1H, 15N, 13C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride

Zhalgas Serimbetov,Jonathan P Waltho,Nicola J Baxter,Matthew J Cliff

doi:10.1007/s12104-017-9758-3

Zhalgas Serimbetov, Jonathan P Waltho + Show 2 more

Open Access

https://doi.org/10.1007/s12104-017-9758-3

Copy DOI

Abstract

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3−) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3−), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1H–15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.

Highlights

Using metal-fluoride approaches, a fully closed human phosphoglycerate kinase (PGK):3PG:MgF3:ADP transition state analogue (TSA) complex was solved to a resolution of 1.47 Å (PDB: 2WZB; Cliff et al 2010), in which clear electron density in the active site places the donor and acceptor oxygen atoms of 3PG and ADP as apical ligands for a central trigonal bipyramidal M gF3− group occupying the position of the transferring P O3− group during the phosphoryl transfer reaction
In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the fully closed PGK:3PG:MgF3:ADP TSA complex, which represents the solution conformation of the X-ray crystal structure reported previously (PDB: 2WZB)
NMR samples containing 0.5 mM 2H,15N,13C-labelled human PGK, 10 mM 3PG, 10 mM ADP, 10 mM MgCl2, 30 mM NH4F in 50 mM Tris–HCl buffer pH 7.4, 20 mM DTT, 1 mM EDTA and 0.04% v/v N aN3 were loaded into 5-mm diameter NMR tubes. 2H2O (10% v/v) and trimethylsilyl propanoic acid (TSP; 0.5% v/v) were added to the NMR samples for the deuterium lock and as a chemical shift reference, respectively. 1H chemical shifts were referenced to the internal TSP signal, whereas 15N and 13C chemical shifts were referenced indirectly using nuclei-specific gyromagnetic ratios

Summary

Biological context

Phosphoglycerate kinase (PGK, EC 2.7.2.3) is a bisubstrate magnesium-dependent enzyme with highly conserved amino acid sequences found in all living organisms In glycolysis, it catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP, while in gluconeogenesis and in the Calvin cycle of photosynthetic species, PGK catalyses the reverse reaction. Using metal-fluoride approaches, a fully closed human PGK:3PG:MgF3:ADP TSA complex was solved to a resolution of 1.47 Å (PDB: 2WZB; Cliff et al 2010), in which clear electron density in the active site places the donor and acceptor oxygen atoms of 3PG and ADP (donor–acceptor distance = 4.3 Å) as apical ligands for a central trigonal bipyramidal M gF3− group occupying the position of the transferring P O3− group during the phosphoryl transfer reaction. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the fully closed PGK:3PG:MgF3:ADP TSA complex, which represents the solution conformation of the X-ray crystal structure reported previously (PDB: 2WZB)

Protein expression and purification

NMR spectroscopy

Findings

Resonance assignments and data deposition