Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.
Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.
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P-糖蛋白半胱氨酸的定向进化揭示了保持多药耐药性的位点特异性、非保守性取代。
DOI：
10.1042/bsr20140062
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发表时间：
2014
影响因子：
4
通讯作者：
Urbatsch,InaL
中科院分区：
文献类型：
--
作者：
Swartz,DouglasJ;Mok,Leo;Botta,SriK;Singh,Anukriti;Altenberg,GuillermoA;Urbatsch,InaL
P-糖蛋白(P-gp)是参与肿瘤多药耐药的ABC(三磷酸腺苷结合盒)转运蛋白的原型。我们用定向进化的方法将Pgp中的6个胞质半胱氨酸残基全部替换为所有20个标准氨基酸，并筛选出活性突变体。从每三个半胱氨酸的75000个转化子池中，我们鉴定了多个保持抗药性和酵母交配活性的突变体。Nbd2区的Cys1223(25%和8%)和连接区的Cys638(24%和16%)是最常见的Walker A基序的甘氨酸和丝氨酸替换(分别为24%和20%)和Cys1070(37%和25%)，而邻近的Cys669对甘氨酸(16%)和丙氨酸(14%)耐受，但丝氨酸(不存在)不存在。Cys1121in Nbd2对带正电荷的精氨酸有明显的偏好(38%)，这表明ICL2(细胞内环2)上与Glu269的盐桥可能稳定结构域相互作用。相反，丙氨酸可以成功地取代跨膜α-螺旋中的三个半胱氨酸残基。得到的CL(Cys-less)PGP在酵母细胞中完全具有活性，纯化的蛋白显示药物刺激的ATPase活性与WT(野生型)PGP没有区别。总体而言，定向进化确定了位点特异的、非保守的半胱氨酸替换，这使得构建强大的CL PGP成为未来功能和结构研究的宝贵新工具，并可能指导丙氨酸和丝氨酸被证明不成功的其他CL蛋白的构建。
Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427(24 and 20%, respectively) and Cys1070(37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223in NBD2 (25 and 8%) and Cys638in the linker region (24 and 16%), whereas close-by Cys669tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.