Characterization of the soluble domain of the ABC7 type transporter Atm1
Characterization of the soluble domain of the ABC7 type transporter Atm1
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DOI：
10.1074/jbc.m306472200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Cowan, JA
中科院分区：
文献类型：
--
作者：
Chen, CA;Cowan, JA
ATM1是一种ABC转运蛋白，位于酵母线粒体中，与胞质铁硫簇蛋白的成熟有关。Atm1的可溶性核苷酸结合区(Atm1-C)已在大肠杆菌中高效表达、纯化和鉴定。用荧光法测定了ATM1-C与ATP(K-D类似于97um，pH 7.3，类似于102um，pH 10.0)和ADP(K-D类似于43um，pH 7.3，92 um，pH 10.0)的解离常数(K-D)。对ADP较高的结合亲和力表明，跨膜结构域可能需要促进核苷酸结合域的结构变化，以促进底物输出和ADP释放。ADP对Atm1-C也有抑制作用，其IC50为10 mM。测得ATM1-C的米氏常数V-max、K-M和k(CAT)分别为1.822微米/分(-1)、513微米和0.055微米(-1)。Atm1-C-ATPase的金属依赖性表现为：Mn2+&Gt；Mg2+&Gt；Co2+；而在较高浓度下，Mg2+和Co2+均表现出抑制作用。PH谱和与Hisp的结构比较与His和Lys在促进ATPase活性方面的作用是一致的。CD谱对Atm1-C的结构分析表明，Atm1-C的二级结构与原核同源物(Hisp)的二级结构相似，而以Hisp为模板对Atm1-C三级结构的建模也与三级结构的相似性一致。在较高浓度下，Atm1-C倾向于形成二聚体或更高的聚集状态；然而，Atm1-C对ATPase活性的浓度依赖性和Hill分析的结果(n(APP)=1.1)表明，在ATP水解过程中基本上没有协同作用，而原核生物Hisp转运蛋白的观察结果表明，全长和可溶性域都表现出完全协同作用。因此，在真核细胞Atm1转运蛋白的情况下，任何合作反应都必须通过跨膜结构域来调节。
Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (K-D) for Atm1-C binding of ATP (K-D similar to97 muM, pH 7.3, and similar to102 muM, pH 10.0) and ADP (K-D similar to43 muM, pH 7.3, and 92 muM, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mM. The Michaelis-Menten constants V-max, K-M, and k(cat) of Atm1-C were measured as 1.822 muM min(-1), 513 muM, and 0.055 min(-1), respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (n(app) = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter.