Fic domain-catalyzed adenylylation: Insight provided by the structural analysis of the type IV secretion system effector BepA
Fic domain-catalyzed adenylylation: Insight provided by the structural analysis of the type IV secretion system effector BepA
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DOI：
10.1002/pro.581
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发表时间：
2011-03-01
期刊：
影响因子：
8
通讯作者：
Schirmer, Tilman
中科院分区：
文献类型：
--
作者：
Palanivelu, Dinesh V.;Goepfert, Arnaud;Schirmer, Tilman
许多细菌病原体通过专用分泌系统注射效应蛋白来破坏真核宿主细胞的细胞功能。来自汉赛巴尔通体的IV型分泌系统（T4 SS）效应蛋白BepA由N-末端Fic结构域和C-末端巴尔通体胞内递送结构域组成，后者负责T4 SS介导的易位到宿主细胞中。包括Fic结构域的蛋白水解抗性片段（残基10-302）显示出自身腺苷酸化活性和腺苷酰转移到Hela细胞提取物蛋白上，如通过与α-[P-32]-ATP孵育的放射自显影所证明的。通过SeMet-SAD方法确定其晶体结构为2.9埃分辨率，显示出典型的Fic折叠，包括HPFxxGNGRxxR特征基序，在环区域中具有几个精细结构，并且在C末端具有额外的富含β的结构域。在用ATP/Mg 2+浸泡的晶体上，额外的电子密度表明在签名基序的阴离子结合巢中存在PPi/Mg 2+部分，其为腺苷酸化反应的副产物。在此基础上的信息和最近的结构的IbpA（Fic 2）的复合物与真核生物的靶蛋白Cdc 42，我们提出了一个详细的模型的三元复合物的Fic与两个基板，ATP/Mg 2+和目标酪氨酸。该模型与去质子化的侧链羟基对核苷酸的α-磷进行在线亲核攻击以实现AMP转移一致。此外，一般的，序列独立的机制，通过酶和目标之间的反平行β链相互作用的目标定位的建议。
Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with alpha-[P-32]-ATP. Its crystal structure, determined to 2.9-angstrom resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional beta-rich domain at the C-terminus. On crystal soaking with ATP/Mg2+, additional electron density indicated the presence of a PPi/Mg2+ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg2+ and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the alpha-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel beta-strand interactions between enzyme and target is suggested.