Structures of the Phosphorylated and VO3-Bound 2H-Phosphatase Domain of Sts-2

The 2H-phosphatase superfamily of enzymes, also referred to as the phosphoglycerate mutase (PGM) superfamily, is so called because the majority of its members are phosphatases that use two catalytic histidine residues to dephosphorylate substrates (1, 2). The first His residue belongs to the ‘RHGE’ signature motif that is conserved among family members and is the nucleophilic residue during catalysis. The second His residue together with two conserved Arg residues are scattered in the primary sequence, complete the active site, and give it a basic potential character that attracts and stabilizes phosphorylated substrates. Outside the His and Arg residues, the primary sequence of these enzymes shows little conservation. Despite the low sequence homology, the overall tertiary fold of these enzymes is overall maintained; more importantly the structure of the active site is highly conserved. Family members include the diverse acid phosphatases (AcPs), the cofactor dependent phosphoglycerate mutase (dPGM), the fructose 2,6-bisphosphatase (F2,6BP), the TIGAR protein (3), the Sts proteins (4), and many others. The diversity among family members is reflected in substrate diversity and specificity: substrates range from small-phosphorylated molecules to phospho-proteins, and some enzymes are very specific (F2,6BP) while others are promiscuous (AcPs). The dephosphorylation reaction is believed to be a two-step reaction (Scheme) (5). In the first step, the nucleophilic His of the ‘RHGE’ signature motif attacks the phosphorus atom of the substrate resulting in the transient transfer of the phosphate to the His and the release of the dephosphorylated substrate. In the second step, the phospho-His is hydrolyzed by an activated water molecule and the enzyme returns to its resting state. The second His and the Arg residues are believed to stabilize the negative charges that appear on the structure of the transition state species. The nucleophilic attacking water molecule is activated by general base catalysis usually by the side chain carboxylate of an Asp or Glu residue (6, 7). As in many phospho-transferases, determining the crystal structures of family members in the presence of phosphate and phosphate-like species including vanadate and tungstate has been very useful in understanding the reaction mechanism and highlighting the catalytic amino acids and water molecules. However, except for the structures of the phospho-histidine activated form of the Escherichia coli dPGM (8) and human bisphosphoglycerate mutase (BPGM) (9), little is known about the phospho-histidine transient intermediate state of the 2H-phosphatases. More generally, there are few high-resolution structures of phosphohistidine-containing proteins, those of the Drosophila and Dictyostelium nucleoside diphosphate (NDP) kinase (10), the pig heart GTP-specific succinyl-CoA synthetase (11), and the Bacillus subtilis histidine-containing protein (HPr) (12) being the exception. Scheme Schematic representation of the phosphatase reaction catalyzed by 2H-phosphatase family members. Only drawn are the conserved active site residues. The phosphorylated substrate binds to the active site such that the phosphorus atom is within an attacking ... To shed additional light on the dephosphorylation reaction carried out by the 2H-phosphatases, we focused on determining the structures of the transition states for hydrolysis of the Sts proteins. Sts-1 and -2 proteins are multidomain proteins that contain a C-terminal 2H-phosphatase homology domain (13-16). Functionally, they play an important role in downregulating the activity of the TCR. Despite the sequence homology between the 2H-phosphatase domains of the two isoforms, the phosphatase activity of Sts-2PGM is by far the weaker one despite conservation of all catalytic residues. We have recently determined the crystal structure of Sts-1PGM and Sts-2PGM alone or in complex with phosphate and tungstate (4, 17). Here we present the crystal structures of Sts-2PGM in the active phosphorylated state and bound to vanadium oxide (VO3).