Unraveling the Allosteric Mechanism of Macrophage Migration Inhibitory Factor with Molecular Resolution

用分子分辨率揭示巨噬细胞迁移抑制因子的变构机制

基本信息

批准号：
10521825
负责人：
GEORGE LISI
金额：
$ 33.82万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-23 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10521825
关键词：
Active Sites Affect Affinity Allosteric Site Amino Acids Animal Model Asthma Binding Binding Sites Biochemical Biochemical Pathway Biochemical Reaction Biochemistry Biological Biological Assay Biological Process Biology Biophysics Catalysis Cell surface Chemicals Child Chronic Childhood Arthritis Communication Coupling Crystallization Data Disease Environment Enzymatic Biochemistry Enzymes Equilibrium Event Functional disorder Glucocorticoids Human Hydrogen Bonding Immunosuppression In Vitro Inflammation Inflammatory Ligand Binding Link Measurement Mediating Migration Inhibitory Factor Modification Molecular Molecular Bank Molecular Conformation Motion Mutagenesis Mutation NMR Spectroscopy Nuclear Magnetic Resonance Outcome Oxidation-Reduction Pathway Analysis Pathway interactions Peripheral Property Protein Dynamics Protein Region Proteins Proteomics Publications Receptor Activation Relaxation Reporting Resolution Respiratory distress Roentgen Rays Role Sampling Scheme Signal Pathway Signal Transduction Site Structure Therapeutic Variant Visualization Work biophysical properties cancer therapy chemical property chemokine receptor cytokine design driving force flexibility human disease in silico in vivo inflammatory milieu inhibitor insight molecular dynamics mutant novel protein protein interaction receptor response simulation small molecule

项目摘要

Project Summary Macrophage migration inhibitory factor (MIF) is critical to the pathophysiology of inflammation through its interaction with the chemokine receptor CD74, while also opposing the immunosuppressive effects of glucocorticoids and catalyzing enzymatic reactions of unknown biological significance. The mechanism by which MIF accommodates these and other biochemical functions within its compact structure is unclear, but we recently identified a network of amino acids that link the enzymatic active site of MIF with peripheral regions of the protein, including the proposed CD74 binding site. These residues, and likely others, allosterically regulate several biochemical functions of MIF, including enzyme catalysis, receptor activation, and protein-protein interaction. Preliminary data showed that multi-timescale dynamics of the MIF structure (and resulting changes to intersubunit hydrogen bonding) contribute to its function, leading us to hypothesize that intrinsic structural flexibility is a major driving force of the allosteric mechanism that enhances spatial-temporal control of MIF. The design of MIF selective inhibitors with therapeutic value for inflammatory diseases would be aided by a more detailed understanding of the biophysical underpinnings of MIF allostery. This proposal will explore how changes to the MIF structure via mutations and pro-inflammatory solution conditions affect its allosteric crosstalk, catalytic activity, and activation of CD74. We will complete three specific aims, beginning with atomic level characterization of the MIF allosteric network using state-of-the-art solution nuclear magnetic resonance (NMR) spectroscopy and molecular simulations. The impact of oxidative solution conditions on the MIF structure and allosteric network will then be assessed with solution NMR and quantitative proteomics. We will mimic inflammatory environments to determine how the MIF structure is modified, and if those modifications result in downstream functional differences. Lastly, we will apply our integrated NMR-MD approach to study the first MIF mutant ever associated with human disease, a Y99C variant found in children with juvenile arthritis. This mutation occurs directly at the allosteric site we identified in earlier publications. Each aim will assess the resulting biological outcomes with measurements of active site chemical properties, catalytic function (in vitro) and CD74 activation (in vivo) function. The project will dissect allosteric pathways through the analysis of differential motions probed by NMR spin relaxation, molecular simulations, and network analysis, mapping the specific amino acids and interactions responsible for transmitting structural or dynamic changes between the allosteric, enzymatic, and CD74 receptor sites. The outcomes of the work can broadly inform the promiscuous mechanisms of cytokines, the role of allostery in the extended MIF superfamily, and focus NMR-guided computational screens of molecular libraries against the MIF allosteric pathway, relevant to asthma, respiratory distress, and cancer therapies.

项目概要巨噬细胞迁移抑制因子（MIF）通过其作用对炎症的病理生理学至关重要与趋化因子受体 CD74 相互作用，同时也对抗免疫抑制作用糖皮质激素和催化具有未知生物学意义的酶反应。其机制 MIF 在其紧凑的结构中容纳这些和其他生化功能尚不清楚，但我们最近确定了一个氨基酸网络，将 MIF 的酶活性位点与蛋白质的外围区域连接起来，包括建议的 CD74 结合位点。这些残基，以及可能的其他残基，变构调节多种 MIF 的生化功能，包括酶催化、受体激活和蛋白质-蛋白质相互作用。初步数据表明，MIF 结构的多时间尺度动态（以及由此产生的变化）亚基间氢键）有助于其功能，使我们假设内在的结构灵活性是变构机制的主要驱动力，增强了 MIF 的时空控制。这设计对炎症性疾病具有治疗价值的 MIF 选择性抑制剂将有助于更多的研究详细了解 MIF 变构的生物物理基础。该提案将探讨如何改变通过突变和促炎溶液条件对 MIF 结构的影响，影响其变构串扰、催化活性和 CD74 的激活。我们将完成三个具体目标，从原子层面开始使用最先进的溶液核磁共振 (NMR) 表征 MIF 变构网络光谱学和分子模拟。氧化溶液条件对MIF结构的影响然后将通过溶液核磁共振和定量蛋白质组学评估变构网络。我们会模仿炎症环境来确定 MIF 结构如何被修饰，以及这些修饰是否会导致下游功能差异。最后，我们将应用我们的集成 NMR-MD 方法来研究第一个 MIF 与人类疾病相关的突变体，一种在患有幼年关节炎的儿童中发现的 Y99C 变体。这个突变直接发生在我们在早期出版物中确定的变构位点。每个目标都会评估由此产生的结果通过测量活性位点化学性质、催化功能（体外）和 CD74 来获得生物学结果激活（体内）功能。该项目将通过差异分析来剖析变构途径通过 NMR 自旋弛豫、分子模拟和网络分析探测运动，绘制特定的氨基酸和负责传递变构之间的结构或动态变化的相互作用，酶和 CD74 受体位点。工作成果可以广泛地为混杂机制提供信息细胞因子、变构在扩展 MIF 超家族中的作用以及焦点 NMR 引导的计算筛选针对 MIF 变构途径的分子文库，与哮喘、呼吸窘迫和癌症相关疗法。