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首页> 外文期刊>Journal of Molecular Biology >Structural basis of substrate binding specificity revealed by the crystal structures of polyamine receptors SpuD and SpuE from pseudomonas aeruginosa
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Structural basis of substrate binding specificity revealed by the crystal structures of polyamine receptors SpuD and SpuE from pseudomonas aeruginosa

机译：铜绿假单胞菌多胺受体SpuD和SpuE的晶体结构揭示了底物结合特异性的结构基础

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The type III secretion system (T3SS) of Pseudomonas aeruginosa is a key virulence determinant whose expression is induced by polyamine signals from mammalian host. SpuD and SpuE were postulated to be spermidine-preferential binding proteins, which regulate the polyamine content in this bacterial pathogen. In this study, we found that SpuD is a putrescine-preferential binding protein, while SpuE binds to spermidine exclusively. We have determined the crystal structures of SpuD in free form and in complex with putrescine and SpuE in free form and in complex with spermidine. Upon ligand binding, SpuD and SpuE undergo an "open-to-closed" conformational switch with the resultant closed ligand-bound forms, SpuD-putrescine and SpuE-spermidine, similar to their Escherichia coli counterparts PotF-putrescine and PotD-spermidine, respectively. Structural comparison suggested that two aromatic residues, Trp271 of SpuE and Phe273 of SpuD in segment II region, are the key structural determinants for putrescine/spermidine recognition specificity. Mutagenesis combined with isothermal titration calorimetry showed that substitution of Trp271 by Phe enabled SpuE to gain substantial binding affinity for putrescine, while replacement of Phe273 by Trp reduced the binding affinity of SpuD toward putrescine by 250-fold. Altogether, these results revealed the molecular mechanism governing polyamine recognition specificity by SpuD and SpuE and provide the basis for further structural and functional studies of polyamine signal importation system in P. aeruginosa.
机译：铜绿假单胞菌的III型分泌系统（T3SS）是关键的毒力决定簇，其表达被来自哺乳动物宿主的多胺信号诱导。 SpuD和SpuE被假定为亚精胺优先结合蛋白，可调节该细菌病原体中的多胺含量。在这项研究中，我们发现SpuD是一种腐胺优先结合蛋白，而SpuE仅与亚精胺结合。我们已经确定了游离形式和与腐胺复合的SpuD的晶体结构，以及游离形式和与亚精胺复合的SpuE的晶体结构。配体结合后，SpuD和SpuE经历“开放到封闭”的构象转换，生成的封闭配体结合形式SpuD-putrescine和SpuE-亚精胺，分别类似于其大肠杆菌对应物PotF-putrescine和PotD-亚精胺。 。结构比较表明，II区中两个芳香残基SpuE的Trp271和SpuD的Phe273是腐胺/亚精胺识别特异性的关键结构决定因素。诱变结合等温滴定热分析表明，用Phe取代Trp271使SpuE获得对腐胺的实质结合亲和力，而用Trp取代Phe273则使SpuD对腐胺的结合亲和力降低了250倍。总之，这些结果揭示了通过SpuD和SpuE控制多胺识别特异性的分子机制，并为进一步研究铜绿假单胞菌中多胺信号输入系统的结构和功能研究提供了基础。

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