Urbatsch I L, Beaudet L, Carrier I, Gros P
Department of Biochemistry, McGill University, Montréal, Québec, Canada, H3G 1Y6.
Biochemistry. 1998 Mar 31;37(13):4592-602. doi: 10.1021/bi9728001.
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.
利用钒酸盐捕获核苷酸和定点诱变技术，研究了两个核苷酸结合（NB）位点在P-糖蛋白（小鼠Mdr3）调节ATP水解中的作用。带有六组氨酸尾巴的Mdr3在毕赤酵母中过表达，并通过镍亲和层析纯化至约90%的纯度。该方案得到了纯化、重组的Mdr3，其对ATP酶活性具有高维拉帕米刺激作用，Vmax为4.2微摩尔·分钟-1·毫克-1，KM为0.7毫摩尔，表明从毕赤酵母中纯化的Mdr3具有高度功能性。在两个NB位点的每个位点的沃克A或B基序的核心共有序列中引入点突变。突变体K429R、K1072R（沃克A）和D551N、D1196N（沃克B）功能受损，无法赋予酿酒酵母对杀真菌剂FK506的细胞抗性。单突变体和双突变体（K429R/K1072R、D551N/D1196N）在毕赤酵母中表达，并对这些突变对Mdr3的ATP酶活性的影响进行了表征。与从阴性对照中纯化的蛋白脂质体相比，纯化的重组Mdr3突变体未显示出可检测到的ATP酶活性（<野生型Mdr3的5%）。孵育10秒后，钒酸盐很容易在野生型酶中诱导8-叠氮核苷酸的捕获，并在紫外线照射后对Mdr3进行特异性光标记。即使在37℃下捕获60分钟后，在任何突变体中都未观察到这种钒酸盐诱导的捕获/光标记。由于用8-叠氮-ATP进行钒酸盐捕获需要核苷酸水解，数据表明所有突变蛋白中的8-叠氮-ATP水解均显著受损（<0.3%活性）。这些结果表明，任一NB位点的突变都会阻止非突变位点中核苷酸的单周转和钒酸盐捕获。这些结果进一步表明，在完整分子中两个NB位点不能作为催化位点独立发挥作用。此外，N端或C端的NB位点在功能上似乎没有区别，ATP水解绝对需要的协同相互作用可能源于两个位点。
