The subunit structure of human macrophage migration inhibitory factor: evidence for a trimer

Abstract

The subunit structure of human macrophage migration inhibitory factor (MIF) has been studied by preliminary X-ray analysis of wild-type and selenomethionine-MIF and dynamic light scattering. Crystal form I of MIF belongs to space group P2(1)2(1)2(1) and is grown from 2 M ammonium sulfate at pH 8.5. A native data set has been collected to 2.4 A resolution. Self-rotation studies and Van values indicate that three molecules per asymmetric unit are present. A data set to 2.8 A resolution has been collected for crystal form II, which belongs to space group P3(1)21 or P3(2)21 and grows from 2 M ammonium sulfate, 2% polyethylene glycol (average molecular mass 400) 0.1 M HEPES, pH 7.5. Three, four, five or six monomers in the asymmetric unit are consistent with Van values for this crystal form. Analysis of crystal form II containing selenomethionine-MIF indicates nine selenium sites are present per asymmetric unit. Dynamic light scattering of MIF suggests that the major form of the protein in solution is a trimer. The results of these studies are in contrast to previous reports indicating that MIF is a monomer or dimer. The subunit arrangement of MIF is similar to that of tumor necrosis factor and suggests that signal transduction might require trimerization of receptor subunits.

Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

“…The human MIF was prepared as previously described (17). For NMR experiments, minimal media that contained 15 NH 4 Cl (Cambridge Isotope Laboratories, Inc.) was used as the nitrogen source to express 15 N-rhMIF.…”

“…Once released, MIF activates the receptor CD74 in a complex with CD44 (13), or the chemokine receptors CXCR2 (14) and CXCR4 (15), initiating a cascade of intracellular signaling (16). One aspect of MIF that remains enigmatic is the presence of a catalytic site, highly conserved among all MIFs and present between subunits of the trimer (16)(17)(18)(19). No physiologic substrate for MIF has been identified, but "pseudosubstrates" have been found that allow for screening inhibitors at this site (20,21).…”

A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)

“…Nevertheless, the uniqueness of an enzymatic activity associated with a cytokine led to studies elucidating the structure-activity relationship between the two activities. These studies revealed that in vitro, MIF exists as a 37.5-kDa homotrimer (26,27). The three monomers that form the trimer exist as a ␤-␣-␤ structure composed of two anti-parallel ␣-helices packed against a fourstranded ␤-sheet.…”

Amyloid fibril formation by macrophage migration inhibitory factor

“…Each monomer consists of 114 amino acid residues and has a molecular weight of 12,343 Da. As revealed by X-ray crystallography [19,20], the tertiary structure of MIF defines a novel protein fold, which is characterized by the packing of an extended 4-stranded b-sheet and two antiparallel a-helices (Fig. 1A).…”

“…1B). Although MIF crystallizes as a trimer [19], experimental studies employing NMR spectroscopy [21], size-exclusion chromatography [22], chemical cross-linking [23,24], and analytical ultracentrifugation support the existence of dimeric and monomeric forms in solution [24].…”