Multiple low-affinity interactions support binding of human osteopontin to integrin α_xβ₂

Integrin α_Xβ₂ (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α_Xβ₂ 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_Xβ₂. Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α_Xβ₂. Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α_Xβ₂ remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α_Xβ₂ 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_Xβ₂ 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_Xβ₂ 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_Xβ₂, which seem to differ in principles considerably from other OPN receptors.