Origins of Ligand Binding and Selectivity in Methyllysine Reader and Writer Proteins

抽象的。 组蛋白赖氨酸(Lys)甲基化是基因表达的表观遗传调控。组蛋白赖氨酸 甲基转移酶(HKMT或“写入者”)在特定位置安装甲基化的赖氨酸(KMAN，n=1-3)，而赖氨酸 甲基化招募了一个不同的“阅读器蛋白”家族，将这些动态的翻译后翻译 修饰并诱导下游事件，导致转录的启动或沉默。 这些事件的调节失调与包括癌症在内的一系列疾病有关。虽然这些阅读器 和Writer蛋白是潜在的药物靶点，但很少有研究探讨它们的作用机制。 识别天然的kman底物或通过哪些小分子或组蛋白突变导致它们的抑制。 这项建议旨在确定提供结合亲和力、选择性和催化性的力量的平衡 以及最近发现的这些蛋白质-蛋白质相互作用的抑制剂，目的是获得 洞察力将进一步努力开发这些蛋白质的抑制剂。为此，可持续发展的机制 识别将通过蛋白质和配体导向的结构活性的组合进行研究 两性关系。将制定补充方法，以便有选择地纳入 电子调谐的非天然氨基酸，包括取代苯丙氨酸和酪氨酸残基以及 含氟芳香族残留物。将这一方法与既定的技术结合使用， 芳香族和带电残基的电子学将被系统地改变，以确定 阳离子-pI相互作用，范德华相互作用，疏水效应，以及盐桥对亲和力和 通过微妙不同的结合标记的二甲基和三甲基赖氨酸阅读器蛋白的选择性 口袋里。此外，还将研究芳香族残基在HKMT活性中心中的作用。 既有催化作用又有抑制作用。在所有情况下，X射线结晶学将被用来提供对 认知机制。此外，与甲基赖氨酸模拟物结合的机制已知 将对阅读器和写入器蛋白的抑制剂进行表征，以确定是否存在新的作用机制 结合和抑制是可行的。总而言之，这些综合研究将提供一个量化框架 用于开发高质量的分子探针和具有亲和力程度的下一代抑制剂 以及应用于疾病所必需的选择性。此外，这项工作应随时扩展到其他 重要的蛋白质家族，包括甲基赖氨酸擦除器和写入器以及甲基精氨酸读取器， 作家和橡皮擦。

Abstract. Histone protein lysine (Lys) methylation is an epigenetic regulator of gene expression. Histone Lys methyltransferases (HKMTs or “writers”) install methylated Lys (KMen, n = 1-3) at specific positions, and Lys methylation recruits a diverse family of “reader proteins” that bind these dynamic post translational modifications and induce downstream events leading to either initiation or silencing of transcription. Dysregulation in these events is associated with a wide range of diseases including cancer. While these reader and writer proteins are potential medicinal targets, few studies have probed the mechanism by which they recognize native KMen substrates or by which small molecules or histone mutations lead to their inhibition. This proposal aims to determine the balance of forces that provide binding affinity, selectivity, and catalysis for KMen as well as recently discovered inhibitors of these protein-protein interactions with the aim of gaining insights that will further the effort to develop inhibitors for these proteins. To this end, the mechanism of KMen recognition will be investigated through a combination of protein- and ligand-directed structure-activity relationships. Complementary methodology will be developed for the site-selective incorporation of electronically tuned unnatural amino acids, including substituted-phenylalanine and tyrosine residues and fluorinated aromatic residues. Using this methodology in conjunction with established techniques, the electronics of aromatic and charged residues will be systematically altered to determine the contribution of cation-pi interactions, van der Waals interactions, the hydrophobic effect, and salt bridges on affinity and selectivity across a range of di- and tri-methyl lysine reader proteins marked by subtly different binding pockets. Additionally, the role of aromatic residues in the active site of HKMTs will be investigated with respect to both catalysis and inhibition. In all cases, X-ray crystallography will be used to provide structural insights into the mechanism of recognition. Additionally, the mechanism of binding to methyl lysine mimetics and known inhibitors of reader and writer proteins will be characterized to determine whether novel mechanisms for binding and inhibition are feasible. In total, these comprehensive studies will provide a quantitative framework for the development of high quality molecular probes and next-generation inhibitors with the degree of affinity and selectivity necessary for application to disease. Furthermore, this work should readily extend to other important protein families, including methyl lysine erasers and writers as well as methyl arginine readers, writers, and erasers.