Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.

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.

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