Dataset Information

Phosphorylation of cytosolic hPGK1 affects protein stability and ligand binding: implications for its subcellular targeting in cancer

ABSTRACT: Human phosphoglycerate kinase 1 is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus, respectively. These changes in subcellular location drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX). We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX studies structurally confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our work thus support that alterations in protein stability might facilitate import of hPGK1 to the nucleus in cancer, whereas the structural and energetic basis of its mitochondrial import remain unknown. The general implications of our work for protein import to certain subcellular locations are discussed in a wider context.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pavla Vankova

LAB HEAD: Petr Man

PROVIDER: PXD049012 | Pride | 2024-09-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HDX.zip	Other
	PGK-S203D_iN2-200-0C_MSMS_7558.d.zip	Other
	PGK-S203D_iN2-200-0C_MSMS_7558_SumPeak.csv	Csv
	PGK-S203D_iN2-200-0C_MSMS_7558_SumPeak.mgf	Mgf
	PGK-S256D_iN2-200-0C_MSMS_7558.d.zip	Other

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Phosphorylation of cytosolic hPGK1 affects protein stability and ligand binding: implications for its subcellular targeting in cancer

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