of structure outside the Sec7 domain, we had compared the activities of cytohesin-1, a ~55-kDa GTP, and its Sec7 domain (C-1Sec7) toward several ARF and ARL proteins. ARL1, first described by other workers in 1991 as an ARF-related protein that did not activate cholera toxin, is 56% identical in amino acid sequence to ARF1. Both cytohesin-1 and C-1Sec7 activate ARF1, but only the latter activates ARL1, i.e., substrate specificity is influenced by structure outside of the catalytic Sec7 domain. Chimeric proteins composed of sequences from ARF1 and ARL1 were prepared several years ago to show that different regions of ARF1 are responsible for the activation of cholera toxin and phospholipase D and to define regions criticals for the interaction with arfaptin. We used those and additional constructs in recent studies to identify multiple structural elements in ARF1 that participate in functional interactions with cytohesin-1. For example, the ARF N-terminus, which was required for cytohesin-1 action, was not necessary for that of C-1Sec7. The data from these experiments mean that the model based on a crystal structure of the ARNO Sec7 domain (which is 87% identical to C-1Sec7) associated with an ARF1 lacking the first 17 residues does not reflect completely, or perhaps, correctly, interactions in the ARF-cytohesin complex. - GTP-binding proteins, ARF, ARL, phospholipase D, cholera toxin