Structural Characterization of Leukocyte Integrin αM I-Domain



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The aim of this study is to characterize the stability of the ligand-binding site (also called the αI-domain) on the cellular adhesion molecule, integrin αM (ITGAM). This integrin, also known as cluster of differentiation molecule 11b (CD11b), and complement receptor 3 (CR3), is heavily involved in the extravasation of leukocytes and phagocytosis of opsonized molecules, making it an interesting target for pharmaceutical therapies. Within this αI-domain lies a Mg2+ binding site called the Metal Ion Dependent Adhesion Site (MIDAS), which is allosterically linked to the domain’s C-terminal helix. Recent findings on its closest sister analog have shown that activation of this receptor causes the C-terminal helix to reshape and lose much of its helicility. To further investigate the structure and movements of this region, we expressed the αM I-domain with a hexameric His-tag using BL21 Rosetta cells. Subsequently the protein was loaded on a nickel-NTA column, and eluted with imidazole. SDS-PAGE was used to visualize the results of the purification. Differential scanning fluorimetry and NMR experiments will be utilized to characterize how Mg2+ binding alters its structure and stability. The results of our work will allow high throughput screening that could be employed to discover possible allosteric inhibitors for macrophage integrins. Therefore, this protein is a highly suitable target for use in novel immunotherapies.