Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.
Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.
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调节孔蛋白通道中带有负电荷残基的阴离子结合位点的亲和力：OprP 的分子细节
DOI：
10.1021/cb500399j
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发表时间：
--
影响因子：
4
通讯作者：
U. Kleinekathöfer
中科院分区：
文献类型：
--
作者：
N. Modi;I. Bárcena-Uribarri;M. Bains;R. Benz;R. E. Hancock;U. Kleinekathöfer
革兰氏阴性条件致病菌铜绿假单胞菌（Pseudomonasaerosaisosa）的细胞被膜对包括抗生素在内的多种亲水性分子的渗透性差，这是由于存在狭窄的和选择性的孔蛋白。在这里，我们专注于窄通道孔蛋白之一，即OprP，这是负责高亲和力摄取磷酸根离子。它的两个中心磷酸结合位点含有许多带正电荷的氨基酸以及一个带负电荷的残基（D94）。在带负电荷的磷酸根离子的结合位点中存在这种带负电荷的残基是非常令人惊讶的，这是由于可能降低的结合亲和力。本研究的目的是更好地了解D94在磷酸盐结合，选择性和运输中的作用，使用诱变，电生理学和自由能计算的组合。带负电荷的残基的结合位点中的存在是至关重要的，这种特定的孔蛋白OprP所强调的几个阴离子选择性孔蛋白的结合位点中的这种带负电荷的残基的进化保守。OprP中的D94突变为任何带正电荷或中性残基增加了磷酸盐对OprP的结合亲和力。详细的分析表明，这种阴离子残基的磷酸盐结合位点的OprP，尽管其负电荷，保持积极有利的磷酸盐结合位点的中心区域的通道，并在同一时间减少停留时间，从而防止过强的结合磷酸盐，将反对磷酸盐通量通过通道。有趣的是，D94突变为带正电荷的残基，赖氨酸和精氨酸，导致非常不同的结合亲和力和自由能谱，表明这些带正电荷的残基的侧链构象在磷酸盐结合OprP中的重要性。
The cell envelope of the Gram negative opportunistic pathogenPseudomonas aeruginosais poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.
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铜绿假单胞菌磷酸盐特异性孔蛋白 OprP 的插入诱变
影响因子：
3.2
作者：
A. Sukhan;Robert E. W. Hancock
通讯作者：
Robert E. W. Hancock
DOI：
10.1021/jz101461d
发表时间：
2011
期刊：
The journal of physical chemistry letters
影响因子：
--
作者：
Jiang W;Hardy DJ;Phillips JC;Mackerell AD Jr;Schulten K;Roux B
通讯作者：
Roux B
图 2 参与鼠伤寒沙门氏菌主动转运的硫酸盐结合蛋白的解析结构。
影响因子：
5.6
作者：
Pflugrath,JW;Quiocho,FA
通讯作者：
Quiocho,FA
磷酸盐和硫酸盐转运受体精细特异性的原子基础。
影响因子：
19.6
作者：
F. Quiocho
通讯作者：
F. Quiocho
影响因子：
2.9
作者：
Niraj Modi;Iván Bárcena-Uribarri;M. Bains;R. Benz;R. Hancock;U. Kleinekathöfer
通讯作者：
Niraj Modi;Iván Bárcena-Uribarri;M. Bains;R. Benz;R. Hancock;U. Kleinekathöfer