Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Abstract

Non-natural L-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, L-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing L-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to L and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of L-type prodrugs to assure their efficient metabolic processing.

Figure 1.

Superimposition of hPGK structures. Cα trace representations of hPGK·l-ADP (molecule B), orange; hPGK (PG-bound, nucleotide-free molecule A), blue; hPGK·l-CDP (molecule A), red; hPGK·d-ADP, green; hPGK·d-CDP (molecule B), grey. Structures were superimposed on their C-terminal lobe.

Figure 2.

Superimposition of nucleotides with their 2fofc omit maps. Final models are shown. Maps were produced by omitting the nucleotide from the model. Prior to map calculation, model bias was removed by subjecting all atoms of the remaining model to 0.15 Å random shifts, resetting their B factors to the value suggested by the Wilson plot, and refinement of the resulting model without nucleotide.

Figure 3.

Details of the nucleotide interactions. (A, B) Two 90° views of hPGK·l-ADP (molecule B, light grey), hPGK·d-ADP (green). Waters are presented as green and grey spheres. (C, D) Two 90° views of hPGK·l-CDP (light blue), and hPGK·d-CDP (magenta). Waters from are presented as blue and magenta spheres. hPGKs were superimposed on their C-terminal lobe. (C) Two 90°-views as compared to (A). Only the nucleotides are shown to illustrate their spatial dispersion in the binding site. The β-phosphates are indicated.

Figure 4.

Comparison of hPGK, dCK, TMPK, ADH and UMP/CMPK. Secondary structures are coloured according to magenta: β-strands, cyan: α-helices, salmon: loops. Ligands are shown in stick representation, with carbon atoms of ADP moieties coloured in blue, and of substrates or nicotinamide moiety in green. (A) dCK (PDB entry 2NO1), (B) TMPK (PDB entry 1E2D), (C) UMP/CMPK (PDB entry 2UKD), (D) NADH-bound liver alcohol dehydrogenase [PDB entry 2OHX (38)], (E) hPGK C-domain and (F) hPGK N-domain.