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

Borja Mateos,Wiktor Koźmiński,Gerald Platzer,Szymon Żerko,Robert Konrat,Clara Conrad-Billroth,Dorothea Anrather,Marco Sealey-Cardona,Julian Holzinger

doi:10.1021/acs.biochem.1c00050

Borja Mateos, Wiktor Koźmiński + Show 7 more

Open Access

https://doi.org/10.1021/acs.biochem.1c00050

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Journal: Biochemistry	Publication Date: Apr 20, 2021
Citations: 15	License type: CC BY 4.0

Affiliation: Vienna Biocenter, University of Warsaw, Max Perutz Labs

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.

Highlights

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells
Nuclear magnetic resonance (NMR) spectroscopy has matured into an exquisite tool to tackle PTMs and to study the structural dynamics of the protein of interest under nativelike conditions.[37−44] denaturing conditions have been useful for characterizing modified sites,[43,44] it is important to take into account the fact that the conformational ensembles of Intrinsically disordered proteins (IDPs) are drastically affected by the presence of denaturing agents, as IDPs are far from being merely unfolded.[45]
Almost complete assignment of phosphorylated S-x-E/pS motifs has been achieved by a combination of mass spectrometry (MS) and NMR spectroscopy

Summary

Introduction

Protein phosphorylation is an abundant post-translational modification (PTM) and an essential modulator of protein functionality in living cells. With respect to OPN, several important features that account for the modulation of compaction, binding and function of the unphosphorylated form have been identified.[46−48] Here, we present an NMR-based strategy for structurally characterizing the fully phosphorylated protein and the dynamics of the hyperphosphorylated OPN. NMR observables such as chemical shifts or 15N relaxation rates are very informative for IDP structural dynamics.[51,52] Possible changes in the structural dynamics of the protein were studied by a series of 15N relaxation experiments (Figure 3).

Results

Conclusion