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首页> 外文期刊>Journal of Molecular Biology >Probing the electrostatics and pharmacological modulation of sequence-specific binding by the dna-binding domain of the ETS family transcription factor Pu.1: A binding affinity and kinetics investigation
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Probing the electrostatics and pharmacological modulation of sequence-specific binding by the dna-binding domain of the ETS family transcription factor Pu.1: A binding affinity and kinetics investigation

机译：探索ETS家族转录因子Pu.1的dna结合域对序列特异性结合的静电和药理学调节：结合亲和力和动力学研究

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摘要

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤ 105 M-1 s -1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g.; Ets-1, TEL) for which high-affinity binding is driven by rapid association ( 10 7 M-1 s-1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.
机译：ETS转录因子家族的成员调节功能多样的基因。所有ETS蛋白都共享一个结构保守但序列差异的DNA结合结构域，称为ETS结构域。尽管ETS-DNA复合物的结构和热力学是众所周知的，但对于序列识别的动力学知之甚少，该方面为了解其分子机理提供了潜在的见识。我们已经通过生物传感器表面等离振子共振（SPR）表征了PU.1的ETS域与DNA的结合。 SPR分析揭示了PU.1 ETS域与DNA结合的惊人动力学特性。在低盐浓度下，它以非常慢的缔合速率常数（≤105 M-1 s -1）结合高亲和力同源DNA，并通过相应较小的解离速率常数进行补偿。动力学强烈地依赖于盐，但是相互平衡以在平衡常数中产生相对弱的依赖。此配置文件与其他ETS域（例如Ets-1，TEL）的报告数据形成了鲜明对比，其他ETS域通过快速关联（> 10 7 M-1 s-1）驱动高亲和力结合。我们根据ETS-DNA结合的水合特性来解释这种差异，并提出该DNA结合域家族至少采用两种序列识别机制。此外，我们使用SPR来证明药理学抑制序列特异性ETS-DNA结合的潜力，使用次要的沟结合性他斯坦霉素作为模型化合物。我们的工作将SPR确立为一种有价值的技术，可用于扩展我们对ETS-DNA相互作用的分子机制的理解以及开发用于生物技术和治疗目的的潜在小分子药物。

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