Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition

Abstract

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. Intrinsically disordered proteins (IDPs) are particular targets of PTM protein kinases due to their involvement in fundamental protein interaction networks. Despite their dynamic nature, IDPs are far from having random-coil conformations but exhibit significant structural heterogeneity. Changes in the molecular environment, most prominently in the form of PTM via phosphorylation, can modulate these structural features. Therefore, how phosphorylation events can alter conformational ensembles of IDPs and their interactions with binding partners is of great interest. Here we study the effects of hyperphosphorylation on the IDP osteopontin (OPN), an extracellular target of the Fam20C kinase. We report a full characterization of the phosphorylation sites of OPN using a combined nuclear magnetic resonance/mass spectrometry approach and provide evidence for an increase in the local flexibility of highly phosphorylated regions and the ensuing overall structural elongation. Our study emphasizes the simultaneous importance of electrostatic and hydrophobic interactions in the formation of compact substates in IDPs and their relevance for molecular recognition events.

Binding Mode Characterization of Osteopontin on Hydroxyapatite by Solution NMR Spectroscopy

“…In order to further investigate these long‐range interactions of the two protein forms upon binding to HAP surfaces, PRE NMR experiments of the cysteine mutant T185 C of both forms were performed in the apo and the HAP‐bound state (Figures 4 , S6 and S7). As we have reported recently, [39] the apo form of the OPNp shows a remarkable reduction of long‐range interactions within the main compact state and towards the N‐terminal region in comparison to the unphosphorylated OPN (Figure 4 , top), suggesting that the phosphorylated form has a significantly elongated structure. In the presence of HAP nanoparticles, OPN shows a clearly enhanced PRE effect within the central core region, however not towards the N‐terminal region (Figure 4 , middle).…”

“…[ 32 , 33 , 36 , 37 ] Besides that, hyperphosphorylation also has an impact on structural dynamics and molecular recognition of OPN (e. g. the binding to extracellular matrix components and integrin receptors). [ 38 , 39 ] Fam20 C (family with sequence similarity 20, member C) kinase has been identified to phosphorylate serine and threonine residues of secreted proteins in the Golgi apparatus, among others OPN. [ 40 , 41 ] Mutations of this kinase affect its activity and cause a bone dysplasia characterized by ectopic calcifications and osteosclerosis, called Raine syndrome, which in many cases is fatal in the neonatal period.…”

“…[ 42 , 43 ] Fam20 C recognizes and phosphorylates S‐x‐E/pS motifs, whereby phosphorylation of other motifs has been reported as well. [ 38 , 39 , 44 ] OPN contains 22 of these motifs, and thus, in case of a full phosphorylation, it is hyperphosphorylated. Recently we identified the phosphorylation of 28 sites in OPN by Fam20 C via a combined approach of NMR spectroscopy and mass spectrometry.…”

“…Recently we identified the phosphorylation of 28 sites in OPN by Fam20 C via a combined approach of NMR spectroscopy and mass spectrometry. [39] …”

Nanopore Stochastic Sensing Based on Non-covalent Interactions

“…The molecular recognition process, one of the most fundamental processes in chemistry and biology, is very complicated and influenced by many factors. , These factors, also called non-covalent interactions, include electrostatic interaction, aromatic–aromatic interaction, , hydrophobic interaction, and hydrogen bond . Accordingly, in terms of ligand–receptor interactions, the design strategies generally involve the manipulation of hydrophobic, polar, and charged residues .…”