Structure of Nucleophosmin DNA-binding Domain and Analysis of Its Complex with a G-quadruplex Sequence from the c-MYC Promoter
Structure of Nucleophosmin DNA-binding Domain and Analysis of Its Complex with a G-quadruplex Sequence from the c-MYC Promoter
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DOI：
10.1074/jbc.m112.371013
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发表时间：
2012-08-03
影响因子：
4.8
通讯作者：
Federici, Luca
中科院分区：
文献类型：
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作者：
Gallo, Angelo;Lo Sterzo, Carlo;Federici, Luca
核磷蛋白(NPM1)是一种核质穿梭蛋白，主要定位于核仁，在核糖体成熟和输出、中心体复制和对应激刺激的反应等细胞功能中发挥关键作用。到目前为止，在急性髓系白血病中已经发现了NPM1基因末端外显子的50多个突变；由于NPM1 C-末端结构域高度不稳定以及新的核输出信号的出现，突变的蛋白稳定地定位于细胞质中。我们以前已经证明，70个残基的NPM1 C末端结构域(NPM1-C70)能够高亲和力结合c-myc基因启动子上具有平行G-四链结构的特定区域。在这里，我们介绍了NPM1-C70结构域的溶液结构，并对其与c-myc衍生的G-四链相互作用的核磁共振分析。这些数据被用来计算该络合物的实验约束的分子对接模型。NPM1-C70末端三螺旋束通过与G-四链磷酸骨架的静电相互作用，在螺旋H1和H2之间的界面结合G-四链DNA。此外，我们还表明，三螺旋束N末端的17个富含赖氨酸残基的序列是无序的，虽然是必要的，但并不直接参与复合体的接触面。
Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.