Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties
Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties
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DOI：
10.1038/srep42812
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发表时间：
2017-02-20
影响因子：
4.6
通讯作者：
Dasgupta, Jhimli
中科院分区：
文献类型：
--
作者：
Agarwal, Shubhangi;Dey, Sanjay;Dasgupta, Jhimli
外源铁载体和血红素收购利用周质结合蛋白（PBP）依赖的ATP结合盒转运蛋白的必需营养铁的分子机制是难以捉摸的，但在霍乱弧菌。我们目前的研究描绘了两个III型PBP的结构，动力学和配体结合特性的霍乱弧菌，VcFhuD和VcHutB。通过晶体结构和荧光猝灭研究，我们证明了VcFhuD结合异羟肟酸盐和儿茶酚酸盐型异铁载体的独特功能。像E. coli FhuD，VcFhuD利用保守的色氨酸和R102结合铁色素和去铁醛。然而，与EcFhuD不同，VcFhuD的微碱性配体结合口袋可能有利于铁肠杆菌素结合，其中R203与R102一起沿着参与，就像它发生在儿茶酚结合PBP中一样。结合分光光度和非变性PAGE分析的结构研究表明，两个血红素分子以pH依赖性方式与VcHutB平行结合，而突变分析建立了Y65和H164在血红素结合中的相对重要性。MD模拟研究显示出一个不可预见的叶间摆动运动在III型PBPs，其幅度是负相关的接头螺旋与其相邻的螺旋的包装。发现VcFhuD中的小叶间运动或VcHutB中的剧烈扭曲影响配体结合。
Molecular mechanisms of xenosiderophore and heme acquisitions using periplasmic binding protein (PBP) dependent ATP-binding cassette transporters to scavenge the essential nutrient iron are elusive yet in Vibrio cholerae. Our current study delineates the structures, dynamics and ligand binding properties of two Type III PBPs of V. cholerae, VcFhuD and VcHutB. Through crystal structures and fluorescence quenching studies we demonstrate unique features of VcFhuD to bind both hydroxamate and catecholate type xenosiderophores. Like E. coli FhuD, VcFhuD binds ferrichrome and ferri-desferal using conserved Tryptophans and R102. However, unlike EcFhuD, slightly basic ligand binding pocket of VcFhuD could favour ferri-enterobactin binding with plausible participation of R203, along with R102, like it happens in catecholate binding PBPs. Structural studies coupled with spectrophotometric and native PAGE analysis indicated parallel binding of two heme molecules to VcHutB in a pH dependent manner, while mutational analysis established the relative importance of Y65 and H164 in heme binding. MD simulation studies exhibited an unforeseen inter-lobe swinging motion in Type III PBPs, magnitude of which is inversely related to the packing of the linker helix with its neighboring helices. Small inter-lobe movement in VcFhuD or dramatic twisting in VcHutB is found to influence ligand binding.