Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.
Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.
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通过定向进化取代十一种天然色氨酸，产生具有位点特异性、非保守取代的活性 P-糖蛋白。
DOI：
10.1038/s41598-020-59802-w
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发表时间：
2020
影响因子：
4.6
通讯作者：
Urbatsch,InaL
中科院分区：
文献类型：
--
作者：
Swartz,DouglasJ;Singh,Anukriti;Sok,Narong;Thomas,JoshuaN;Weber,Joachim;Urbatsch,InaL
P-糖蛋白（P-glycoprotein，Pgp）是一种将疏水性化合物泵出细胞外的蛋白质，在药物代谢动力学和肿瘤多药耐药中起重要作用。然而，Pgp中的多特异性药物结合和ATP水解驱动的药物输出知之甚少。利用色氨酸（Trp）在关键位置插入的荧光光谱是研究配体结合的重要工具。在Pgp中，该方法将需要去除11种内源性Trps，包括可能对功能、蛋白质-脂质相互作用和/或蛋白质稳定性重要的高度保守的Trps。在这里，我们开发了一种定向进化的方法，首先取代所有8个跨膜Trps和选择的运输活性的突变体在酿酒酵母。令人惊讶的是，许多色氨酸的位置含有非保守的取代，这使体内活性，并优于芳香族氨基酸。最活跃的构建体，W（3Cyto），为定向进化的三个细胞质的Trps，其中两个位置显示强大的功能偏向酪氨酸。W（3Cyto）和Trp-少Pgp保留野生型样蛋白表达，定位和运输功能，纯化的蛋白保留药物刺激ATP水解和药物结合亲和力。这些数据表明，优选的色氨酸取代特定于当地的情况下，通常由蛋白质结构要求和/或膜脂质相互作用，这些新的见解将提供指导膜蛋白工程。
P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supportedin vivoactivity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.