Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4.

This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.

人 P-糖蛋白 (P-gp) 或 ABCB1 在许多癌症中过度表达，并且由于其外流而改变化疗药物的生物利用度，从而导致化疗耐药性的发展。为了阐明 P-gp 的机制和结构功能关系，我们之前使用了富含酪氨酸 (Y) 的 P-gp 突变体 (15Y)，并证明 P-gp 的药物结合口袋中至少有 15 个保守残基负责最佳的底物相互作用和运输。为了进一步了解这15个残基的作用，产生了两个新的突变体，即跨膜结构域（TMD）1中的6个残基（F72、F303、I306、F314、F336和L339）被Y取代的6Y和9个残基的9Y TMD2 中的替换（F732、F759、F770、F938、F942、M949、L975、F983 和 F994）。尽管两种突变体在细胞表面均以正常水平表达，但6Y突变体未能转运除Bodipy-维拉帕米之外的所有测试底物，而9Y突变体以与野生型蛋白非常相似的方式流出所有测试底物。进一步的突变分析显示，两个第二位点突变，一个位于细胞内螺旋（ICH）4（F916Y）和一个位于核苷酸结合结构域（NBD）1（F480Y）的Q环中，恢复了6Y的转运功能。6Y 和 6Y+F916Y 突变体的其他生化数据和比较分子动力学模拟表明，P-gp 的 NBD1 的 Q 环通过 ICH4 与跨膜区的底物结合位点进行通讯。

这是人类 P-gp 中存在第二位点抑制子的第一个证据，该抑制子可以恢复由初级突变引起的转运功能丧失。对此类突变的进一步研究可能有助于绘制底物结合袋与 P-gp 和可能的其他 ABC 药物转运蛋白的 NBD 之间的通讯途径。