Kinch Lisa N, Yarbrough Melanie L, Orth Kim, Grishin Nick V
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
PLoS One. 2009 Jun 5;4(6):e5818. doi: 10.1371/journal.pone.0005818.
The Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes.
METHODOLOGY/PRINCIPAL FINDINGS: We show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate.
CONCLUSIONS/SIGNIFICANCE: The demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates.
副溶血性弧菌III型分泌效应蛋白VopS含有一个fic结构域，该结构域可将AMP共价修饰到Rho GTPase苏氨酸上，以抑制宿主细胞中的下游信号事件。VopS fic结构域包含一个对AMP化有贡献的保守序列基序（HPFx[D/E]GN[G/K]R）。Fic结构域存在于多种物种中，包括细菌、少数古细菌和后生动物真核生物。
方法/主要发现：我们发现AMP化活性扩展到果蝇CG9523中的一个真核fic结构域，并使用基于序列和结构的计算方法来鉴定doc毒素和III型效应蛋白AvrB中的相关结构域。对AMP化有贡献的保守序列基序将fic与doc统一起来。尽管AvrB缺乏这个基序，但其结构显示出与fic和doc折叠相似的拓扑结构。AvrB与其宿主毒力靶标的肽片段结合的方式与fic结合肽底物的方式相似。AvrB还将结合的ADP配体中的磷酸基团定位在肽结合位点附近，且位置与结合的fic磷酸基团相似。
结论/意义：已证明的真核fic结构域AMP化活性表明VopS效应蛋白利用了一种新的宿主翻译后修饰。与Fic结构域相关的结构为AMP化活性位点以及VopS fic结构域与其宿主GTPase靶标的相互作用提供了见解。这些结果表明fic、doc和AvrB起源于一个共同的祖先，该祖先已进化为对蛋白质底物进行AMP化。
