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

•Integrin αXβ2 binds polyanions, but the preferred anions remain uncertain.•Osteopontin contains many phosphorylations and aspartate and glutamate residues.•We show that phosphorylation does not contribute to αXβ2 binding.•In αX I domain binding to osteopontin, glutamate is preferred over aspartate.

Integrin αXβ2 (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. αXβ2 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 αXβ2. Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin αXβ2. Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by αXβ2 remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of αXβ2 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 αXβ2 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 αXβ2 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 αXβ2, which seem to differ in principles considerably from other OPN receptors.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (167 K)Download as PowerPoint slide