Investigating Interactions between the Lectin-Like Domain of Thrombomodulin and Complement Component 3

Protein-protein interactions are vital to the proper functioning of numerous biological systems. Thrombomodulin (TM) is a protein that is involved in the down-regulation of coagulation induced by the clotting protein thrombin. Complement component 3 (C3) is a vital component of the complement system, which is involved in innate immunity against bacteria and viruses. However, dysregulation of C3 can lead to the degradation of host cells. Evidence suggests that the lectin-like domain of TM (TMD1) may interact with active C3 (C3b) to inactivate it, thus preventing host cell degradation. The research conducted herein required the expression and purification of TMD1 in yeast cells and the isolation and purification of C3 from bovine blood plasma. A protein pull-down assay was used to verify that the two proteins interact. To aid in specific immobilization of the TMD1, two lysine residues were converted to methionine by site-directed mutagenesis via polymerase chain reaction leaving only one free amine available for future reactions. The wild type and mutant TMD1 proteins were characterized by mass spectrometry and urea-induced unfolding. The proteins were also both used in pull-down assays with C3. Once interactions are confirmed, hydrogen/deuterium exchange followed by matrix assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) will be performed to determine which regions of each protein are involved in binding. Additionally, interactions between the two proteins will be characterized using fluorescence resonance energy transfer (FRET) experiments.