Eren Elif, Kennedy David C, Maroney Michael J, Argüello José M
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
J Biol Chem. 2006 Nov 10;281(45):33881-91. doi: 10.1074/jbc.M605218200. Epub 2006 Sep 13.
HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-type ATPases, plant Zn2+-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn2+ K(1/2) for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 +/- 3 nM). Circular dichroism spectral analysis indicated the presence of 8% alpha-helix, 45% beta-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% alpha-helix, 39% beta-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn2+ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn2+ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn2+ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn2+ binding to the C terminus, pointing out the importance of His residues in Zn2+ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn2+ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)4 coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn2+-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P1B-type ATPases, they also appear to regulate enzyme turnover rate.
HMA2是来自拟南芥的一种锌离子ATP酶。它通过驱动锌离子外流，有助于维持细胞内的金属稳态。与P1B型ATP酶不同，植物锌离子ATP酶具有富含半胱氨酸（Cys）和组氨酸（His）的长C末端序列。去除HMA2的244个氨基酸C末端会导致酶周转率降低43%，而对酶激活的锌离子K(1/2)没有显著影响。对分离出的HMA2 C末端的表征表明，该片段以高亲和力（Kd = 16 +/- 3 nM）结合三个锌离子。圆二色光谱分析表明，C末端肽中存在8%的α-螺旋、45%的β-折叠和48%的无规卷曲，金属结合后结构有明显变化（8%的α-螺旋、39%的β-折叠和52%的无规卷曲）。在存在一当量锌离子的情况下，锌-C-金属结合结构域（Zn-C-MBD）的锌K边X射线吸收光谱（XAS）表明，形成的平均锌配合物由三个组氨酸残基和一个半胱氨酸残基组成组成。每个C-MBD再添加两个额外的锌离子后，这些锌离子主要由组氨酸残基配位，因此表明三个锌离子结合结构域可能不相同。用焦碳酸二乙酯修饰组氨酸残基完全抑制了锌离子与C末端的结合，指出了组氨酸残基在锌离子配位中的重要性。相比之下，用碘乙酸烷基化半胱氨酸并不能阻止锌离子与HMA2 C末端的结合。半胱氨酸烷基化蛋白的锌K边XAS与锌位点的（N/O）4配位一致，其中三个配体适合组氨酸残基。总之，植物锌离子ATP酶在其细胞质C末端含有新型金属结合结构域。它们在结构上与大多数P1B型ATP酶中特征明确的N末端金属结合结构域不同，似乎也能调节酶的周转率。
