Conformational Changes in IpaD from Shigella flexneri upon Binding Bile Salts Provide Insight into the Second Step of Type III Secretion
Conformational Changes in IpaD from Shigella flexneri upon Binding Bile Salts Provide Insight into the Second Step of Type III Secretion
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DOI：
10.1021/bi101365f
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发表时间：
2011-01-18
期刊：
影响因子：
2.9
通讯作者：
Picking, William D.
中科院分区：
文献类型：
--
作者：
Dickenson, Nicholas E.;Zhang, Lingling;Picking, William D.
福氏志贺菌使用其III型分泌装置（TTSA）将改变宿主的蛋白质注入靶向真核细胞。TTSA由基体和暴露的针头组成，针头与侵袭质粒抗原D（iPad）形成控制分泌的尖端复合物。胆盐脱氧胆酸盐（DOG）刺激转运蛋白IpaB募集到成熟的TTSA针尖复合物中。这一过程似乎是由DOC和iPad之间的直接相互作用触发的。荧光光谱和NMR光谱在这里被用来确认DOC iPad的相互作用，并揭示iPad的构象变化后，DOG绑定触发IpaB的出现在针尖。在此使用iPad上的特定位点之间的福斯特共振能量转移来鉴定由于DOG结合而导致的iPad结构域之间的距离的变化。为了进一步探索DOG结合对iPad结构的影响，采用NMR化学位移映射。建议的DOG结合位点内的残基和“远端”球状结构域内的其他残基的环境在DOG结合后被扰动，进一步表明DOG结合后iPad内发生构象变化。这些事件被认为是TTSA针尖处IpaB募集的原因。突变分析结合额外的光谱分析证实，由DOG结合诱导的iPad的构象变化有助于IpaB向S. flexneri TTSA针尖。这些发现为确定环境因素如何促进TTSA针尖在宿主细胞接触之前成熟奠定了基础。
Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOG) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC IpaD interaction and to reveal that IpaD conformational changes upon DOG binding trigger the appearance of IpaB at the needle tip. Forster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOG binding. To further explore the effects of DOG binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOG binding site and additional residues within the "distal" globular domain were perturbed upon DOG binding, further indicating that conformational changes occur within IpaD upon DOG binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOG binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.