Rothnie Alice, Storm Janet, McMahon Roisin, Taylor Andrew, Kerr Ian D, Callaghan Richard
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.
FEBS Lett. 2005 Jul 18;579(18):3984-90. doi: 10.1016/j.febslet.2005.06.030.
The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.
P-糖蛋白（P-gp）中的跨膜（TM）结构域包含药物结合位点，并在核苷酸催化驱动下发生构象变化以实现转运。然而，我们对于究竟哪些区域参与此类事件仍不清楚。采用定点标记方法，将硫醇反应性探针连接到引入跨膜片段6（TM6）的半胱氨酸上，以干扰功能并推断特定残基在药物结合和/或结构域间通讯中的作用。在TM6内的339C残基处共价连接香豆素-马来酰亚胺会导致P-gp的ATP水解受损。这种效应的本质是减少由转运底物、调节剂和强效抑制剂XR9576引起的基础活性的特征性调节。用[³H]-叠氮平对P-gp进行光亲和标记表明，339C残基本身不会改变药物结合。然而，该残基的共价修饰似乎会阻止导致药物刺激ATP水解的构象变化。
