Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin
Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin
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DOI：
10.1107/s0907444907068217
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发表时间：
2008-04-01
影响因子：
2.2
通讯作者：
Mueller-Tiemann, Beate
中科院分区：
文献类型：
--
作者：
Bandeiras, Tiago M.;Hillig, Roman Christian;Mueller-Tiemann, Beate
丝氨酸/苏氨酸蛋白Polo-like kinase1(PLK-1)作为有丝分裂的关键调控因子，在肿瘤治疗中是一个有效的药物靶点。为了能够进行结构导向的药物设计，尝试确定PLK-1的激动域的晶体结构。采用多重平行克隆和表达方法，筛选出一组可在昆虫细胞中大量表达并可纯化至高纯度的长度变异体。然而，所有使这些结构具体化的尝试都失败了。最终通过产生针对PLK-1的设计的锚蛋白重复序列(DARPins)并将其用于共结晶而获得晶体。在这里，野生型载脂蛋白PLK-1的激动域的第一晶体结构，与DARPin 3H10，强调了选择性DARPins作为结晶工具的力量。结构被细化到2.3埃分辨率，显示了PLK-1的活性构象。它拓宽了药物设计的建模和共结晶研究的基础。3H10的结合表位富含精氨酸、谷氨酰胺和赖氨酸残基，表明DARPin通过掩盖不利于晶体接触形成的表面补丁而实现结晶。基于在晶体中观察到的堆积，设计了一个截断的DARPin变体，该变体具有更好的结合特性。
As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 angstrom resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.