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: 9742021
• 负责人: MARCEY L WATERS
• 金额: $ 3.77万
• 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/9742021
• 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; Regulator Genes; Reporting; Role; Site; Sodium Chloride; Source; Structure; Structure-Activity Relationship; Sulfur; Techniques; Tyrosine; Work; X-Ray Crystallography; alkyl group; base; cancer type; druggable target; gain of function; inhibitor/antagonist; insight; interdisciplinary approach; mimetics; molecular recognition; next generation; novel; protein protein interaction; recruit; 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剂。组蛋白lys 甲基转移酶（HKMTS或“作家”）在特定位置安装甲基化的Lys（Kmen，n = 1-3），Lys安装 甲基化报告了一个不同的“读取器蛋白”的家族，该家族结合了这些动态后翻译后 修改和影响下游事件导致主动或沉默转录。 这些事件中的失调与包括癌症在内的多种疾病有关。而这些读者 作者蛋白是潜在的医学靶标，很少有研究证明它们的机制 公认的天然KMEN底物或小分子或组蛋白突变导致其抑制作用。 该建议旨在确定提供结合亲和力，选择性和催化的力量的平衡 Kmen以及最近发现了这些蛋白质蛋白相互作用的抑制剂，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，Kmen的机制 将通过蛋白质和配体指导结构活性的结合来研究识别 关系。将开发互补方法，以用于现场选择性就业 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 氟芳族的保留。将此方法与既定技术结合 芳香和充电残差的电子设备将系统地改变，以确定 阳离子-PI相互作用，范德华相互作用，疏水作用以及对亲和力和 在一系列二甲基赖氨酸读取器蛋白上的选择性，标志着微妙的结合 口袋。此外，将研究芳香族残留物在HKMT的活性部位中的作用 催化和抑制。在所有情况下，X射线晶体学都将用于提供结构性见解 识别机制。另外，与甲基赖氨酸模仿和已知的结合机制 读者和作家蛋白的抑制剂将被表征以确定新的机制是否具有 结合和抑制是可行的。总的来说，这些全面研究将提供定量框架 为了发展高质量分子问题和具有亲和力程度的下一代抑制剂 以及适用疾病所需的选择性。此外，这项工作应该很容易扩展到其他 重要的蛋白质家族，包括甲基赖氨酸橡皮擦和作家以及甲基精氨酸读取器， 作家和橡皮擦。