title: MIF intersubunit disulfide mutant antagonist supports activation of CD74 by endogenous MIF trimer at physiologic concentrations.
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.
authors.affiliation: Department of Pharmacology, Yale University, New Haven, CT 06510, USA.
authors.forename: Chengpeng
authors.initials: C
authors.lastname: Fan
authors.forename: Deepa
authors.initials: D
authors.lastname: Rajasekaran
authors.forename: Mansoor Ali
authors.initials: MA
authors.lastname: Syed
authors.forename: Lin
authors.initials: L
authors.lastname: Leng
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Loria
authors.forename: Vineet
authors.initials: V
authors.lastname: Bhandari
authors.forename: Richard
authors.initials: R
authors.lastname: Bucala
authors.forename: Elias J
authors.initials: EJ
authors.lastname: Lolis
mesh_terms: D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D018360:Crystallography, X-Ray; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011971:Receptors, Immunologic / Q000378:metabolism
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D008263:Macrophage Migration-Inhibitory Factors; D050505:Mutant Proteins; D021122:Protein Subunits; D011971:Receptors, Immunologic; C045084:invariant chain; C033184:macrophage migration inhibitory factor receptor; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; C076231:Mif protein, mouse
keywords: ERK-1/2 activation; mechanism; thermostable variant