2.1. His39 Determines tPphA Substrate Specificity
Wild-type tPphA and H39A were crystallized under previously described conditions [11
]. The crystal structure parameters are listed in Table 1
. The overall structures were similar to published tPphA structures [12
]. However, there were variations in all of the structures, especially in the region housing the FLAP domain (Thr138 → Leu163) and loops connecting α helixes and β strands (Figure 1
The loop (Gly37
) connecting β3 and α1, which contains His39, was disordered in three of the tPphA structures (PDB codes: 2J82, 2XZV and 5ITI). However, the structure of this loop in the H39A structure (PDB code: 5D2U) could be determined (Figure 1
B). These results indicate that the mutation of His39 to alanine made the loop more rigid than the corresponding loop in wild-type tPphA. Moreover, H39A exhibited a different conformation than D119A (Figure 1
B). Overall, the different conformation and flexibility of this loop in H39A compared to the wild-type enzyme may underlie the change in the substrate specificity of H39A. This relationship could explain our previous enzymatic assay results in which H39A could not recognize the natural substrate of tPphA, i.e.
, the PII protein [5
In the plant PP2Cs ABI1 and HAB1, there is a glycine residue at the position equivalent to tPphA His39, in the loop connecting β3 and α1 [6
]. The mutation of this glycine to aspartate inhibited the abilities of ABI1 and HAB1 to dephosphorylate casein but did not greatly affect the abilities of these two phosphatases to dephosphorylate their other physiological substrates [19
]. These results indicate that this glycine residue in plant PP2Cs also plays a role in helping phosphatases recognize substrates.
2.2. New Insights into the Catalytic Center of tPphA
Three calcium ions were identified in each of the catalytic centers of the two tPphA structures solved here. This ion composition is different from that found in previously published tPphA structures [11
]. In one published tPphA crystal structure (PDB: 2J86), three magnesium ions were identified in the catalytic center. This structure was solved from a crystal grown in 0.2 M CaCl2
, and the crystal was soaked with p
NPP and 0.2 M MgCl2
prior to diffraction. In another tPphA structure (PDB: 2J82), one fully occupied magnesium ion (M3) was present from the purification procedure used, one fully occupied calcium ion (M2) was present from the crystallization conditions used, and one metal ion (M1) was present that comprised a mixture of magnesium and calcium. Although the ion compositions of the different structures varied, the positions of the three divalent cations in the tPphA structures were nearly identical (Figure 1
C). The mutual distance of the same divalent cations in different tPphA structures was less than 0.5 Å.
Five conserved aspartate residues present in the structures of all high-resolution tPphA structures (better than 2.0 Å) were merged with each other (Figure 1
C). Both tPphA structures solved here and a 1.28 Å tPphA structure (PDB: 2J82) were generated from crystals grown under the same conditions. The positions of the five aspartate residues in these three structures were found to be nearly identical (all-atom RMSD value: 0.25). By contrast, the positions of these aspartate residues in wild-type tPphA (PDB: 2J82 and 5ITI) and H39A structures showed differences (all-atom RMSD values: 0.86, 0.81, 0.73, and 0.98) compared to the aspartate residues in another structure (PDB: 2J86) that contains two tPphA subunits in the asymmetric unit. In the 2J86 structure, three Mg2+
are present in the catalytic center. The crystal used to generate this structure was soaked with p
NPP and 0.2 M MgCl2
prior to diffraction. These results imply that the catalytic center of the 2J86 structure may be in an “active state”, whereas the tPphA structures bound with Ca2+
may be in an “inhibited state”. Ca2+
have different geometries, and Ca2+
is larger than Mg2+
serves as a ligand of tPphA, whereas Ca2+
serves as an inhibitor of tPphA (See 2.4). When tPphA is complexed with Mg2+
may induce aspartate residues at suitable positions of tPphA to adopt conformations that enable the enzyme to dephosphorylate substrates. By contrast, if tPphA is coordinated with Ca2+
, then Ca2+
may force the aspartate residues to adopt conformations that prevent tPphA from dephosphorylating its substrates. Therefore, Ca2+
may inhibit the catalytic activity of tPphA not only because of its unique characteristics (size and geometry) but also by slightly changing the structure of the catalytic center.
2.3. Relationship between the FLAP Subdomain and M3
The most flexible region of tPphA is the FLAP subdomain. The FLAP subdomain shows variable conformations in different tPphA structures (Figure 1
A). The RMSD values between parts of FLAP subdomains (Ile151 → Leu163) are listed in Table 2
. Most of the RMSD values are higher than 1. Two exceptions are the RMSD values (0.681 and 0.707) between two tPphA structures solved here and between D119A and D193A. Although these RMSD values are lower than other RMSD values, they are nonetheless high. The structure of most of the FLAP subdomain in one tPphA structure (PDB: 2J82) could not be determined. Therefore, the RMSD values of this FLAP domain relative to other FLAP domains could not be determined. The bottom of the FLAP subdomain in this tPphA structure (PDB: 2J82) is visible, and this region adopts a significantly different conformation compared to those found in other tPphA structures (Figure 1
A). In addition, the B factor of the FLAP subdomain is significantly higher than those in other parts of tPphA (Figure 1
D). Overall, these results indicate that the FLAP subdomain is a flexible region of tPphA.
Asp119 and Asp193 are the only two residues found to coordinate with M3. The mutation of Asp119 and Asp193 to alanine caused tPphA to lose catalytic activity. The crystal structures of D119A and D193A only show M1 and M2 in the catalytic center. This result indicates that M3 is essential for the dephosphorylation reaction catalyzed by tPphA [12
]. It has been suggested that the FLAP subdomain and M3 can influence each other’s positions and regulate PP2C catalytic activity and substrate specificity [7
]. Here, the FLAP subdomain showed variable conformations in different structures, while the mutual distance of M3 in different tPphA structures was less than 1 Å. (Figure 1
A,C). Moreover, in a previous study, His161 of the FLAP subdomain, a residue that is located within the vicinity of the catalytic core and occupies the same position of Ser160 of M. tuberculosis
], was replaced with either serine or alanine. However, the mutation of His161 only slightly affected tPphA activity. This result indicates that the FLAP subdomain does not influence the position or function of M3 and vice versa
. Overall, the FLAP subdomain and M3 are independent of each other.
2.4. Inhibition Effects of Nine Chemicals on tPphA Activity
The catalytic constants of tPphA and H39A towards casein were determined (Table 3
). The Km
of H39A towards casein are higher than those of tPphA. However, the Kcat
of H39A is almost equivalent to the wild-type enzyme. This result indicates that the H39A variant has the same catalytic efficiency as tPphA.
A small screen of tPphA inhibitors was performed. Nine chemicals were used to inhibit tPphA activity. The inhibitory mechanisms of these chemicals on tPphA were different. By comparing the inhibitory effects of these chemicals on tPphA, a better inhibitory mechanism could potentially be identified, and a new effective inhibitor could be designed based on this information.
We previously showed that H39A does not dephosphorylate the PII protein because this variant could not recognize it. Therefore, a substitute protein, casein, was used in this assay. Casein has been used in many PP2C studies [19
]. We found that H39A could dephosphorylate this protein. Therefore, we used casein as a substitute for the PII protein to investigate the inhibitory effects of different chemicals on H39A. The IC50s of nine chemicals on tPphA and H39A with regard to the dephosphorylation of p
NPP and casein were determined (Figure 2
and Table 4
Preliminary pre-clinical in vitro
and in vivo
studies have demonstrated that sanguinarine causes apoptosis in human cancer cells [24
]. Recently, sanguinarine was identified as inhibiting the dephosphorylation of casein by human PP2Cα with an IC50 value of 2.5 μM [23
], although the inhibitory mechanism is unknown. Here, sanguinarine also showed good inhibition of tPphA (Table 4
). The IC50 value of sanguinarine on tPphA with regard to the dephosphorylation of casein was approximately 30 times higher than that obtained for PP2Cα. However, the IC50 value of sanguinarine on tPphA with regard to the dephosphorylation of p
NPP was only four times higher than that obtained for PP2Cα. These results imply that the structural differences between PP2Cα and tPphA may cause sanguinarine to produce different inhibitory effects on these enzymes. Future work should be focused on explaining how this compound inhibits PP2Cs, as well as how chemical modifications of this compound may improve its specific inhibitory effect on PP2Cs.
The replacement of Mn2+
in the catalytic center of tPphA with two divalent cations (Ni2+
) could also inhibit tPphA activity. The low IC50 value of Ni2+
measured in this report suggests that this divalent cation is a stronger inhibitor of tPphA than Ca2+
, which also shows inhibitory effects on other PP2Cs [30
]. Other divalent cations have also been reported to inhibit PP2C catalytic activity [32
]. The inhibitory effects of these divalent cations on PP2Cs may come from the replacement of Mg2+
with the above ions. It is assumed that the different sizes and preferred geometries of Mg2+
these divalent cations are responsible for the loss of activity in PP2C [38
Several inhibitors of Wip1 have previously been synthesized [13
]. These inhibitors specifically inhibit Wip1 by binding to the FLAP subdomain. Because the FLAP subdomain helps PP2Cs recognize their specific substrates, its primary and tertiary structures should be considered in the design of new inhibitors. Based on the present results, a good strategy for the design of PP2C inhibitors is the creation of compounds which contain both divalent cation moieties and other moieties that could specifically bind to the FLAP subdomain: first, the FLAP subdomain interaction moieties bind to the unique FLAP subdomain of PP2C; second, divalent cation moieties disturb the catalytic center of PP2C and inhibit its activity.
In previous studies, we showed that Mg2+
could activate tPphA to dephosphorylate the PII protein and other phosphopeptides but could not induce tPphA to dephosphorylate p
]. This characteristic of Mg2+
has also been observed in other p
NPP assays of PP2Cs [34
]. The IC50 values of Mg2+
on tPphA and H39A when p
NPP was used as substrate were determined as 10.98 and 9.64 mM, respectively.
EDTA, NaF and AlF3
are general inhibitors of phosphatase. EDTA effectively chelates divalent cations from proteins. The IC50s of EDTA on tPphA with regards to the dephosphorylation of p
NPP and casein are 0.93 and 2.29 mM, respectively. PP2Cs dephosphorylate substrates via an SN
2 mechanism. A nucleophilic hydroxide ion activated by M1 and M2 is essential for the dephosphorylation reaction [12
mimics the nucleophilic hydroxide ions present in phosphatase active sites [40
]; therefore, F−
could be used as an inhibitor of tPphA. The IC50 value of NaF on tPphA with regard to the dephosphorylation of casein is 1.24 mM, which is much lower than the value obtained for H39A. Thus, the histidine at position 39 may help tPphA correctly place a substrate so that hydroxide ions cannot easily diffuse into the catalytic center of the enzyme; in this case, a low concentration of NaF would be sufficient to inhibit 50% of tPphA’s activity. By contrast, the substitution of His39 with an alanine may alter the contact points that form between tPphA and casein such that hydroxide ions could easily reach the catalytic center; in this case, a high concentration of NaF would be needed to reduce H39A activity. Indeed, NaF concentrations of 21.08 mM and 23.73 mM are required to reduce 50% of tPphA and H39A’s activities, respectively, when using p
NPP as a substrate. AlF3
structurally mimics the transition state of phosphate. It is assumed that phosphate groups on a given substrate can be replaced with AlF3
, which will inhibit dephosphorylation reactions [41
]. However, AlF3
could not effectively inhibit tPphA’s dephosphorylation of its substrates.
Sodium phenyl phosphate, a compound structurally related to pNPP, could not be dephosphorylated by tPphA (according to a malachite green assay). The main difference between sodium phenyl phosphate and pNPP is that sodium phenyl phosphate lacks one nitro group. This indicates that the nitro group of pNPP is required for tPphA to dephosphorylate pNPP. Sodium phenyl phosphate could inhibit tPphA dephosphorylation of pNPP. In addition, glycerol-phosphate, which has three phosphate groups, showed some inhibition of tPphA activity.
2.5. An Active tPphA Dimer
In tPphA structures, one single tPphA molecule forms an asymmetric unit. The FLAP subdomain of a tPphA molecule in one asymmetric unit forms an interaction with the FLAP subdomain of a tPphA molecule in another asymmetric unit via crystal-packing contact (Figure 3
A). However, tPphA only showed one single peak on size exclusion chromatography [5
]. This result indicates that tPphA can also exist in a monomeric form. Thus, to stabilize the tPphA dimer, two chemicals (glutaraldehyde and DTSSP) were used to cross-link tPphA. Glutaraldehyde is a classic cross-linker for the immobilization of enzymes and can be used in many types of medium [42
]. DTSSP has dual N
-hydroxysulfosuccinimide esters that can react with primary amines on proteins. A disulfide bond present in the center of DTSSP can be cleaved with reducing agents. The dimeric form of tPphA was stabilized by cross-linking with glutaraldehyde and DTSSP (Figure 3
B,C). The tPphA dimer cross-linked by glutaraldehyde was a stable dimer: neither SDS nor DTT could destroy this complex (Figure 3
B). By contrast, the tPphA dimer that was cross-linked by DTSSP could be decomposed by DTT (Figure 3
After the glutaraldehyde cross-linking reaction, tPphA monomers and dimers were separated over a gel filtration column. There were two main peaks in following size exclusion chromatography (Figure 3
D): A 15 mL peak that mainly contained dimeric tPphA and an 18 mL peak that mainly contained monomeric tPphA. The activities of the protein fractions collected from the gel filtration were assayed using p
NPP as a substrate. After cross-linking, both tPphA monomers and tPphA dimers could dephosphorylate p
NPP (Figure 3
tPphA is highly soluble and easy to purify. It also maintains reaction activity at 65 °C [11
]. Even when crosslinked by glutaraldehyde, tPphA still maintains activity. These characteristics of tPphA may allow it to be immobilized on several media through glutaraldehyde crosslinking and it is used in the chemical industry for dephosphorylating particular compounds under high temperature conditions. In addition, because tPphA is inhibited by several divalent cations, such as Ni2+
, it can also be immobilized in a biosensor to detect divalent cations.