Amor J Carlos, Swails Jennifer, Zhu Xinjun, Roy Craig R, Nagai Hiroki, Ingmundson Alyssa, Cheng Xiaodong, Kahn Richard A
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2005 Jan 14;280(2):1392-400. doi: 10.1074/jbc.M410820200. Epub 2004 Nov 1.
The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the "glutamic finger," conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.
嗜肺军团菌蛋白RalF通过Dot/Icm IV型转运体分泌到宿主细胞质中，在那里它将ADP核糖基化因子（Arf）招募到含病原体的吞噬体上，以建立复制细胞器。所有Arf鸟嘌呤核苷酸交换因子中都存在的Sec7结构域存在于RalF中，这表明Arf的招募是发病机制中的早期步骤。我们已经确定了RalF和分离的Sec7结构域的晶体结构，发现RalF由两个结构域组成。Sec7结构域与哺乳动物的Sec7结构域同源。C端结构域在Sec7结构域的活性位点上形成一个帽，并包含一个保守的折叠基序，以前在囊泡包被复合物的衔接子亚基中观察到。使用三种不同的测定方法研究了帽结构域和所有Sec7结构域中保守的“谷氨酸指”中的谷氨酸对RalF功能的重要性。这些数据突出了Arf鸟嘌呤核苷酸交换因子中Sec7以外的结构域对生物活性的功能重要性，并提出了调节这些活性的新机制。
