Determining Ligand Binding and Specificity Within the β 2 -Integrin Family with a Novel Assay Platform

The family of the β 2 -integrin receptors is critically involved in host defense and homeostasis, by mediating immune cell adhesion, migration, and phagocytosis. Due to their key roles in immune surveillance and inflammation, their modulation has been recognized as an attractive drug target. However, the development of therapeutics has been limited, partly due to the high promiscuity of endogenous ligands, their functional responses, and gaps in our understanding of their disease-related molecular mechanisms. The delineation of the molecular role of β 2 integrins and their ligands has been hampered by a shortage of validated assay systems. To facilitate molecular and functional studies on the β 2 -integrin family, and to enable screening of modulators, this study provides a uniform and validated assay platform. For this purpose, the major ligand-binding domains (αI) of all four β 2 integrins were recombinantly expressed in both low- and high-affinity states. By optimizing the expression parameters and selecting appropriate purification tags, all αI-domain variants could be produced with high yield and purity. Direct binding studies using surface plasmon resonance (SPR) confirmed the expected activity and selectivity profiles of the recombinant αI domains towards their reported ligands, validating our approach. In addition, the SPR studies provided additional insights into ligand binding, especially for the scarcely described family member CD11d. Alongside characterizing endogenous ligands, the platform can be employed to test pharmacologically active compounds, such as the reported β 2 -integrin antagonist simvastatin. In addition, we established a bead-based adhesion assay using the recombinant αI domains, and a cell-based adhesion assay underlining most findings generated with the isolated αI domains. Interestingly, the binding of ligands to the recombinant α D I is not dependent on divalent cation, in contrast to the full integrin CD11d/CD18, suggesting a binding mode distinct of the metal ion-dependent adhesion site (MIDAS). The setup highlights the applicability of recombinant αI domains for first screenings and direct or competitive interaction studies, while the full integrin is needed to validate those findings.