Bal Wojciech, Schwerdtle Tanja, Hartwig Andrea
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
Chem Res Toxicol. 2003 Feb;16(2):242-8. doi: 10.1021/tx025639q.
Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (Asmuss, M., Mullenders, L. H. F., Elker, A., and Hartwig, A. (2000) Carcinogenesis 21, 2097-2104). To study the possible molecular mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding constants were determined using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding constants for Zn(II) and Ni(II) are (8.5 +/- 1.5) x 10(8) (log value 8.93(7)) and (1.05 +/- 0.07) x 10(6) M(-)(1) (6.02(3)), respectively, in 10 mM phosphate buffer, pH 7.4, and (6 +/- 4) x 10(9) (9.8(2)) and (2.9 +/- 0.5) x 10(6) M(-)(1) (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding constant ratios Zn(II)/Ni(II) of 800 +/- 100 and 2300 +/- 500, respectively. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-saturated XPAzf is remarkably resistant to air oxidation and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H(2)O(2) in a concentration-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H(2)O(2) concentrations tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.
着色性干皮病A互补蛋白（XPA）是DNA修复核苷酸切除修复（NER）途径蛋白复合物的成员，参与切口复合物的组装。XPA的4S锌指结构域参与与其他NER蛋白的相互作用。如先前所示，XPA的活性受到几种与DNA修复抑制有关的金属离子的损害，包括镍（II）、镉（II）和钴（II）（阿斯穆斯，M.，穆伦德斯，L.H.F.，埃尔克，A.，和哈特维希，A.（2000年）《癌变》21，2097 - 2104）。为了研究XPA抑制的可能分子机制，我们研究了锌（II）和镍（II）与代表XPA锌指序列AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam的合成37肽（XPAzf）的相互作用。使用荧光光谱和紫外 - 可见光谱测定结合常数，通过圆二色光谱提供结构见解，并用高效液相色谱研究XPAzf的氧化损伤。在pH 7.4的10 mM磷酸盐缓冲液中，锌（II）和镍（II）的结合常数分别为（8.5±1.5）×10⁸（对数8.93（7））和（1.05±0.07）×10⁶ M⁻¹（6.02（3）），在pH 7.4的50 mM磷酸盐缓冲液中分别为（6±4）×10⁹（9.8（2））和（2.9±0.5）×10⁶ M⁻¹（6.46（8）），锌（II）/镍（II）的结合常数比分别为800±100和2300±500。镍（II）离子与XPAzf的硫形成平面正方形配合物，与天然锌（II）配合物的四面体结构相反。因此，在镍（II）取代的肽中整体锌指结构丧失。锌（II）饱和的XPAzf对空气氧化具有显著抗性，并且仅以浓度依赖的方式被浓度为0.01 mM、0.1 mM和1 mM的过氧化氢缓慢氧化。然而，仅10倍摩尔过量的镍（II）的存在就足以加速所有三种测试过氧化氢浓度下的这一过程。总体而言，我们的结果表明XPAzf在特定条件下会受到镍（II）的攻击。
