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

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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.