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We solved the crystal structure of Streptococcus agalactiae serine/threonine phosphatase (SaSTP) using a combination of single-wavelength anomalous dispersion phasing and molecular replacement. The overall structure resembles that of previously characterized members of the PPM/PP2C STP family. The asymmetric unit contains four monomers and we observed two novel conformations for the flap domain among them. In one of these conformations, the enzyme binds three metal ions, whereas in the other it binds only two. The three-metal ion structure also has the active site arginine in a novel conformation. The switch between the two- and three-metal ion structures appears to be binding of another monomer to the active site of STP, which promotes binding of the third metal ion. This interaction may mimic the binding of a product complex, especially since the motif binding to the active site contains a serine residue aligning remarkably well with the phosphate found in the human STP structure.
Protein phosphatases are primarily classified on the basis of the type of the amino acid they dephosphorylate, serine/threonine phosphatases (STPs) act specifically on phosphoserine and phosphothreonine residues. Evolution has developed two main families of metalloenzymes for this purpose, phosphoprotein phosphatase P (PPP) and phosphoprotein phosphatase M (PPM) . Based on sequence similarity, Streptococcus agalactiae STP (SaSTP) studied here belongs to a PPM subfamily called PP2C, because members of this subfamily resemble human phosphoprotein phosphatase 2C .
Serine/threonine phosphorylation/dephosphorylation is intimately linked with signaling events inside the cell. Many STPs expand the scope of signaling by recruiting additional domains into their structures. This is most common in PPP family enzymes, where both regulatory and targeting domains occur; for example, in phosphoprotein phosphatase 5, the STP domain is fused to four tetracotripeptide repeat protein–protein interaction modules . PPM/PP2C enzymes may also have additional domains, for example, Arabidopsis thaliana ABI1 in which the catalytic domain is fused to an EF-hand motif, and human STP (HsSTP), which has an additional 8 kDa α-helical domain at the C-terminus [3,4].
The sizes of the catalytic domains of both PPP and PPM families are well conserved and structural studies have revealed significant similarities between them [4–9]. Both utilize two β sheets to help the enzymes orient their active site residues in a conformation where they bind active-site metal ions. The active-site ligands are, however, different; in PPP enzymes, histidine, aspartate and asparagine side chains bind the metal ions, whereas in PPM enzymes, aspartates and a glycine backbone carbonyl coordinate the metal ions . The identity of metal ions within the groups varies and studies have sometimes shown slightly controversial results . The PPM/PP2C studied to date have been shown to contain either Mg2+ or Mn2+[1,4]. In addition, in the crystal structure of Toxoplasma gondii STP (TxSTP; PDB code 2I44), the metals are modeled as Ca2+ ions (unpublished).
Detailed biochemical analysis has revealed differences between these enzymes. Only PPPs are inhibited by the classical STP inhibitor okadaic acid . Although similar, the mechanisms of these enzymes are not identical, because PPM and PPP class enzymes bind their substrates differently. In the PPP family, the substrate phosphoryl group is bound directly to the two metal ions via its oxygen residues, whereas PPM/PP2C family enzymes bind the substrate indirectly, via hydrogen-bonding interactions between the phosphoryl group and water molecules liganded to the metal ions [4,10,11].
The biochemistry of PPM/PP2C has been studied extensively using the human enzyme as a model [4,10,11]. It relies on two divalent metal ions and an activated bridging water molecule with a pKa of 7.5  to achieve catalysis, a common feature in hydrolytic metalloenzymes . One residue that appears to take part in catalysis in HsSTP is His62, which may act as a general acid and protonate the phosphate as it leaves , but this residue is missing from the prokaryotic Mycobacterium tuberculosis STP (MtSTP) ; and from SaSTP. This implies that, in these enzymes, some other residue or a water molecule would act as the general acid. HsSTP Arg33, conserved among STPs, has been proposed to take part in binding the phosphorylated protein substrate. The function of other conserved residues near the active site remains unclear . Interestingly, MtSTP has been shown to bind a third metal ion near the active site . A serine residue that takes part in binding the third metal ion is not conserved and the function of the third metal ion in MtSTP is unknown.
Recently, serine/threonine phosphorylation/dephosphorylation has been shown to occur in many prokaryotes, where it modulates cellular activities analogously to events found in eukaryotes. In Bacillus subtilis a PPM/PP2C STP activates sporulation transcription factor [13,14]. M. tuberculosis contains many serine/threonine kinases (STKs), including serine/threonine protein kinase G, which mediates survival of the bacteria . Yersinia pseudotuberculosis and Yersinia enterocolitica YpkA STKs induce the secretion of many Yop virulence effector proteins [16,17], the Streptococcus pneumoniae stkP– strain has reduced infectivity in mice , and the Pseudomonas aeruginosa ppkA STK is needed for virulence in mice . S. agalactiae has an active STP/STK system, which affects both the virulence and morphology of the bacteria [20–22].
The above-mentioned studies have thus shown that serine/threonine signaling cascades are linked to the virulence of organisms, leading to interesting possibilities for rational drug design against these pathogens. Drugs targeting S. agalactiae signaling enzymes may cure many severe diseases, such as sepsis and meningitis, which threaten the lives of newborn babies and immunocompromised adults. To support drug design, we need detailed structural information about the signaling proteins (STKs and STPs), and their complexes with their downstream targets, which in S. agalactiae include the response regulator CovR, adenylosuccinate synthase and a family II inorganic pyrophosphatase (SaPPase) [20–22]. We have previously crystallized one of the substrate molecules (SaPPase) . Here we report the crystal structure of SaSTP. The structure revealed a third metal ion, as in MtSTP. However, unlike MtSTP, its presence correlates with binding of another STP monomer over the active site. This interaction may resemble the dephosphorylated product complex.