Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3)
Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3)
复制标题
DOI：
10.1016/j.febslet.2006.12.030
复制
发表时间：
2007-01-23
期刊：
影响因子：
3.5
通讯作者：
Gros, Philippe
中科院分区：
文献类型：
--
作者：
Carrier, Isabelle;Urbatsch, Ina L.;Gros, Philippe
a环是最近在ABC转运体的nbd中描述的一个保守区域[Ambudkar， SN]。金姆，我。a环是ABC转运体中Walker a基序上游的一个新的保守芳香酸亚结构域，对ATP结合至关重要。FEBS Lett. 580,1049-1055；Kim I.W, Peng, x.h., Sauna， Z.E, FitzGerald, p.c., Xia， D, Muller， M, Nandigama， K. and Ambudkar， SN。（2006）人类p糖蛋白ATP位点上的保守酪氨酸残基401和1044对ATP结合和水解至关重要：证据表明ATP结合盒中存在一个保守的亚结构域，即a环。生物化学45,7605-76161。在小鼠p -糖蛋白（Abcb1a）中，两种NBDs中Aloop的芳香残基都是酪氨酸：NBDI为Y397， NBD2为Y1040。另一个酪氨酸残基（NBDI中的618和NBD2中的1263）似乎也位于ATP分子附近。我们将Y397、Y618、Y1040和Y1263突变为色氨酸，并分析了这些突变对Abcb1a（小鼠Mdr3）转运特性、ATP结合和ATP水解的影响。Y618W和Y1263W酶具有与WT Abcbla相似的催化特性。另一方面，Y397W和Y1040W表现出运输受损和atp酶活性大大降低，包括MgATP的K-m增加了10倍。由此可见，Y397和Y1040在Abcb1a催化中起着重要的作用。(c) 2006年欧洲生化学会联合会。Elsevier B.V.版权所有。
The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, SN., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580,1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, SN. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-76161. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the Aloop in both NBDs is a tyrosine: Y397 in NBDI and Y1040 in NBD2. Another tyrosine residue (618 in NBDI and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcbla. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a similar to 10-fold increase in K-m for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.