Zhang Da-Wei, Nunoya Kenichi, Vasa Monika, Gu Hong-Mei, Theis Ashley, Cole Susan P C, Deeley Roger G
Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, K7L 3N6, Canada.
Biochemistry. 2004 Jul 27;43(29):9413-25. doi: 10.1021/bi0495230.
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.
人类多药耐药蛋白1（MRP1）是一种ATP结合盒（ABC）转运蛋白，它赋予细胞对许多天然产物化疗药物的抗性，并且能够转运结构多样的共轭有机阴离子。MRP1有三个多跨膜结构域（TMDs），总共17个跨膜螺旋。对MRP1的光标记和诱变研究表明，TMD2中的最后一个螺旋TM11可能构成该蛋白底物结合口袋的一部分。我们已经证明，多个跨膜螺旋中的某些极性残基，包括TM11中的Arg(593)，是MRP1底物特异性或整体活性的决定因素。我们现在扩展了这些分析，以评估突变TM11内部和附近其余七个极性残基的功能后果。螺旋预测的外小叶区域中的突变Q580A、T581A和S585A对功能没有可检测到的影响，而靠近膜/细胞质界面的三个残基的突变改变了底物特异性。其中两个突变仅影响耐药性。N597A分别增加和降低了对长春新碱和依托泊苷（VP-16）的抗性，而S605A降低了对长春新碱、VP-16和阿霉素的抗性。第三个突变S604A选择性地增加了17β-雌二醇17-(β-D-葡萄糖醛酸苷)（E(2)17βG）的转运。相反，消除590位残基（野生型蛋白中的Asn）的极性特征会一致损害MRP1转运潜在生理底物以及赋予对三类不同天然产物药物抗性的能力。动力学和光标记研究表明，突变N590A不仅降低了MRP1对半胱氨酰白三烯4（LTC(4)）的亲和力，还大幅降低了ATP与核苷酸结合结构域1（NBD1）的结合。因此，涉及TM11中残基的极性相互作用不仅影响MRP1的底物特异性，还影响该蛋白提议的催化循环中的早期步骤。
