SH2结构域和包括翻译后磷酸化酪氨酸残基(pY)的蛋白质片段之间的蛋白质-蛋白质相互作用支持许多信号转导级联,使细胞对其环境做出反应。写作失调,擦除,阅读这些翻译后修饰是人类疾病的标志,尤其是癌症。阐明含有SH2结构域的衔接蛋白Crk和CrkL在肿瘤细胞迁移和侵袭中的精确作用是具有挑战性的,因为没有特异性和有效的拮抗剂可用。Crk和CrkLSH2与对接蛋白p130Cas的一个区域相互作用,该区域包含15个潜在的含pY的四肽基序。本章总结了针对这种Crk/CrkL-p130Cas相互作用的肽拮抗剂的最新努力。我们描述了用于功能测定的Crk和CrkLSH2的重组表达和纯化的方案,以及我们确定p130Cas序列的共有结合基序的程序。为了开发一种更有效的拮抗剂,我们采用通常与基于结构的药物设计相关的方法。使用RosettaFlexPepDock进行计算对接,这说明肽比通常构成文库的有机小分子具有更多的构象自由度,提供定量对接指标,以优先考虑用于实验测试的候选肽。一系列生物物理分析,包括荧光偏振,差示扫描荧光分析和饱和转移差异核磁共振波谱,被用来评估候选人。并行,GST下拉竞争测定法在体外表征蛋白质-蛋白质结合。一起来看,我们的方法产生的Crk/CrkL-p130Cas轴的肽拮抗剂将用于验证目标,评估可药用性,促进体外试验发展,并可能作为治疗干预的先导化合物。
Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein
p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-
p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the
p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-
p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.