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
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
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DOI：
10.1021/bi060308o
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发表时间：
2006-06-20
期刊：
影响因子：
2.9
通讯作者：
Ambudkar, Suresh V.
中科院分区：
文献类型：
--
作者：
Kim, In-Wha;Peng, Xiang-Hong;Ambudkar, Suresh V.
哺乳动物P-糖蛋白（Pgps）和人ATP结合盒（ABC）B亚家族成员的每个核苷酸结合结构域（NBD）含有类似于步行者A结构域上游25个残基的酪氨酸残基。为了评估人Pgp的保守Y 401和Y1044残基的作用，我们用F、W、C或A单独或一起取代这些残基。突变蛋白在基于牛痘病毒的瞬时表达系统以及杆状病毒感染的HighFive昆虫细胞中表达。Y 401 F、Y 401 W、Y1044 F、Y1044 W或Y 401 F/Y1004 F突变体转运的荧光底物与野生型蛋白相似。另一方面，Y 401 L和Y 401 C表现出部分（30-50%）功能，并且在Y 401 A、Y1044 A和Y 401 A/Y1044 A突变体Pgps中转运被完全消除。类似地，在Y 401 A、Y1044 A和Y 401 A/Y1044 A突变体中，TNP-ATP结合、钒酸盐诱导的核苷酸捕获和ATP水解被完全消除。因此，ABC转运蛋白中步行者A基序上游的芳香族残基对于ATP的结合至关重要。此外，在核苷酸结合形式，数据挖掘和18 514 ABC结构域的一致保守序列在所有非冗余蛋白质的数据库中的对齐的几个NBD的晶体结构表明，一个芳香族残基是高度保守的，类似于85%的ABC蛋白质。虽然这个芳香族残基的作用以前已经研究了在一些ABC蛋白质，我们提供了一个近乎普遍的结构和功能的作用，这个残基的证据，并认识到它的存在作为一个保守的亚结构域类似的25个氨基酸上游的步行者A基序，是ATP结合的关键。我们将这个亚结构域命名为“A环”（与ATP的腺嘌呤环相互作用的芳香残基）。
Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue similar to 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18 514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in similar to 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain similar to 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).