Structural and biochemical studies of TIGAR (TP53-induced glycolysis and apoptosis regulator)

Abstract

Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebra fish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate but not fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.

FIGURE 1.

Overall structure of TIGAR. A, ribbon diagram of the full-length structure. Two bound phosphate molecules and residues in the active site are shown as sticks. B, structure of the tryptic core fragment in the same orientation. The positions of structural elements missing after trypsin digest are indicated with arrows. C, symmetric dimer formation between two monomers in the asymmetric unit of the full-length structure. D, close-up view illustrating the hydrogen-bonding network between the two molecules and the coordination of the two metal ions. E, dimer formation in the H11A mutant structure. Molecule A (colored in blue and cyan) is shown in the same orientation as in C. F, close-up view of the interface region illustrating the substantially different orientation of molecule B (orange) compared with the full-length structure in D.

FIGURE 2.

Phosphate binding in the wild type and H11A mutant active site. A, ribbon diagram for the active site of wild type TIGAR. Residues engaged in hydrogen bonds with the two phosphate molecules are shown as sticks. Solvent water molecules are shown as red spheres. B, final σ_A-weighted 2F_o-F_c electron density for the active site region in the full-length wild type structure contoured at the 1 σ level. C, comparison of phosphate position in wild type (yellow) and H11A mutant (orange). Solvent water molecules are shown for the mutant structure. D, final σ_A-weighted 2F_o-F_c electron density for the H11A mutant structure.

FIGURE 3.

Structural comparison with the PhoE phosphatase and with fructose-2,6-bisphosphatase. A, least squares superposition of TIGAR with PhoE (Protein Data Bank code 1H2E). TIGAR is colored yellow, and PhoE is colored cyan. The positions of significant structural differences are indicated with arrows. B, comparison of phosphate binding in TIGAR and PhoE. C, least squares superposition of TIGAR (yellow) with FBPase-2 (green; Protein Data Bank code 2BIF). D, comparison of phosphate binding in TIGAR and FBPase-2.