Department of Pathology and Molecular Medicine (K.E.W., S.P.C.C.) and Division of Cancer Biology and Genetics (G.C., A.J.R., S.P.C.C.), Queen's University Cancer Research Institute, Kingston, Ontario, Canada; and Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv, Israel (M.A., Y.T.).
Department of Pathology and Molecular Medicine (K.E.W., S.P.C.C.) and Division of Cancer Biology and Genetics (G.C., A.J.R., S.P.C.C.), Queen's University Cancer Research Institute, Kingston, Ontario, Canada; and Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv, Israel (M.A., Y.T.)
Mol Pharmacol. 2018 Sep;94(3):1069-1078. doi: 10.1124/mol.118.112615. Epub 2018 Jul 5.
The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.
人源 190kDaMRP1 是一种 ATP 结合盒多药和多有机阴离子外排转运体。其三个跨膜结构域的 17 个跨膜螺旋，以及两个核苷酸结合结构域（NBD），形成一个稳定的结构域间相互作用网络，确保细胞质中底物的结合与 ATP 结合和水解有效偶联，从而将溶质外排到细胞外环境中。在这里，我们表明转运体两半之间的外向环中苯丙氨酸的 Ala 取代基本上消除了 MRP1 对多种有机阴离子的结合。用色氨酸和酪氨酸进行保守取代几乎没有或没有影响。F583A 突变也导致细胞溶质 NBD 中 orthovanadate 诱导的 azidoADP 与 MRP1 的结合显著增加，尽管 ATP 的结合不受影响。这些观察结果表明，位置 583 的芳香侧链的丢失会损害 ADP 的释放，从而有效地将转运体锁定在低亲和力溶质结合状态。F583 是在 MRP1 中发现的第一个对其转运功能至关重要的外向氨基酸。我们的数据提供了证据，表明在 MRP1 的这个外向区域与转运体的溶质结合和核苷酸结合区域之间存在长程偶联，推测是通过变构相互作用。MRP1 的 cryoelectron microscopy 结构和同源模型表明，ATP 结合会改变苯丙氨酸侧链的取向，但目前无法提供有关这种长程信号传播的分子机制的见解。
