Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden.
Methods Mol Biol. 2021;2318:21-43. doi: 10.1007/978-1-0716-1476-1_3.
The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.
c-MYC 转录因子的 C 端区域由大约 100 个氨基酸组成，在其自然状态下不具有稳定的结构。当这个区域通过耦合折叠和结合机制与必需的伴侣 MAX 结合时，它形成一个碱性-螺旋-环-螺旋-亮氨酸拉链（bHLHZip）异二聚体复合物。MYC 的 C 端区域是许多药物发现计划的目标，这些计划通过阻断二聚化事件和/或与 DNA 结合来直接抑制 MYC，对异二聚体复合物的部分折叠、动态性质的正确理解对于这些努力至关重要。bHLHZip 基序还驱动与辅因子的蛋白质-蛋白质相互作用，这些辅因子对于 MYC 靶基因的转录抑制和激活都至关重要。针对这些相互作用可能提供了一种开发替代方法来阻止 MYC 功能的手段；然而，这些调节相互作用的分子机制尚未被很好地理解。本文提供了生产高质量人 c-MYC C 端自身及其与 MAX 复合物的方法，以及如何使用核磁共振波谱学和 X 射线晶体学研究它们的方法。我们的蛋白质表达和纯化方案已经用于研究与辅因子的相互作用。
