Li Mavis Jiarong, Nath Abhinav, Atkins William M
Department of Medicinal Chemistry, University of Washington , Box 357610, Seattle, Washington 98195-7610, United States.
Biochemistry. 2017 May 16;56(19):2506-2517. doi: 10.1021/acs.biochem.6b01245. Epub 2017 May 4.
The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2-10-fold increases in K in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.
ATP结合盒转运体P-糖蛋白（ABCB1或P-gp）在细胞对药物的抗性及药物相互作用中起主要作用。实验研究支持一种机制，即其在向内构象和向外构象之间存在核苷酸依赖性波动，并与核苷酸水解相偶联。然而，目前尚缺乏对这些构象集合体的药物依赖性调节的详细了解。不同药物可能占据部分重叠但又不同的位点，因此与核苷酸结合和水解的偶联方式也各不相同。许多荧光药物类似物被用于基于细胞的转运模型中；然而，它们与P-gp的特异性相互作用尚未得到研究，这限制了根据分子模型对转运试验结果的解释。在此，我们通过荧光相关光谱法监测了荧光探针底物BODIPY-维拉帕米、BODIPY-长春碱和Flutax-2在低占有率情况下，于脂质纳米盘内处于不同核苷酸结合状态的小鼠P-gp上的结合情况。不同探针底物对不同核苷酸依赖性构象的亲和力变化各不相同。对于BODIPY-维拉帕米和BODIPY-长春碱，与无核苷酸状态相比，在核苷酸结合或钒酸盐捕获状态下，其解离常数（K）增加了2至10倍。相比之下，Flutax-2的亲和力不受核苷酸或钒酸盐捕获的影响。与BODIPY-维拉帕米和BODIPY-长春碱进一步不同的是，尽管Flutax-2在基于囊泡的转运试验中能够被转运，但它不会引起ATP水解的刺激。既定的底物维拉帕米、紫杉醇和长春碱会将BODIPY-维拉帕米或BODIPY-长春碱从其高亲和力位点上置换下来，而转运底物Flutax-2不会被这些底物中的任何一种所置换。结果表明，Flutax-2具有独特的结合位点，能够在不刺激ATP水解的情况下实现转运。
