Structural and Biochemical Characterization of the αI-domain, the Ligand Binding Domain, of Integrins αDβ2 and αXβ2



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β2-integrins are among the most complex cell surface metallo-receptors known, and upon ligand binding, its ligand-binding domain (αI-domain) undergoes allosteric conformational changes. These “shape-shifting” events of the αI-domain are key to the mechanism of integrin action allowing metal-dependent bidirectional regulation of cellular signaling across the cell membrane in events such as migratory behavior and inflammatory responses of myeloid cells. αDβ2 was specifically detected and showed a distinctive contributing role in the inflammatory response in human atherosclerotic lesions and white adipose tissue during metabolic syndrome. αDβ2 appears to be a unique regulatory receptor in macrophage retention and egress in the inflamed tissue and thus, is emerging as a potential drug target. However, perhaps since it was very recently discovered, the recognition specificity and the molecular basis of αDβ2 ligand-binding are essentially unknown. Herein, we determined the structure of the αD I-domain without divalent cation and in the presence of the chloride salts of Mg2+. While overall the αD I-domain structures are highly similar to other αI-domains, several residues in the vicinity of its MIDAS differ from other αI-domain, which, together with change in surface change, revealing why some αI-domains recognize an overlapping set of ligands with different affinity as well as a set of non-overlapping ligands. The thermal stability as well as the αD I-domain affinity are altered by metal ions. Metal ions endow unique ligand-affinity and thermodynamic stability to the αD I-domain structure. We also discovered that αDβ2 binds to iC3b molecule, a complement factor in the immune system, in a metal-dependent manner. The αX I-domain structure has been extensively studied by X-ray crystallography. However, these crystal structures only provide the average positions and arrangement of individual atoms of this protein in either metal-ion free state or open state NMR experiments are able to probe molecular motions at the nanosecond timescale and are ideal for studying the transition between the open and closed states in integrins. Here, we report the triple resonance NMR backbone assignment of the αX I-domain integrin, as a preliminary experiment for future structural and dynamic studies.



Crystallography, Biochemistry, NMR, Integrins, Structure, Ligands, Protein purification