Institute of Medical Chemistry, Medical University of Vienna, Vienna, Austria.
Mol Pharmacol. 2011 Mar;79(3):443-52. doi: 10.1124/mol.110.067611. Epub 2010 Dec 21.
The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.
人多药耐药蛋白（P-gp）是一种多功能药物外排转运体，可导致药物耐药，并调节全身给予的药物的药理学特征。它是由基因复制产生的同源二聚体祖先演变而来的。鼠 MDR1A 的晶体结构表明，P-gp 与两个同源二聚体细菌外排泵 Sav1866 和 MsbA 具有相同的整体结构，这两个外排泵具有完全的旋转对称性。对于 ABC 转运蛋白，核苷酸结合发生在马达结构域的两个对称位置。基于与完全对称的半转运体的同源性，本研究提出了一个关键问题：是否可以在 P-gp 的跨膜结构域中找到双药物转运途径存在的生化证据？用丙戊酸类似物对 P-gp 进行光标记，然后进行纯化和蛋白水解消化，并通过高分辨率质谱鉴定肽片段。通过将数据投影到同源模型中，将标记分配到蛋白质的两个区域。随后，确定了假定转运途径中的对称残基对，并通过定点突变进行了替换。转运实验证实了存在两个拟似对称的转运途径。尽管 rhodamine123 优先选择一条途径，但维拉帕米、丙戊酸类和长春碱类则优先选择另一条途径。这项研究有两个主要发现：P-gp 中存在两种溶质转运途径，这反映了其起源于同源二聚体祖先；不同的 P-gp 溶质选择性但非排他性地使用这些途径中的一种。拟似对称行为调和了早期的动力学和热力学数据，为 P-gp 药物转运提供了一个替代概念，有助于理解 P-gp 相互作用药物的非靶定量构效关系。
