NMR Investigations of the IDP Osteopontin on Bone Material

The aim of this project was to determine a more detailed picture of the interaction of the human protein osteopontin upon binding to a surface of bone-like material. For this purpose kinetics, thermodynamics and possible mobile domains in the bound state were investigated utilizing NMR (nuclear magnetic resonance) spectroscopy techniques, in order to analyze bonding domains of osteopontin on hydroxyapatite nanoparticles. The extracellular matrix protein osteopontin is an intrinsically disordered protein, i.e. a protein which features an extended conformation without a strong driving force towards a tertiary structure. This feature gives intrinsically disordered proteins an enormous potential to interact with multiple binding partners simultaneously. Osteopontin is, among multiple physiological functions, associated with bone mineralization and the modulation of osteogenesis: interaction with osteoclasts; inhibitor of hydroxyapatite (principle component of bone: approx. 65 wt%) formation, growth and proliferation; assistant of intrafibrillar collagen mineralization and moderator of extrafibrillar mineral coating. Phosphorylated osteopontin in particular plays an important role (phosphorylation is an abundant post-translational modification of proteins which can be achieved with a human kinase). NMR studies on the phosphorylated osteopontin reveal both a gain in flexibility in regions, which comprise the phosphorylation sites, and reduced long-range interactions, and hence a decompaction of the structure. NMR experiments of osteopontin binding to hydroxyapatite nanoparticles can take up to several days. The addition of methyl cellulose to the measurement buffer has proven to be an easy and suitable option to keep the nanoparticles in a stable suspension and to avoid their sedimentation, and at the same time to maintain a solution-like environment for the protein. The NMR studies reveal different binding modes of osteopontin (as it is and phosphorylated) upon binding to hydroxyapatite. The unphosphorylated osteopontin binds weakly to the mineral surfaces, while its main compact state is not interacting with the hydroxyapatite surface and remains unaltered. By contrast, the phosphorylated osteopontin, which has a more elongated structure due to a reduction of long-range correlations by the hyperphosphorylation, binds to the mineral surfaces over the full length as phosphorylated residues are distributed broadly over the protein structure. Moreover, the data suggest that the structure of phosphorylated osteopontin remains elongated upon binding, covering the hydroxyapatite surface. For both the un- and phosphorylated osteopontin the binding to the hydroxyapatite surface is of an electronegative nature and no conformational changes can be observed upon binding. However, the different binding modes may explain the distinct biological functions of the unphosphorylated and phosphorylated osteopontin during osteogenesis and biomineralization: the rather "covering" binding mode of the phosphorylated protein may explain its function as a mineralization inhibitor through physically blocking the mineral surface from further growth and its property to stabilize transient pseudo-coacervate phases of calcium phosphate.