Abstract
Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H2O2). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H2O2 exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H2O2–induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.
Highlights
While the residues important for human phosphatase 1 (PHPT1) (hPHPT1) activity and substrate binding have been characterized, there have been no studies concerning the structural and functional outcomes of post-translational modification (PTM) or chemical modification to this protein
H2O2 was determined by LC- tandem mass spectrometry (MS/MS)
Recombinant hPHPT1 was incubated for two hours in a heat bath at 37 °C with 0 μM, 100 μM, 500 μM, and 1 mM H2O2
Summary
While the residues important for hPHPT1 activity and substrate binding have been characterized, there have been no studies concerning the structural and functional outcomes of post-translational modification (PTM) or chemical modification to this protein. We investigate the impact of oxidation, through the known reactive oxygen species (ROS), hydrogen peroxide (H2O2), on hPHPT1 structure and related activity using mass spectrometry and computational methods. Computational studies using molecular dynamics simulations were carried out to investigate structural outcomes from selective methionine oxidation of hPHPT1. The results from this combined experimental and computational study provide new insight into the significance of hPHPT1 as a target of ROS and highlight the importance of elucidating the correlation of structural to functional diversity brought about by protein modifications
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