Multiple low-affinity interactions support binding of human osteopontin to integrin αXβ2.

Integrin α(X)β(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)β(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)β(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)β(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)β(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)β(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)β(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)β(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)β(2), which seem to differ in principles considerably from other OPN receptors.