Modi Niraj, Bárcena-Uribarri Iván, Bains Manjeet, Benz Roland, Hancock Robert E W, Kleinekathöfer Ulrich
School of Engineering and Science, Jacobs University Bremen , Campus Ring 1, 28759 Bremen, Germany.
ACS Chem Biol. 2015 Feb 20;10(2):441-51. doi: 10.1021/cb500399j. Epub 2014 Oct 31.
The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is 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.
革兰氏阴性机会致病菌铜绿假单胞菌的细胞包膜对包括抗生素在内的许多类亲水分子的渗透性很差，这是由于存在狭窄且具有选择性的孔蛋白。在这里，我们聚焦于一种狭窄通道孔蛋白，即OprP，它负责高亲和力地摄取磷酸根离子。其两个磷酸根中央结合位点包含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结合位点存在这样一个带负电荷的残基，由于其可能降低的结合亲和力，这非常令人惊讶。本研究的目的是通过诱变、电生理学和自由能计算相结合的方法，更好地理解D94在磷酸根结合、选择性和转运中的作用。结合位点中带负电荷残基的存在对于这种特定的孔蛋白OprP至关重要，这一点在几种阴离子选择性孔蛋白结合位点中此类带负电荷残基的进化保守性中得到了强调。将OprP中的D94突变为任何带正电荷或中性的残基都会增加磷酸根对OprP的结合亲和力。详细分析表明，OprP磷酸根结合位点中的这个阴离子残基，尽管带负电荷，但在通道中央区域维持了能量上有利的磷酸根结合位点，同时减少了停留时间，从而防止了磷酸根与通道的过度强结合，而这种强结合会阻碍磷酸根通过通道的通量。有趣的是，将D94突变为带正电荷的残基赖氨酸和精氨酸，会导致非常不同的结合亲和力和自由能分布，这表明这些带正电荷残基的侧链构象在磷酸根与OprP结合中具有重要性。
