Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase.
Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase.
复制标题
DOI：
10.1042/bj20091351
复制
发表时间：
2010-02-24
期刊：
影响因子：
--
通讯作者：
van Aalten DM
中科院分区：
文献类型：
--
作者：
Hurtado-Guerrero R;Zusman T;Pathak S;Ibrahim AF;Shepherd S;Prescott A;Segal G;van Aalten DM
军团病是由革兰氏阴性细菌嗜肺军团菌在肺泡巨噬细胞中的致命定植引起的。LpGT（L.嗜肺菌葡糖基转移酶;也称为Lgt 1）最近被鉴定为毒力因子，其通过EF 1A（延伸因子1A）的特异性葡糖基化，使用未知的底物识别模式和糖基转移的保留机制，关闭人细胞中的蛋白质合成。我们已经确定了LpGT的晶体结构与基板的复杂性，揭示了GT-A折叠与两个不寻常的突出域。通过对LpGT的结构导向突变，确定了几个对UDP-葡萄糖-供体和EF 1A-受体底物结合至关重要的残基，这些残基也影响L.嗜肺菌毒力，如通过显微注射研究所证实的。总之，这些结果表明，带正电荷的EF 1A环与LpGT结构上带负电荷的保守沟结合，并且两个天冬酰胺残基对于催化是必需的。此外，我们还证明了两个进一步的L。嗜肺菌糖基转移酶具有保守的UDP-葡萄糖-结合位点和EF 1A-结合沟，并且像LpGT一样，通过Icm/Dot（细胞器运输中的细胞内增殖/缺陷）系统易位到巨噬细胞中。
Legionnaires’ disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.