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节因子。组蛋白赖氨酸 甲基转移酶（HKMT或“writers”）在特定位置安装甲基化的Lys（KMen，n = 1-3），并且Lys 甲基化招募了一个不同的“阅读器蛋白”家族， 修饰并诱导导致转录起始或沉默的下游事件。 这些事件中的失调与包括癌症在内的多种疾病有关。虽然这些读者 和书写蛋白是潜在的药物靶点，但很少有研究探讨其作用机制， 识别天然KMen底物或通过小分子或组蛋白突变导致其抑制。 该提案旨在确定提供结合亲和力，选择性和催化的力的平衡， KMen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 这些见解将进一步努力开发这些蛋白质的抑制剂。为此，KMen的机制 识别将通过结合蛋白质和配体定向的结构-活性来研究 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，包括取代的苯丙氨酸和酪氨酸残基， 氟化芳族残基。使用这种方法结合已建立的技术， 芳香族和带电残基的电子将被系统地改变，以确定 阳离子-π相互作用、货车范德华相互作用、疏水效应和盐桥对亲和力和 在一系列二甲基和三甲基赖氨酸阅读器蛋白中的选择性， 口袋此外，芳香族残基在HKMT活性中心的作用将被研究， 催化和抑制作用。在所有情况下，X射线晶体学将被用来提供结构的见解， 识别机制。此外，结合甲基赖氨酸模拟物和已知的 阅读器和写入器蛋白的抑制剂将被表征，以确定是否有新的机制， 结合和抑制是可行的。总的来说，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下一代抑制剂， 和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。

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.