Mechanistic insights into DDT-CD74 interactions via crystallization of DDT variants

Human D-dopachrome tautomerase (DDT), also known as Macrophage Migration Inhibitory Factor 2 (MIF-2), is a novel immunomodulatory protein with key implications in cancer development, proliferation, and metastasis. Although it is known that DDT is involved in important processes in immunoregulation, its actual pathogenic role is poorly understood. The upregulation of DDT expression has been found in several solid and hematological (blood) cancers. By understanding the structure of DDT and the binding mechanism of its sole selective and reversible inhibitor, 4-CPPC, we will further understand how it activates its natural cell surface receptor, CD74, causing its undesirable pathological effects. Understanding the structure and function of DDT will also open the door to creating optimal inhibitors with drug-like properties that can be used for therapeutic purposes. Recent findings demonstrate that the C-terminus of DDT possesses unique properties that were previously unseen for the MIF superfamily and may be critical for binding and activation of CD74. To explore this further, three C-termini truncated versions of DDT will be produced, expressed and crystallized in the presence or absence of 4-CPPC. In these experiments, the wild type DDT will be used as a control. Crystallization trials of the DDT variants will be carried out at UOP using hundreds of previously established crystallization conditions. The crystals will be flash-frozen and tested in a synchrotron facility (the Advanced Light Source, Berkeley, California) for diffraction. Complete data sets will be analyzed and the crystal structures of the DDT variants will be determined at UOP, using the known crystal structure of wild type DDT as the model. We anticipate that our findings will be of great interest to the scientific community and provide key information on the DDT induced activation of CD74