使用荧光偏振和分子动力学模拟分析ErbB2的MEMO1结合特异性,Biochemistry

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使用荧光偏振和分子动力学模拟分析ErbB2的MEMO1结合特异性
Biochemistry ( IF 2.9 ) Pub Date : 2018-08-01 00:00:00 , DOI: 10.1021/acs.biochem.8b00582
Madeline L. Newkirk ₁ , Kristen J. Rubenstein ₁ , Jessica Y. Kim ₁ , Courtney L. Labrecque ₁ , Justin Airas ₁ , Cooper A. Taylor ₁ , Hunter D. Evans ₁ , Quincy McKoy ₁ , Carol A. Parish ₁ , Julie A. Pollock ₁

Affiliation

ErbB2信号通路与乳腺癌的形成，生长和侵袭有关。因此，了解与这些途径相关的蛋白质的行为以及影响ErbB2功能的调节因子至关重要。MEMO1是一种氧化还原活性蛋白，显示与磷酸化的ErbB2缔合并介导细胞运动。我们已经开发了一种荧光偏振测定法，以探测MEMO1和含有磷酸化酪氨酸残基的ErbB2衍生肽之间的相互作用。如蛋白质与蛋白质的相互作用所预期的，这种相互作用被证明是pH依赖性的，并且与更长的肽结合更强。我们已经使用荧光偏振测定和分子动力学模拟将MEMO1的结合界面定量映射到了肽上。我们已经证实，肽的磷酸化对于结合是必不可少的，并且通过诱变已经鉴定出有助于有利相互作用的残基。我们的结果突出了MEMO1的蛋白质与蛋白质相互作用的重要性，这种相互作用补充了氧化酶的活性。将来，这些研究将提供一种筛选MEMO1选择性调节剂的方法，这将允许进行其他生物学研究。

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Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations

ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH-dependent and stronger with longer peptides as would be expected for protein–protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein–protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.

更新日期：2018-08-01

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