DNA as a Molecular Ruler: Interrogation of a Tandem SH2 Domain with Self-Assembled, Bivalent DNA-Peptide Complexes
DNA as a Molecular Ruler: Interrogation of a Tandem SH2 Domain with Self-Assembled, Bivalent DNA-Peptide Complexes
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DOI：
10.1002/anie.201007593
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
--
作者：
Eberhard, Hendrik;Diezmann, Franziska;Seitz, Oliver
DNA的识别特性使得能够通过自组装按顺序编程构建定义的DNA结构。[1]化学修饰的DNA分子已被用于精确控制发色团、金属、纳米颗粒和蛋白质的空间排列。[2]绝大多数研究都与材料性能的设计有关。相对较少的研究已经调查了用于生命科学的生物活性配体的DNA支架排列。[3]其中值得注意的例子是DNA组装的糖簇[4]和编码的自组装文库。[5]其目的是模仿生物配体展示和/或促进药物发现过程。[6]在这里，我们提出了一种新的方法。我们制备了自组装的DNA-肽复合物，并探讨了DNA控制的肽呈递是否可以用于探测同源靶蛋白的结构特性。具体来说，我们研究了Syk激酶的蛋白结合域，Syk激酶是一种参与调节淋巴细胞活化的蛋白激酶。我们证明，重要的结构参数，如蛋白质结合口袋的首选安排和连接域间的灵活性，可以通过使用一组DNA复合物进行评估。它示出的自组装三元复合物中的单链和双链段的组合提供了一种方便的手段来系统地改变距离约束以及配体显示的灵活性。许多信号传导酶使用两个或更多个同源蛋白结合结构域来实现与同源靶标的牢固相互作用。例如，Syk和Zap-70激酶[7]包含串联的SH 2结构域，其选择性地识别蛋白质底物的两个适当间隔的四磷酸肽结合基序。[8]在缺乏蛋白质-肽复合物的解析结构的情况下，很难评估高亲和力蛋白质-蛋白质结合的要求。使用二价合成结合剂的化学探测已被广泛使用，例如，在Syk激酶的串联SH 2结构域的研究中。[9，10]由于这种二价蛋白质被充分表征，我们选择它作为基于DNA的结构进行空间筛选的靶标。与二价肽复合的Syk-tSH 2结构域的晶体结构显示，两个识别的磷酸酪氨酸残基彼此排列在34内（图1a）。[11]发现不对称单元包含6个tSH 2分子，其显示SH 2结构域的不同排列。化学探测显示
The recognition properties of DNA enable the sequenceprogrammed construction of defined DNA architectures by self-assembly.[1] Chemically modified DNA molecules have been used to precisely control the spatial arrangement of chromophores, metals, nanoparticles, and proteins.[2] The vast majority of studies have been concerned with the design of materials properties. Comparatively few studies have investigated the DNA-scaffolded arrangement of bioactive ligands for use in the life sciences.[3] Among the notable examples are DNA-assembled glycoclusters [4] and encoded self-assembled libraries.[5] The aim was to mimic biological ligand display and/or to facilitate the drug-discovery process.[6] Herein we present a new approach. We prepared self-assembled DNA–peptide complexes and explored whether the DNA-controlled presentation of peptides can be used to probe the structural properties of the cognate target protein. Specifically, we studied a protein-binding domain of the Syk kinase, a protein kinase that is involved in the regulation of lymphocyte activation. We demonstrate that important structural parameters, such as the preferred arrangement of protein-binding pockets and the flexibility of the connecting interdomain, can be assessed by using a set of DNA complexes. It is shown that the combination of single-stranded and double-stranded segments in self-assembled ternary complexes provides a convenient means to systematically vary the distance constraint as well as the flexibility of the ligand display. Many signaling enzymes use two or more homologous protein-binding domains to achieve firm interactions with cognate targets. For example, the Syk and Zap-70 kinases,[7] among others, contain tandem SH2 domains, which selectively recognize two appropriately spaced tetraphosphopeptide binding motifs of the protein substrate.[8] In the absence of a solved structure of the protein–peptide complex, it is difficult to assess the requirements for high-affinity protein–protein binding. Chemical probing with bivalent synthetic binders has been used extensively, for example, in studies of the tandem SH2 domain of the Syk kinase.[9, 10] Since this bivalent protein is well-characterized, we selected it as a target for spatial screening with DNA-based architectures. The crystal structure of the Syk-tSH2 domain in a complex with a bivalent peptide shows that the two recognized phosphotyrosine residues are arranged within 34 of one another (Figure 1a).[11] The asymmetric unit was found to contain six tSH2 molecules, which showed varied arrangements of the SH2 domains. Chemical probing has revealed