The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites.

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)ß(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state.

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.

Backbone assignment of osteopontin, a cytokine and cell attachment protein implicated in tumorigenesis.

OPN is an RGD-containing protein overexpressed in cells transformed by v-myc and v-mil(raf) oncogenes. Here we report the resonance assignment of recombinant quail OPN and provide NMR evidence that quail OPN is an intrinsically unstructured protein in solution.

Monitoring the effects of antagonists on protein-protein interactions with NMR spectroscopy.

We describe an NMR method that directly monitors the influence of ligands on protein-protein interactions. For a two-protein interaction complex, the size of one component should be small enough (less than ca. 15 kDa) to provide a good quality (15)N((13)C) HSQC spectrum after (15)N((13)C) labeling. The size of the second unlabeled component should be large enough so that the molecular weight of the preformed complex is larger than ca. 40 kDa. When the smaller protein binds to a larger one, broadening of NMR resonances results in the disappearance of most of its cross-peaks in the HSQC spectrum. Addition of an antagonist that can dissociate the complex would restore the HSQC spectrum of the smaller component. The method directly shows whether an antagonist releases proteins in their wild-type folded states or whether it induces their denaturation, partial unfolding, or precipitation. We illustrate the method by studying lead compounds that have recently been reported to block the MDM2-p53 interaction. Activation of p53 in tumor cells by inhibiting its interaction with MDM2 offers new strategy for cancer therapy.