Spectroscopic and functional determination of the interaction of Pb2+ with GATA proteins
Spectroscopic and functional determination of the interaction of Pb2+ with GATA proteins
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DOI：
10.1021/ja0464544
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发表时间：
2005-03-23
影响因子：
15
通讯作者：
Godwin, HA
中科院分区：
文献类型：
--
作者：
Ghering, AB;Jenkins, LMM;Godwin, HA
加塔蛋白是通过Cys（4）结构锌结合域结合加塔DNA元件的转录因子，并且在神经和泌尿生殖发育以及心脏疾病的发展中发挥关键的调节作用。为了评估加塔蛋白作为铅的潜在靶点，使用光谱监测的金属结合滴定来测量Pb 2+对来自鸡加塔-1（CF）的C-末端锌结合结构域和来自人加塔-1（DF）的双指结构域的亲和力。利用这种方法，通过在光谱的近紫外区域（250-380 nm）出现强谱带，直接观察到Pb 2+与CF和DF的配位。从这些实验中收集的吸收数据最适合1：1 Pb 2 +-CF模型和2：1 Pb 2 +-DF模型。使用Zn 2+的竞争实验用于确定Pb 2+对这些蛋白质的绝对亲和力。这些研究表明，Pb 2+与加塔蛋白中锌结合位点的半胱氨酸残基形成紧密的复合物，β（Pb）（1）= 6.4 CF为（+/- 2.0）x 10（9）M-1，β（2）= 6.3（+/-6.3）× 10（19）M-2，并且在Zn 2+对这些蛋白质的亲和力的一个数量级内。此外，Pb 2+能够从CF和DF中置换结合的Zn 2+。加入Pb 2+后，加塔显示出与DNA结合并随后激活转录的能力降低。因此，加塔蛋白的DNA结合和转录活性最有可能被体内螯合Pb 2+的细胞和组织（包括大脑和心脏）中的Pb 2+靶向。
GATA proteins are transcription factors that bind GATA DNA elements through Cys(4) structural zinc-binding domains and play critical regulatory roles in neurological and urogenital development and the development of cardiac disease. To evaluate GATA proteins as potential targets for lead, spectroscopically monitored metal-binding titrations were used to measure the affinity of Pb2+ for the C-terminal zinc-binding domain from chicken GATA-1 (CF) and the double-finger domain from human GATA-1 (DF). Using this method, Pb2+ coordinating to CF and DF was directly observed through the appearance of intense bands in the near-ultraviolet region of the spectrum (250-380 nm). Absorption data collected from these experiments were best fit to a 1:1 Pb2+-CF model and a 2:1 Pb2+-DF model. Competition experiments using Zn2+ were used to determine the absolute affinities of Pb2+ for these proteins. These studies reveal that Pb2+ forms tight complexes with cysteine residues in the zinc-binding sites in GATA proteins, beta(Pb)(1) = 6.4 (+/- 2.0) x 10(9) M-1 for CF and beta(2) = 6.3 (+/- 6.3) x 10(19) M-2 for Pb-2(2+)-DF, and within an order of magnitude of the affinity of Zn2+ for these proteins. Furthermore, Pb2+ was able to displace bound Zn2+ from CF and DF. Upon addition of Pb2+, GATA shows a decreased ability to bind to DNA and subsequently activate transcription. Therefore, the DNA binding and transcriptional activity of GATA proteins are most likely to be targeted by Pb2+ in cells and tissues that sequester Pb2+ in vivo, which include the brain and the heart.