Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.
Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.
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P-糖蛋白 (Mdr3) 任一核苷酸结合位点的突变都会阻止这两个位点上核苷酸的钒酸盐捕获。
DOI：
10.1021/bi9728001
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Philippe Gros
中科院分区：
文献类型：
--
作者：
I. Urbatsch;Lucille Beaudet;Isabelle Carrier;Philippe Gros
用钒酸捕获法和定点突变法研究了这两个核苷酸结合位点在P-糖蛋白（小鼠Mdr 3）调节ATP水解中的作用。用六聚组氨酸尾标记的Mdr 3在酵母Pichia pastoris中过表达，并通过Ni亲和层析纯化至约90%的均一性。该方案产生纯化的重构Mdr 3，其表现出对ATP酶活性的高维拉帕米刺激，Vmax为4.2 μ mol min-1 mg-1，KM为0.7 mM，表明从巴斯德毕赤酵母纯化的Mdr 3是高度功能性的。在两个NB位点的每一个中，将点突变引入到步行者A或B基序的核心共有序列中。突变体K429 R、K1072 R（步行者A）和D551 N、D1196 N（步行者B）功能受损，不能赋予酿酒酵母细胞对杀真菌剂FK 506的抗性。在毕赤酵母中表达单突变体和双突变体（K429 R/K1072 R，D551 N/D1196 N），并表征这些突变对Mdr 3的ATP酶活性的影响。与从阴性对照纯化的蛋白脂质体相比，纯化的重构Mdr 3突变体显示出不可检测的ATP酶活性（野生型Mdr 3的<5%）。钒酸盐容易诱导捕获的野生型酶中的8-叠氮基-核苷酸后，短短的10秒的孵育，和特定的光标记的Mdr 3紫外线照射后。在任何突变体中都没有观察到这种钒酸盐诱导的捕获/光标记，即使在37 ℃下60分钟的捕获期后也是如此。由于用8-叠氮基-ATP捕获钒酸盐需要水解核苷酸，数据表明8-叠氮基-ATP水解在所有突变体蛋白中显著受损（<0.3%活性）。这些结果表明，在任何NB网站的突变防止单周转和钒酸捕获的核苷酸在非突变位点。这些结果进一步表明，这两个NB网站不能独立的功能作为催化位点在完整的分子。此外，N-或C-末端NB网站出现功能上难以区分，和合作的相互作用绝对需要ATP水解可能源于这两个网站。
Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.