Functional role of the linker region in purified human P-glycoprotein
Functional role of the linker region in purified human P-glycoprotein
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DOI：
10.1111/j.1742-4658.2009.07072.x
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发表时间：
2009-07-01
期刊：
影响因子：
5.4
通讯作者：
Kato, Hiroaki
中科院分区：
文献类型：
--
作者：
Sato, Tomomi;Kodan, Atsushi;Kato, Hiroaki
人P-糖蛋白（P-gp）是一种ATP依赖性的药物外排泵，可将多种结构上不相关的化合物转运出细胞，具有多重耐药性。P-gp具有类似于75个氨基酸的“接头区”，其连接两个同源的半部分，每个半部分包含跨膜结构域，随后是核苷酸结合结构域。为了研究连接区的作用，纯化的人P-gp在连接区被蛋白酶切割，然后与天然P-gp进行比较。基于维拉帕米刺激的ATP水解酶测定、尺寸排阻色谱分析和热稳定性测定，P-gp连接体的裂解不直接影响ATP水解的整体结构或催化过程的保留。然而，连接体裂解增加了具有底物（k（sub））和不具有底物（k（basal））的k（cat）值，但降低了所有10种测试底物的k（sub）/k（basal）值。前一个结果表明，切割接头激活P-gp，而后一个结果表明，接头区域保持ATP水解酶反应和底物识别之间的耦合的紧密性。P-gp同源物MsbA的结构检查表明，接头切割的P-gp具有增加的ATP水解酶活性，因为接头干扰伴随ATP水解酶反应的构象变化。此外，连接体裂解影响某些底物的特异性常数[k（sub）/K-m（D）]（即连接体裂解可能改变P-gp的底物特异性特征）。因此，这一结果也表明，接头区调节P-gp的固有底物特异性。
Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a 'linker region' of similar to 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, size-exclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the k(cat) values both with substrate (k(sub)) and without substrate (k(basal)), but decreased the k(sub)/k(basal) values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [k(sub)/K-m(D)] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp.