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

甲基赖氨酸读取和写入蛋白中配体结合和选择性的起源

• 批准号: 9309407
• 负责人: Eric Michael Brustad
• 金额: $ 27.73万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9309407
• 关键词: Active Sites; Affinity; Ammonium; Arginine; Aromatic Amino Acids; Benchmarking; Binding; Binding Proteins; Biochemical; Biological Assay; Biology; Catalysis; Catalytic Domain; Cations; Charge; Complement; Computing Methodologies; Coupled; Data; Development; Discrimination; Disease; Drug Design; Electronics; Epigenetic Process; Equilibrium; Event; Family; Future; Gene Expression; Gene Silencing; Generations; Genetic Transcription; Histone-Lysine N-Methyltransferase; Histones; Human; Hydrophobic Interactions; Hydrophobicity; Individual; Lead; Ligand Binding; Ligands; Lysine; Malignant Childhood Neoplasm; Malignant Neoplasms; Measures; Methodology; Methods; Methylation; Methyltransferase; Molecular; Molecular Probes; Mutagenesis; Mutation; Norleucine; Outcome; Phenylalanine; Positioning Attribute; Post-Translational Protein Processing; Protein Family; Protein Methyltransferases; Proteins; Proteome; Pyrrolidines; Reader; Recruitment Activity; Regulator Genes; Reporting; Role; Site; Sodium Chloride; Source; Structure; Structure-Activity Relationship; Sulfur; Techniques; Tyrosine; Work; X-Ray Crystallography; alkyl group; base; cancer type; gain of function; inhibitor/antagonist; insight; interdisciplinary approach; mimetics; molecular recognition; next generation; novel; protein protein interaction; small molecule; small molecule inhibitor; therapeutic target; tool; unnatural amino acids

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.

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 Lys（KMen，n = 1-3），并且 Lys 甲基化招募了一个多样化的“阅读蛋白”家族，这些蛋白结合了这些动态的翻译后蛋白 修饰并诱导导致转录启动或沉默的下游事件。 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医学靶点，但很少有研究探讨它们的机制 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、选择性和催化作用的力量平衡 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 将通过蛋白质和配体指导的结构活性的结合来研究识别 关系。将开发补充方法用于选择性地纳入 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以及 氟化芳族残基。将这种方法与现有技术结合使用， 芳香族和带电残基的电子将被系统地改变以确定的贡献 阳离子-π相互作用、范德华相互作用、疏水效应和盐桥对亲和力和 对一系列二甲基和三甲基赖氨酸阅读器蛋白的选择性，其结合略有不同 口袋。此外，还将研究芳香族残基在 HKMT 活性位点中的作用 具有催化和抑制作用。在所有情况下，X 射线晶体学都将用于提供结构见解 识别机制。此外，与甲基赖氨酸模拟物结合的机制和已知的 读者和作者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定量框架 用于开发具有亲和力的高质量分子探针和下一代抑制剂 以及应用于疾病所需的选择性。此外，这项工作应该很容易扩展到其他领域 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。