Cobalt(II)-Responsive DNA Binding of a GCN4-bZIP Protein Containing Cysteine Residues Functionalized with Iminodiacetic Acid
Cobalt(II)-Responsive DNA Binding of a GCN4-bZIP Protein Containing Cysteine Residues Functionalized with Iminodiacetic Acid
复制标题
DOI：
10.1002/anie.200902888
复制
发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Futaki, Shiroh
中科院分区：
文献类型：
--
作者：
Azuma, Yusuke;Imanishi, Miki;Futaki, Shiroh
设计具有可由外部刺激控制的功能的蛋白质是肽/蛋白质工程中的一个挑战。[1]许多天然蛋白质利用金属离子来稳定其结构和调节其生物活性。因此，具有有效金属配位的蛋白质的结构工程使得能够在两种不同结构之间切换将增加开发新型蛋白质机器的可行性。[2，3]我们以前的研究表明，当Ida部分位于螺旋肽中的位置i和i+ 2时，亚氨基二乙酸（Ida）部分的金属配位产生螺旋度的显著降低。[3]这种方法对于具有稳定螺旋结构的蛋白质的功能调节特别有用，如通过衍生自Jun和Fos癌蛋白的亮氨酸拉链肽之间的识别切换以及通过人工离子通道的膜外门控控制所例示的。[3]然而，由于缺乏在蛋白质的特定位置有效引入Ida部分的方法，该概念的应用仅限于合成肽。[4]我们现在提出了一种新的方法，用于将Ida部分引入蛋白质。该方法涉及通过用新的官能化试剂N-（2-甲苯磺酰基硫代乙基）亚氨基二乙酸（Ts-S-IDA，5）处理来特异性修饰半胱氨酸残基。我们证明了这种方法的实用性，通过修改来自酵母转录因子GCN 4与艾达部分的蛋白质。对于所得蛋白质之一，观察到响应于金属的DNA结合的成功切换。GCN 4-bZIP蛋白（bZIP=碱性亮氨酸拉链）由两个螺旋片段组成。通过亮氨酸拉链片段的二聚化对于碱性片段与靶DNA片段（AP-1位点）的结合至关重要。[5]该蛋白质的结构已被充分研究，并已被用作通过二聚体形成的切换来设计DNA结合的可逆控制的模型。[6]在本研究中，设计了在亮氨酸拉链片段（bZIP-1a，bZIP-1b）的i和i+ 2位含有一对Ida残基的bZIP蛋白，通过Ida与金属的相互作用来改变DNA结合，从而使bZIP蛋白的螺旋结构不稳定（图1）。如图Ic所示选择Ida修饰的位置，以便在不存在金属的情况下对二聚体形成具有最小的影响。[七]《中国日报》
The design of proteins with functions that can be controlled by external stimuli is a challenge in peptide/protein engineering.[1] Many natural proteins utilize metal ions to stabilize their structure and regulate their bioactivity. The structural engineering of proteins with effective metal coordination to enable switching between two different structures would thus increase the feasibility of developing novel protein machineries.[2, 3] Our previous studies showed that metal coordination of iminodiacetic acid (Ida) moieties yielded a significant decrease in helicity when the Ida moieties were placed at positions i and i+ 2 in helical peptides.[3] This approach is particularly useful for the functional regulation of proteins with stable helical structures, as exemplified through recognition switching between leucine zipper peptides derived from Jun and Fos oncoproteins and through extramembrane gating control of artificial ion channels.[3] However, due to the lack of methodology for the effective introduction of Ida moieties at specific positions in proteins, the application of this concept has been limited to synthetic peptides.[4] We now present a new method for introducing Ida moieties into proteins. The method involves the specific modification of cysteine residues by treatment with a new functionalization agent, N-(2-tosylthioethyl) iminodiacetic acid (Ts-S-IDA, 5). We demonstrated the practicability of this approach by modifying a protein derived from the yeast transcription factor GCN4 with Ida moieties. Successful switching of the DNA binding in response to a metal was observed for one the resulting proteins. The GCN4-bZIP protein (bZIP= basic leucine zipper) consists of two helical segments. Dimerization through the leucine zipper segment is critical for binding to the target DNA segment (AP-1 site) by the basic segment.[5] The wellstudied structure of the protein has been employed as a model for the design of reversible control of DNA binding through switching of the dimer formation.[6] In the present study, bZIP proteins containing a pair of Ida residues at positions i and i+ 2 in the leucine zipper segment (bZIP-1a, bZIP-1b) were designed to destabilize the helical structure of the bZIP protein by the interaction of Ida with metals to switch DNA binding (Figure 1). The positions of the Ida modification were selected as shown in Figure 1 c so as to have a minimum effect on dimer formation in the absence of metals.[7]