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

> **NIH NIH R01** · BROWN UNIVERSITY · 2022 · $338,197

## Abstract

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 molecula...

## Key facts

- **NIH application ID:** 10521825
- **Project number:** 1R01GM144451-01A1
- **Recipient organization:** BROWN UNIVERSITY
- **Principal Investigator:** GEORGE LISI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $338,197
- **Award type:** 1
- **Project period:** 2022-09-23 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10521825

## Citation

> US National Institutes of Health, RePORTER application 10521825, Unraveling the Allosteric Mechanism of Macrophage Migration Inhibitory Factor with Molecular Resolution (1R01GM144451-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10521825. Licensed CC0.

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