Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations
Analysis of MEMO1 Binding Specificity for ErbB2 Using Fluorescence Polarization and Molecular Dynamics Simulations
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DOI：
10.1021/acs.biochem.8b00582
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发表时间：
2018-08-28
期刊：
影响因子：
2.9
通讯作者：
Pollock, Julie A.
中科院分区：
文献类型：
--
作者：
Newkirk, Madeline L.;Rubenstein, Kristen J.;Pollock, Julie A.
ERBB2信号通路与乳腺癌的形成、生长和侵袭有关；因此，了解与这些通路相关的蛋白质的行为以及影响ErbB2功能的调节因素是至关重要的。MEMO1是一种氧化还原活性蛋白，被证明与磷酸化的ErbB2有关，并介导细胞运动。我们开发了一种荧光偏振分析方法来探测MEMO1与含有磷酸化酪氨酸残基的ErbB2衍生多肽之间的相互作用。这种相互作用是pH依赖的，并且与蛋白质相互作用的预期一样，随着多肽的增加，这种相互作用更强。我们利用荧光偏振分析和分子动力学模拟对MEMO1与多肽的结合界面进行了定量映射。我们已经证实了多肽的磷酸化对于结合是必不可少的，并且通过突变已经确定了有助于有利相互作用的残基。我们的结果强调了MEMO1的蛋白质相互作用的重要性，该相互作用补充了氧化酶的活性。在未来，这些研究将提供一种筛选MEMO1选择性调节子的方法，这将允许进行更多的生物学研究。
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 ErbB2derived 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.