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MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic
Chengpeng Fan1, Deepa Rajasekaran, Mansoor Ali Syed
1Department of Pharmacology, Yale University, New Haven, CT 06510, USA.
Abstract:
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. In addition to its known receptor-mediated biological activities, MIF possesses a catalytic site of unknown function between subunits of a homotrimer. Each subunit contributes three β-strands to adjacent subunits to form a core seven-stranded β-sheet for each monomer. MIF monomers, dimers, or trimers have been reported, but the active form that binds and activates the MIF receptor (CD74) is still a matter of debate. A cysteine mutant (N110C) that covalently locks MIF into a trimer by forming a disulfide with Cys-80 of an adjacent subunit is used to study this issue. Partial catalytic activity and receptor binding to CD74 are retained by N110C (locked trimer), but there is no cellular signaling. Wild-type MIF-induced cellular signaling, in vivo lung neutrophil accumulation, and alveolar permeability are inhibited with a fivefold excess of N110C. NMR and size-exclusion chromatography with light scattering reveal that N110C can form a higher-order oligomer in equilibrium with a single locked trimer. The X-ray structure confirms a local conformational change that disrupts the subunit interface and results in global changes responsible for the oligomeric form. The structure also confirms these changes are consistent for the partial catalytic and receptor binding activities. The absence of any potential monomer and the retention of partial catalytic and receptor binding activities despite changes in conformation (and dynamics) in the mutant support an endogenous MIF trimer that binds and activates CD74 at nanomolar concentrations. This conclusion has implications for therapeutic development.
Insights
The active form of macrophage migration inhibitory factor (MIF) is likely a trimer, as a locked trimer mutant retained receptor binding and partial catalytic activity, suggesting therapeutic potential.
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with a catalytic site of unknown function.
- The oligomeric state of MIF, whether monomer, dimer, or trimer, and its role in receptor binding and activation are debated.
- The MIF receptor is CD74.
Purpose of the Study:
- To determine the active oligomeric form of MIF responsible for binding and activating its receptor, CD74.
- To investigate the functional consequences of locking MIF into a trimeric state using a cysteine mutant.
Main Methods:
- Utilized a cysteine mutant (N110C) to covalently lock MIF into a trimer.
- Employed NMR and size-exclusion chromatography with light scattering to analyze oligomeric states.
- Determined the X-ray structure of the mutant MIF to understand conformational changes.
Main Results:
- The locked trimer mutant (N110C) retained partial catalytic activity and CD74 binding but lacked cellular signaling.
- N110C inhibited wild-type MIF-induced signaling, neutrophil accumulation, and alveolar permeability.
- Structural analysis revealed conformational changes in N110C leading to higher-order oligomers in equilibrium with the locked trimer.
Conclusions:
- The findings support the hypothesis that an endogenous MIF trimer is the active form that binds and activates CD74.
- The locked trimer mutant's properties suggest implications for developing MIF-targeted therapeutics.
- Absence of monomer and retained partial activities in the mutant reinforce the trimer as the functional unit.
