ATP binding to the KTN/RCK subunit KtrA from the K+- uptake system KtrAB of Vibrio alginolyticus -: Its role in the formation of the KtrAB complex and its requirement in vivo
ATP binding to the KTN/RCK subunit KtrA from the K+- uptake system KtrAB of Vibrio alginolyticus -: Its role in the formation of the KtrAB complex and its requirement in vivo
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DOI：
10.1074/jbc.m609084200
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发表时间：
2007-05-11
影响因子：
4.8
通讯作者：
Bakker, Evert P.
中科院分区：
文献类型：
--
作者：
Kroening, Nadine;Willenborg, Marion;Bakker, Evert P.
细菌Na+依赖性K+转位KtrAB系统的亚基KtrA属于调节蛋白和蛋白结构域的KTN/RCK家族。它们位于细胞膜的细胞质侧。通过结合配体，它们调节许多 K+ 转运蛋白和 K+ 通道的活性。为了研究溶藻弧菌 (VaKtrA) 细菌 KtrA 的功能，以 His 标记形式 (His(10)-VaKtrA) 过量生产该蛋白质，并通过亲和层析进行分离。 VaKtrA 含有富含 G 的 ADP 部分结合 β-α-β 折叠（“罗斯曼折叠”）。使用光交联和流动透析测定[P-32] ATP 和[P-32] NAD(+) 与His(10)-VaKtrA 的结合。其他核苷酸的结合通过这些化合物对P-32标记的核苷酸与蛋白质的结合的竞争来估计。 [gamma-P-32] ATP 以高亲和力结合 His(10)-VaKtrA（K-D 为 9 μM）。所有其他测试的核苷酸均表现出 30 μM 或更高的 K-D (K-i) 值。胰蛋白酶的有限蛋白水解表明 ATP 是唯一改变 VaKtrA 构象的核苷酸。 ATP 特异性促进 VaKtrA 与其 K+ 易位伴侣 VaKtrB-His(6) 的 His 标记形式形成复合物，这一点在重叠实验和将 VaKtrA 添加到与 Ni2+-琼脂糖结合的 VaKtrB-His(6) 中的实验中均检测到。在完整的大肠杆菌细胞中，VaKtrAB 活性需要高膜电位和高细胞质 ATP 浓度。 VaKtrA 中的 C 端缺失表明，对于体内活性，总共 220 个 N 端氨基酸残基中至少需要 169 个，而其 40 个 C 端残基是可有可无的。
Subunit KtrA of the bacterial Na+-dependent K+-translocating KtrAB systems belongs to theKTN/RCKfamily of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K+ transporters and K+ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His(10)-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding beta-alpha-beta-fold ("Rossman fold"). Photocross-linking and flow dialysis were used to determine the binding of [P-32] ATP and [P-32] NAD(+) to His(10)-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the P-32-labeled nucleotides to the protein. [gamma-P-32] ATP bound with high affinity to His(10)-VaKtrA (K-D of 9 mu M). All other nucleotides tested exhibited K-D (K-i) values of 30 mu M or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K+-translocating partner, VaKtrB-His(6), as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His(6) bound to Ni2+-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.