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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：
10708796
负责人：
GEORGE LISI
金额：
$ 31.12万
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/10708796
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 Data Disease Environment Enzymatic Biochemistry Enzymes Equilibrium Event Functional disorder Glucocorticoids Heterozygote Human Hydrogen Bonding Immunosuppression In Vitro Inflammation Inflammatory Ligand Binding Link Maps 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 transmission process

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.

项目摘要巨噬细胞移动抑制因子(MIF)在炎症的病理生理过程中起重要作用。与趋化因子受体CD74的相互作用，同时也对抗糖皮质激素和催化酶反应的生物学意义未知。这一机制通过它 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将MIF的酶活性部位与蛋白质外围区域联系起来的氨基酸网络，包括建议的CD74结合位点。这些残基，以及可能的其他残基，变构地调节着几个 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。初步数据显示，MIF结构的多时间尺度动力学(以及由此产生的变化亚基间氢键)有助于其功能，使我们假设内在结构灵活性是变构机制的主要驱动力，变构机制增强了MIF的时空控制。这个设计对炎症性疾病有治疗价值的MIF选择性抑制剂将得到更多的帮助详细了解MIF变构的生物物理基础。这份提案将探讨如何改变通过对MIF结构的突变和促炎溶液条件影响其变构串扰，催化 CD74的活性和活化。我们将完成三个具体目标，从原子水平开始用最新溶液核磁共振表征MIF变构网络光谱学和分子模拟。氧化溶液条件对MIF结构和性能的影响然后用溶液核磁共振和定量蛋白质组学对变构网络进行评估。我们将模仿炎性环境以确定如何修改MIF结构，以及这些修改是否会导致下游功能差异。最后，我们将应用我们集成的核磁共振-分子动力学方法来研究第一个MIF 曾与人类疾病有关的突变，一种在幼年关节炎儿童中发现的Y99C突变。这种突变直接发生在我们在早期出版物中确定的变构位置。每个目标都将评估由此产生的活性部位化学性质、催化功能(体外)和CD74测定的生物学结果激活(体内)功能。该项目将通过差异分析来剖析变构途径。通过核磁共振自旋松弛、分子模拟和网络分析来探测运动，绘制特定的氨基酸和相互作用负责传递变构之间结构或动态变化，酶和CD74受体位点。这项工作的结果可以广泛地为混杂机制提供信息细胞因子，变构在扩展的MIF超家族中的作用，以及焦点核磁共振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治疗。