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

项目摘要 巨噬细胞移动抑制因子（MIF）通过其在炎症病理生理学中的作用， 与趋化因子受体CD 74相互作用，同时也对抗免疫抑制作用， 糖皮质激素和催化未知生物学意义的酶促反应。的机制 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 鉴定了将MIF的酶活性位点与蛋白质的外围区域连接的氨基酸网络， 包括提出的CD 74结合位点。这些残基和其他可能的残基，变构调节几种 MIF的生物化学功能包括酶催化、受体活化和蛋白质-蛋白质相互作用。 初步数据显示，MIF结构的多时间尺度动态（以及由此产生的 亚基间氢键）有助于其功能，导致我们假设其内在结构 柔性是增强MIF的时空控制的变构机制的主要驱动力。的 设计对炎症性疾病有治疗价值的MIF选择性抑制剂， 详细了解MIF变构的生物物理基础。本提案将探讨如何改变 通过突变和促炎性溶液条件对MIF结构产生影响，影响其变构串扰，催化 活性和CD 74的活化。我们将完成三个具体目标，从原子水平开始 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。氧化性溶液条件对MIF结构的影响， 然后将用溶液NMR和定量蛋白质组学评估变构网络。我们将模仿 炎症环境，以确定MIF结构如何被修饰，以及这些修饰是否导致 下游功能差异。最后，我们将应用我们的综合NMR-MD方法来研究第一个MIF Y 99 C是一种与人类疾病相关的突变体，在青少年关节炎儿童中发现的Y 99 C变异体。这种突变 直接发生在我们在早期出版物中鉴定的变构位点。每个目标将评估结果 生物学结局，测量活性部位化学性质、催化功能（体外）和CD 74 激活（体内）功能。该项目将通过分析不同的 通过NMR自旋弛豫、分子模拟和网络分析探测的运动，绘制了特定的 氨基酸和负责传递变构之间的结构或动力学变化的相互作用， 酶和CD 74受体位点。这项工作的结果可以广泛地告知混杂机制 细胞因子，变构在扩展的MIF超家族中的作用，以及聚焦NMR引导的计算筛选 针对与哮喘、呼吸窘迫和癌症相关的MIF变构途径的分子库 治疗

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.