Structural Study of the Dynamics of the Phenylisothiocyanate Binding to MIF

Intermediate states of the complex of the macrophage migration inhibitory factor (MIF) with the covalent inhibitor phenylisothiocyanate (PITC) were studied by X-ray diffraction analysis. It was demonstrated that the covalent modification of the N-terminal proline is preceded by the non-covalent binding of the inhibitor in the previously unknown holding site. The holding site was identified due to the use of short-term soaking of a MIF crystal in a ligand-containing cryo-solution followed by flash freezing in a nitrogen stream to collect the X-ray diffraction data at 100 К. A comparison of this structure with the crystal structure of the pre-modified protein revealed the details of the dynamics of the PITC binding.