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

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

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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.

项目摘要巨噬细胞迁移抑制因子（MIF）对于炎症的病理生理至关重要与趋化因子受体CD74的相互作用，同时还与糖皮质激素和未知生物学意义的催化酶促反应。该机制 MIF在其紧凑的结构中适应这些和其他生化功能尚不清楚，但是我们最近确定了将MIF的酶促活性位点与蛋白质的外围区域联系起来的氨基酸网络包括拟议的CD74结合位点。这些残留物以及其他可能的残留物会对几个 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。初步数据表明，MIF结构的多时间计算动力学（以及结果变化为亚基间的氢键有助于其功能，导致我们假设固有结构灵活性是变构机制的主要驱动力，可增强MIF的空间控制。这具有炎症性疾病治疗价值的MIF选择性抑制剂的设计将得到更多的帮助详细了解MIF变构的生物物理基础。该建议将探讨如何改变通过突变和促炎溶液条件到MIF结构影响其变构串扰，催化 CD74的活性和激活。我们将完成三个特定目标，从原子水平开始使用最先进的溶液核磁共振（NMR）对MIF变构网络的表征光谱和分子模拟。氧化溶液条件对MIF结构和然后，将使用溶液NMR和定量蛋白质组学评估变构网络。我们会模仿炎症环境以确定MIF结构如何修饰，以及这些修改是否导致下游功能差异。最后，我们将应用我们集成的NMR-MD方法来研究第一个MIF 与人类疾病有关的突变体是在少年关节炎儿童中发现的一种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网站。每个目标都会评估结果活性位点化学特性，催化功能（体外）和CD74的生物结果激活（体内）功能。该项目将通过分析差分分析变构途径通过NMR自旋松弛，分子模拟和网络分析探测的运动，绘制特定氨基酸和负责在变构之间传递结构或动态变化的相互作用酶促和CD74受体位点。作品的结果可以广泛地告知滥交机制细胞因子，变构在扩展的MIF超家族中的作用，并将NMR引导的计算筛选聚焦针对MIF变构途径的分子文库，与哮喘，呼吸窘迫和癌症有关疗法。