Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding
Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding
复制标题
DOI：
10.1073/pnas.1612927114
复制
发表时间：
2017-02-28
影响因子：
11.1
通讯作者：
Vrielink, Alice
中科院分区：
文献类型：
--
作者：
Anandan, Anandhi;Evans, Genevieve L.;Vrielink, Alice
多药耐药(MDR)革兰氏阴性细菌增加了现代致命脓毒症的患病率。粘菌素是一种阳离子抗菌肽(CAMP)抗生素，可穿透细菌外膜(OM)，已被用于治疗这些感染。OM外层小叶由含有脂类A的内毒素组成，可对其进行修饰以增加对cAMP的抵抗力，并防止先天性免疫反应清除。一种类型的脂质A修饰包括通过磷乙醇胺转移酶将磷乙醇胺加到1和4‘头基位置。先前关于这种酶的截短形式的结构工作表明，全长蛋白是正确的脂底物结合和催化所必需的。我们现在报道脑膜炎奈瑟氏菌的全长脂质A磷酸乙醇胺转移酶的晶体结构，其分辨率为2.75-a。该结构揭示了一个先前未描述的螺旋膜域和一个面向周质的可溶性结构域。这些结构域由沿着膜表面与磷脂头部基团相互作用的螺旋连接。位于两个跨膜螺旋之间的周质环中的两个螺旋含有保守的带电残基，并与底物结合有关。本征荧光、有限的蛋白质分解和分子动力学研究表明，该蛋白质可能会采样不同的构象状态，以使两种大小非常不同的脂类底物结合。这些结果为内毒素修饰的机制提供了洞察力，并将有助于以结构为导向的合理药物设计方法来治疗多重耐药细菌感染。
Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-a resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.