Bacterial PhyA protein-tyrosine phosphatase-like myo-inositol phosphatases in complex with the Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃ second messengers

Abstract

myo-Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃. Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo-inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP₆ or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P₅, have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P₅. These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended Ω-loop, and a β-hairpin turn of the Phy-specific domain.

Introduction

myo-Inositol phosphates (IPs)² containing between one and eight phosphoryl groups are ubiquitous in eukaryotic species and have diverse biological activities (1). The most abundant, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP₆ or phytate), has multiple important roles in eukaryotic cellular processes including the regulation of plant hormone receptors, DNA repair, RNA processing, mRNA export, plant development, apoptosis, and pathogenicity (2–9). Less-abundant, less-phosphorylated IPs have also been implicated in many important biological processes including second messenger activities (1, 10). For example, the role of Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ in Ca²⁺ mobilization has been well-characterized (1, 11). Ins(1,4,5)P₃ stimulates the release of Ca²⁺ from the endoplasmic reticulum resulting in further cellular responses, and Ins(1,3,4,5)P₄ acts to increase sensitivity and generate a longer-lasting signal. In general, highly phosphorylated IPs (InsP₆ and InsP₅) serve as cofactors, whereas less-phosphorylated IPs are utilized as second messengers in signal transduction pathways (10). In contrast to eukaryotic cells, IPs are typically absent from prokaryotes (12), despite the presence of enzymes specific for IPs (IPases) in many organisms. Known microbial IPases have few characterized roles in prokaryotes (13). Most serve as phosphate-scavenging proteins, and more recently others have been implicated in pathogenesis (4, 14, 15).

Microbial IPases that specifically hydrolyze the C3- and C4-phosphoryl groups of InsP₆ (d-myo numbering) are common, and experimental structures of both classes of enzyme are available (16–18). Structural features that determine C3-phosphoryl group specificity of IPases (3-phytases) have been identified for two distinct enzyme families: protein-tyrosine phosphatase-like myo-inositol phosphatase (PTPLPs) and histidine-acid phosphatases (16, 17). In each of these cases, the InsP₆ substrate adopts its lowest energy conformation, and steric interactions restrict the only axial phosphoryl group (C2) to a specific location adjacent to the scissile phosphoryl group (16, 17).

PTPLPs and histidine-acid phosphatases hydrolyze InsP₆to produce myo-inositol-2-monokisphosphate (Ins(2)P) and phosphate after prolonged incubation (18–22). Characterized PTPLPs have been shown to hydrolyze InsP₆ to Ins(2)P via different dephosphorylation pathways (19–22). An example is PhyA from Selenomonas ruminantium (PhyAsr), which first removes the C3-phosphoryl group (P3) followed (in order) by P1, P6, P5, and P4 (19). PTPLPs such as PhyA from Mitsuokella multacida (PhyAmm) and Legionella pneumophila (LppA) have demonstrated activity toward the Ins(1,4,5)P₃ second messenger (23, 24). It is likely that these enzymes can also hydrolyze additional IP second messengers and that this activity is shared by other PTPLPs.

Here we have presented the first structures of bacterial PTPLPs in complex with eukaryotic IP second messengers, representing crystallographic evidence that PTPLPs bind and hydrolyze these compounds. These IPs bind in a different conformation than InsP₆ while utilizing a subset of the previously determined phosphoryl-binding sites (17). Further, we have identified several variable loops adjacent to the active site that influence their dephosphorylation pathways. Consequently, this work both refines and extends previous studies aimed at understanding the function and specificity of these enzymes and likely applies to other IPase families.

Results

Active sites of PTPLPs are pre-formed

The overall fold of PTPLPs is composed of a catalytic α-β-α sandwich PTP domain and an antiparallel α-β sandwich Phy domain. The active site includes the general acid loop (GA-loop) and phosphate-binding loop (P-loop) from the catalytic PTP domain as well as residues from both domains that participate in substrate binding (17). The fold and active site of PhyAsr are shown in Fig. 1A. Least squares (LSQ) superpositions of PhyAsr crystal structures in the presence and absence of ligand yielded r.m.s.d. of ∼0.2 Å (over 1248 main-chain atoms), clearly demonstrating that there are no large-scale main-chain conformational changes associated with ligand binding (supplemental Table S1) (17, 19). The same is true within the active site, as the main- and side-chain conformations of the residues alone or in complex with InsP₆, Ins(1,3,4,5)P₄ or Ins(1,4,5)P₃ are essentially identical (supplemental Table S2).

Figure 1.

Ribbon diagrams of the overall fold of PhyAsrC252S (gray) and PhyAmmC250S/C548S (brown/blue) in complex with Ins(1,3,4,5)P₄. A, PhyAsrC252 is oriented so that the Ins(1,3,4,5)P₄ ring and phosphoryl groups are clearly visible, with the C1-phosphoryl group bound to the phosphate-binding loop (P-loop, yellow). B, the individual N-terminal (brown) and C-terminal (blue) repeats of PhyAmm are shown in the same orientation as in A. C, PhyAmm oriented to view the tandem repeats and linker resulting in the Ins(1,3,4,5)P₄ ligand viewed from the top with the P-loop below the ligand. The ligand atoms are shown as sticks with carbon shown in gray, oxygen in red, and phosphorus in orange. The variable loops are shown in green. These include the extended loops prior to the penultimate helix, the α-turn (N-terminal repeat) and β-hairpin turns (PhyAsr and C-terminal repeat), and the extended Ω-loops. The P-loop (yellow) and the general acid loop (GA-loop, cyan) contribute catalytic residues. Mutation of the cysteine nucleophile to the isosteric serine prevents thiolate formation and renders the enzyme inactive.

The PhyAmm monomer is an example of a tandemly repeated IPase that contains two copies of the catalytic PTP and Phy domains (Fig. 1B). Although the N- and C-terminal repeats of PhyAmm have different IP substrate specificities, they are both active toward the Ins(1,4,5)P₃ second messenger (23). Presented here is the first complex structure of PhyAmm: PhyAmmC252S/C548S in complex with Ins(1,3,4,5)P₄, a related second messenger. The PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ structure was solved in a different space group (P1) than PhyAmm without ligand (P2₁). Between the two structures, the individual repeats of PhyAmm are nearly identical as judged by LSQ superpositions (supplemental Table S3) (23). Small movements in the linker region between repeats subtly alter the relative orientation of the tandem repeats and account for the bulk of the observed differences between the individual monomers and dimers. As reported previously, the dimer interface of PhyAmm is almost exclusively formed between the N-terminal repeats, and these interactions are an extensive network of hydrogen bonds, salt bridges, and van der Waals contacts (23). As seen with PhyAsr, the C-terminal active site of PhyAmmC250S/C548S is virtually identical in the presence or absence of Ins(1,3,4,5)P₄ (supplemental Table S4). Taken together, the structures of both PhyAsr and PhyAmm support previous suggestions that the PTPLP family of enzymes have pre-formed active sites (17).

Alternate binding modes of IPs

The electron density for bound ligand is clearly visible in the initial 2mF_o − DF_c (1.5 σ) and mF_o − DF_c (3.5 σ) maps of each PhyAsrC252S structure. Refined electron density maps (2mF_o − DF_c) for the Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃ ligands at 1 σ are shown in Fig. 2, A and B. Obvious electron density for the axial C2-hydroxyls and each phosphoryl group clearly identify the conformation of each ligand bound within the active site. We determined two separate PhyAsrC252S^.Ins(1,3,4,5)P₄ structures that differ in how the ligand was soaked: 1 mm Ins(1,3,4,5)P₄ for 1 h and 10 mm Ins(1,3,4,5)P₄ for 15 min. The resulting structures are virtually identical, apart from an additional Ins(1,3,4,5)P₄ conformer (0.25 occupancy) modeled in the 1 mm soak, as it reduces the difference density within the active site. The major conformer of the 1 mm soak is identical to the single conformer modeled at full occupancy in the 10 mm soak. Interestingly, PhyAsrC252S binds Ins(1,4,5)P₃ in a position identical to that of Ins(1,3,4,5)P₄ less one phosphate (Fig. 3).

Figure 2.

Clear electron density for the phosphoryl groups and C2-hydroxyl allows for an unambiguous fit of the ligands and places the C1-phosphoryl group (P1) above the cysteine to serine mutations at positions 252 (PhyAsr) and 548 (PhyAmm). The refined 2mF_o − DF_c electron density is contoured at 1 σ (blue mesh) for PhyAsrC252S in complex with Ins(1,3,4,5)P₄ (A) and Ins(1,4,5)P₃ (B) and for the PhyAmmC252S/C548S C-terminal repeat in complex with Ins(1,3,4,5)P₄ (C). Ligand and protein are shown as sticks, with oxygen shown in red, nitrogen in blue, phosphorus in orange, and carbon in gray.

Figure 3.

PhyAsrC252S and the C-terminal repeat of PhyAmmC250S/C548S bind the IP substrates nearly identically using a subset of the contacts identified in the PhyAsrC252·InsP₆ (PDB 3MMJ) structure (17). The phosphoryl-binding sites are labeled according to the InsP₆ structure (P_s, P_b, P_a′, and P_c). A, stereo view of the superposition of PhyAsrC252S (gray) and the C-terminal repeat of PhyAmmC250S/C548S (blue) in complex with Ins(1,3,4,5)P₄. B, PhyAsrC252S in complex with Ins(1,4,5)P₃ in the same conformation as in A less one phosphoryl group. C, the N-terminal active site does not bind Ins(1,3,4,5)P₄ in a catalytically competent manner. Instead, the ligand is more than 8 Å (long dashed line) from the inorganic phosphate bound by the P-loop. The short dashed lines represent the distances between the phosphoryl group bound by the P-loop and the serine hydroxyl. Residues that interact with the ligands are derived from the P-loop (yellow), GA-loop (cyan), Phy domain, and penultimate helix. Oxygen is shown in red, nitrogen in blue, phosphorus in orange, and carbon in gray.

The modeled IPs adopt the lowest-energy chair conformation with five equatorial hydroxyl/phosphoryl groups and an axial C2-hydroxyl. The C1-phosphoryl groups of the ligands are positioned for hydrolysis in the P_s phosphoryl-binding site by forming extensive interactions with the P-loop (Fig. 3 and Table 1) (17, 25). The P-loop (residues 252–259), GA-loop (residues 222–225), and Lys-312 are the only interactions that originate from the catalytic PTP domain. The remaining contacts are mediated by residue side chains derived from the Phy domain (residue ranges of 51 to 59 and 136 to 203) and the Phy-specific extension of the penultimate helix (residues 291–307). The less-phosphorylated IPs utilize a subset of the phosphoryl-binding sites identified previously in the PhyAsrC252S·InsP₆ structure (17).

Table 1.

Electrostatic and hydrogen bond distances in the PhyAsrC252S·Ins(1,3,4,5)P₄, PhyAsrC252S·Ins(1,4,5)P₃, and PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ structures are highly similar

Contact distances (<3.4 Å) between PhyA and the ligand phosphoryl (Phos) and hydroxyl (-OH) groups are shown. Bolded distances are main-chain interactions.

Residue	Site	PhyAsrC252S				Residue	Site	PhyAmmC250S/C548S
		Ins(1,3,4,5)P4		Ins(1,4,5)P3				Ins(1,3,4,5)P4
		Phos/-OH	Distance (Å)	Phos/-OH	Distance (Å)			Phos/-OH	Distance (Å)
Arg-57	Pa′	P3	2.80/3.17			Arg-351	Pa′	P3	3.16/2.85/2.64
Asp-153	Pa′					Asp-449	Pa′	P3	3.24
Lys-189	Pa′	P3	2.60			Lys-485	Pa′	P3	2.73
	Pc	P4	2.96	P4	2.33		Pc	P4	3.07/3.03
Asp-223	Pa′	P3	3.16/2.81			Asp-519	Pa′	P3	2.67
His-224	Pa	P3	2.95			His-520	Pa	P3	3.02
		P4	3.4
		P5	2.51
Ser-252	Ps	P1	2.51	P1	2.27	Ser-548	Ps	P1	2.67
Glu-253	Ps	P1	3.06	P1	3.00	Gln-549	Ps	P1	3.22
Ala-254	Ps	P1	3.17	P1	3.24	Ala-550	Ps
Gly-255	Ps	P1	2.91	P1	2.79	Gly-551	Ps	P1	3.15
Val-256	Ps	P1	2.69	P1	2.92	Ala-552	Ps	P1	2.90
Glu-257	Pa	O6	3.16	O6	3.08	Gly-553	Pa	O6	3.34
								P1	3.31
Arg-258	Ps	P1	2.92/3.00/2.99	P1	2.72/2.90/2.90	Arg-554	Ps	P1	2.73/3.11/2.76
Lys-305	Pc	P4	2.52/2.51	P4	2.41	Lys-600	Pc	P4	2.90/3.10/3.15
	Pb	P5	2.82	P5	3.02		Pb	P5	2.95/2.99
Tyr-309	Pb	P5	2.82/2.47/3.18	P5	2.68/3.13	Tyr-604	Pb	P5	2.31/3.20/3.38

The myo-inositol rings of the less-phosphorylated IP complexes have different relative orientations within the active site when compared with the PhyAsrC252S·InsP₆ structure. In particular, the myo-inositol rings are rotated by 180°, resulting in the opposite face contacting the enzyme. For the C1-phosphoryl group to maintain contact with the P-loop, the rotated myo-inositol rings tilt toward the GA-loop (Fig. 4). Overall, the IPs shift by more than 1.2 Å (center of mass to center of mass; supplemental Table S5) and fill the space occupied by ordered solvent in the InsP₆ complex structure.

Figure 4.

The superposition of PhyAsrC252S in complex with InsP₆ (blue) (PDB 3MMJ) and Ins(1,3,4,5)P₄ (orange) demonstrates the 180° rotation and tilt of the myo-inositol ring toward the GA-loop of the less-phosphorylated IPs relative to InsP₆. The P-loop, GA-loop, and Tyr-309 of the protein are shown. The myo-inositol ring and only two of the phosphoryl group are displayed to simplify the diagram. Despite the rotation and tilt of the myo-inositol ring, the C1-phosphoryl group of Ins(1,3,4,5)P₄ remains bound by the P-loop, and the remaining phosphoryl groups are bound by equivalent residues. For example, the C5-phosphoryl groups in these structures form similar hydrogen bonds with Tyr-309 that originates from the opposite side of the residue.

The N- and C-terminal active sites of PhyAmmC250S/C548S are non-equivalent and have different substrate specificities (23). The C-terminal active site of PhyAmm is highly active toward InsP₆, and 12 of the 14 residues contacting the ligand are conserved when compared with PhyAsr (Table 1). Not unexpectedly, Ins(1,3,4,5)P₄ binding within the PhyAmmC250S/C548S C-terminal active site is nearly identical to that observed in the PhyAsrC252S^.Ins(1,3,4,5)P₄ structure. In contrast, the N-terminal active site of PhyAmm does not bind ligand in a catalytically competent manner. Instead, an inorganic phosphate is bound by the catalytic P-loop, and a partially occupied (0.6) Ins(1,3,4,5)P₄ is bound at a novel site more than 8 Å (phosphorus to phosphorous) from the inorganic phosphate (Fig. 3C). The N-terminal active site has residue substitutions as well as a two-residue insertion. The insertion is in the Phy-specific domain and generates a larger α-turn (residues 182–188) that extends into the active site. The equivalent region of PhyAsr and the C-terminal repeat of PhyAmm have smaller β-hairpin turns. The mutations in the N-terminal repeat of PhyAmm reduce the positive electrostatic surface potential and introduce additional steric limitations.

PTPLP specificity differences

The active sites of PhyAsr and the C-terminal repeat of PhyAmm have conserved residues that contact the ligands (Fig. 3 and Table 1) (23). However, they contain significant differences in the main-chain conformation of the three loops that contribute to the active sites of these enzymes: the extended loop prior to the penultimate helix (PhyAsr(287–305) and PhyAmm(583–600)); the extended Ω-loop (PhyAsr(73–102) and PhyAmm(367–398)); and a β-hairpin turn of the Phy-specific domain (PhyAsr(186–189) and PhyAmm(482–485)) (Fig. 5). As the observed differences in these loop conformations may affect substrate access to the active site, we determined the InsP₆ hydrolysis pathway for these enzymes. The time courses of the InsP₆ hydrolysis products separated by high-performance ion chromatography (HPIC) clearly demonstrate that even though the enzymes are highly specific for the C3-phosphoryl group of InsP₆, they have different specificities for the Ins(1,2,4,5,6)P₅ substrate (Fig. 6, A and B). Comparisons with standard chromatograms demonstrate that PhyAmm is specific for the C4-phosphoryl group of Ins(1,2,4,5,6)P₅, whereas PhyAsr hydrolyzes the C1-phosphoryl (supplemental Fig. S1).

Figure 5.

Variable loops implicated in the substrate specificity of PTPLPs. A, stereo view of the superposition of PhyAsrC252S^.Ins(1,3,4,5)P₄ (blue), PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ (red), and PhyAbb (green, PDB 4NX8) as a ribbon diagram with the ligand as sticks. The variable loops (colored segments) that influence substrate specificity include the extended loop prior to the penultimate helix, the extended Ω-loop, and the β-hairpin turn within the Phy-specific domain. B, the PhyAsr (blue) active site is relatively occluded (RO) on the P_a/P_b side and relatively accessible (RA) on the P_a′/P_b′ side. C, in the case of PhyAmm (red), the relatively occluded and relatively accessible sides are reversed. D, PhyAbb (green) has a 13-residue deletion, which removes the loop that contains the β-hairpin turn, resulting in an accessible (RA) active site on the P_a′/P_b′ side. Additionally, the position of the extended loop prior to the penultimate helix of PhyAbb is similar in position to the equivalent loop of PhyAmm, and the extended Ω-loop is deleted, leaving the P_a/P_b side more accessible (RA). As a result, PhyAbb produces four different InsP₄ products in contrast to PhyAsr and PhyAmm.

Figure 6.

HPIC chromatograms of the PhyAsr (A), PhyAmm (B), and PhyAbb InsP₆ (C) hydrolysis products demonstrating that the hydrolysis pathways diverge to produce alternate InsP₄ products. InsP₆ (5 mm) was incubated with 10 nm PhyAsr and PhyAmm at room temperature and 100 nm PhyAbb with 10 mm InsP₆. Samples were taken at 0 min (blue), 20 min (orange), 30 min (red), 40 min (green), 50 min (purple), and 60 min (cyan) and separated using a CarboPac PA-100 analytical column with a methanesulfonic acid gradient (37). IPs were visualized using a post-column reactor with 0.1% (m/v) Fe(NO₃)₃ in a 2% (m/v) HClO₄ solution (0.2 ml/min).

To further assess the contribution of these variable loops to the substrate specificity of PTPLPs, we examined the structure and hydrolysis pathway of PhyA from Bdellovibrio bacteriovorus (PhyAbb, previously Bd1204). PhyAbb is one of the smallest known PTPLPs and has large deletions in the extended loop prior to the penultimate helix, the extended Ω-loop, and the Phy-specific domain (Fig. 5) (26). As a result, the active site of PhyAbb is more open and accessible than in PhyAsr or PhyAmm. Taken together, it is expected that PhyAbb has an altered pathway and would have a broader specificity for IPs than either PhyAsr or PhyAmm, which is confirmed by the hydrolysis pathway (Fig. 6C). Although PhyAbb is also specific for the C3-phosphoryl group of InsP₆, making it a 3-phytase, PhyAbb produces four different InsP₄, which include the PhyAsr (Ins(2,4,5,6)P₄) and PhyAmm (Ins(1,2,5,6)P₄) products. The much broader specificity of PhyAbb for IPs indicates that the loops that contribute to the active site influence substrate specificity.

Discussion

myo-Inositol ring movements compensate for PTPLP active-site rigidity

There are no changes in the conformation of the active site upon binding of IP ligands to either PhyAsr or PhyAmm (supplemental Tables S2 and S4). The apparent rigidity of these enzymes suggests this may be a feature of the PTPLPs, and their specificity can be understood in simple structural terms. Further, the PhyAmmC250S/C548S C-terminal active site binds Ins(1,3,4,5)P₄ using the same phosphoryl-binding sites identified in PhyAsr (Fig. 3A and Table 1) (17). In the PhyAsrC252S·InsP₆ structure, the orientation of InsP₆ was such that in the P_a′-binding site, only a hydroxyl or an axial phosphoryl group could be accommodated. Consequently, the P_a′ site was identified as the primary structural determinant giving rise to the specificity of PhyAsr for the C3-phosphoryl group of InsP₆ (19, 17). Assuming the ring orientation remains constant, the inability of the P_a′-binding site to accommodate an equatorial phosphoryl group is sufficient to explain the known hydrolysis pathway of PhyAsr, but it does not account for all minor hydrolysis products (19). For example, PhyAsr produces small amounts of Ins(1,2,4,6)P₄ by removing the C5-phosphoryl group from Ins(1,2,4,5,6)P₅, which requires an equatorial phosphoryl group adjacent to the scissile phosphoryl group to bind within the P_a′ site.

This work identifies a significant shift and a 180° rotation of the myo-inositol ring in the less-phosphorylated Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃ complex structures compared with the PhyAsrC252S·InsP₆ structure (Fig. 4). The ring shift and rotation allows smaller substrates to utilize a different subset of phosphoryl-binding sites. For example, in the PhyAsrC252S·InsP₆ structure and the less-phosphorylated IP complexes, hydrogen bonds to the C5-phosphoryl group originate from opposite sides of Tyr-309 (Fig. 4). The myo-inositol ring shift also allows phosphoryl groups not adjacent to the scissile phosphoryl group to occupy the P_a′ site. Importantly, simple modeling studies based on the observed ring shift suggest that, provided the P_b′ site contains a hydroxyl, the P_a′-binding site can accommodate equatorial phosphoryl groups adjacent to the scissile phosphate. Alternatively, both the P_a′ and P_b′ sites may be able to accommodate equatorial phosphoryl groups if the ring is allowed to adopt ring orientations that are intermediate to those observed in the InsP₆ and less-phosphorylated IP complex structures. These observations are sufficient to rationalize the formation of minor products of the PhyAsr hydrolysis pathway and suggest that PTPLPs may be able to hydrolyze a wide range of less-phosphorylated IPs.

Structural determinants of the substrate specificity of PTPLPs

PhyAsr and PhyAmm hydrolyze Ins(1,2,4,5,6)P₅ to different InsP₄ products despite having the same specificity for the C3-phosphoryl group of the InsP₆ and binding Ins(1,3,4,5)P₄ nearly identically (Figs. 3 and 6) (19). The residues that contact the ligands are conserved between PhyAsr and PhyAmm except for residue substitutions in the P-loop (Table 1). In the PhyAmm C-terminal repeat, Gln-549 immediately follows the catalytic Cys-548 (Ser-548 in our structure), whereas the equivalent residue in the PhyAsr is Glu-253. The Gln-549 side chain of PhyAmm is directed toward the active site and may directly or indirectly contact highly phosphorylated IPs providing an additional favorable electrostatic interaction in the P_a′ site. In contrast, both in the presence and absence of ligand, the side chain of Glu-253 (PhyAsr) is directed away from the active site. The charge difference and the spatial orientation in the P_a′ site are capable of influencing the difference in the activity and specificity of these enzymes but do not fully account for the pathway divergence.

The extended loop prior to the penultimate helix, extended Ω-loop, and β-hairpin in the Phy domain are loops around the active site that are different in PhyAsr and PhyAmm and contribute to the divergent pathways (Fig. 5). The main-chain conformational differences in these connecting segments result from residue substitutions, insertions/deletions, and the distinct homodimers formed by each enzyme. The extended loop prior to the penultimate helix of PhyAsr located on the P_a/P_b side of the active site contains a single residue insertion, and the C-terminal end of the loop folds into the active site of the enzyme. In contrast, the equivalent loop in PhyAmm is pulled away from the active site and participates in a 2-fold symmetric, homodimer interface. Also located on the P_a/P_b side of the active sites are the extended Ω-loops. These loops have similar conformations, and there is a structurally equivalent lysine (PhyAsr Lys-83 and PhyAmm Lys-379) pointing toward the active site. However, the extended Ω-loop of PhyAmm has a 2-residue insertion that leaves the active site more accessible on the P_a/P_b side than PhyAsr. On the P_a′/P_b′ side of the active site is the β-hairpin turn, which differs both in type (PhyAsr type II and PhyAmm type I) and orientation between the two active site. The conformational difference is primarily a result of homodimer formation even though the turn and associated β-strands have several residue substitutions. In particular, PhyAsr residues 191–193 are part of the 2-fold symmetric homodimer interface that features an intermolecular antiparallel β-sheet, whereas the equivalent residues in PhyAmm interact with solvent. As a consequence of these differences, the β-hairpin and following residues are pulled away from the active site in PhyAsr to facilitate homodimer formation, whereas the equivalent residues of PhyAmm extend into the active site on the P_a′/P_b′ side.

Taken together, the residue substitution and main-chain conformational differences indicate that the PhyAsr active site is relatively occluded on the P_a/P_b side of the active site and relatively accessible on the P_a′/P_b′ side (Fig. 5). In the case of the PhyAmm active site, the converse is true. This is consistent with the specificity of PhyAsr for the C1-phosphoryl group of Ins(1,2,4,5,6)P₅, as it would place the C3-hydroxyl on the relatively occluded side of its active site. Likewise, the specificity of PhyAmm for the C4-phosphoryl group of Ins(1,2,4,5,6)P₅ would place the C3-hydroxyl on the opposite side of its active site, which corresponds to its occluded side.

The structure and pathway of PhyAbb supports the occlusion theory as an explanation of the divergent pathways of PhyAsr and PhyAmm. PhyAbb lacks the β-hairpin in the Phy-specific domain on the P_a′/P_b′ side of the active site, leaving it more accessible than in either PhyAsr or PhyAmm (Fig. 5). Further, the deletions of the extended Ω-loop, in the extended loop prior to the penultimate helix, and of two turns of the penultimate helix result in a more accessible P_a/P_b side of the active site. The result is a more open and accessible active site that allows Ins(1,2,4,5,6)P₅ to bind in multiple orientations and gives rise to four distinct InsP₄.

Ins(1,3,4,5)P₄ is not bound at the catalytic site of the PhyAmm N-terminal repeat

The PhyAmm N-terminal repeat shares 36 and 34% sequence identity with the C-terminal repeat and PhyAsr, respectively (23). Not surprisingly, the main-chain conformation of the N-terminal active site shares a high degree of similarity with both PhyAsr and the C-terminal repeat of PhyAmm (0.71 and 0.28 Å, respectively, 128 atoms). Despite the closely similar main-chain conformations, in our structure only the C-terminal repeat binds Ins(1,3,4,5)P₄ in a catalytically competent manner. Differences between the N- and C-terminal repeat active sites have been discussed previously (23). At present, there is no clear and unambiguous rationale for the lack of binding of Ins(1,3,4,5)P₄ to the N-terminal repeat P-loop. We note that Ins(1,3,4,5)P₄ is not a natural substrate for this enzyme, and the observed binding sterically prevents another Ins(1,3,4,5)P₄ from binding to the P-loop. Further, the natural InsP₄ substrates, which contain a C2-phosphoryl, cannot bind in the same manner because of steric clashes involving this axial phosphoryl group. This suggests that what we observed was a binding site that is preferred by non-native substrates. Alternatively, the structure was produced by soaking the substrate into a pre-formed crystal, which may prevent binding if there is an induced fit associated with IP binding to the N-terminal active site.

Biological implications

PhyAsr, PhyAmm, and PhyAbb all function as 3-phytases and yet have different specificities for Ins(1,2,4,5,6)P₅, thus producing alternate InsP₄. In the case of PhyAsr and PhyAmm, we were unable to predict their different specificities by identification of residues directly interacting with the substrates, as they are essentially identical. This suggests that substrate specificity is influenced by structural features that do not directly interact with the bound ligand. We identified three variable loops unique to PTPLPs that alter substrate access to the PhyAsr and the PhyAmm C-terminal repeat active sites, which may explain their observed specificities. This role of the variable loops is supported by our ability to predict and demonstrate that PhyAbb, an enzyme with large deletions in these loops, has a broad specificity toward IP substrates.

The activity of PTPLPs toward second messengers has been demonstrated, and this work confirms that two second messengers, Ins(1,3,4,5)P₄ and Ins(1,4,5)P₃, are capable of binding to the PTPLP active sites in a catalytically competent manner (23, 24). Further, various IPases have been demonstrated as important for the function and survival of pathogenic bacteria in host systems (4, 14, 15). They function by either providing the phosphate for growth or by derangement of the host phosphatidylinositol signaling pathway (4, 14). This suggest that PTPLP virulence factors disrupt phosphatidylinositide or inositol phosphate signaling pathways as opposed to phosphotryosine-mediated pathways (27, 28, 15).

Experimental procedures

Expression and purification

The phyA genes of the S. ruminantium (phyAsrC252S), M. multacida (phyAmmC250S/C548S), and B. bacteriovorus HD100 (PhyAbb, previously Bd1204), minus the putative signal peptide, were previously cloned into the NdeI site of the pET28b^Kan expression vector (EMD Biosciences) (19, 23, 26). The resulting PhyAsrC252S and PhyAmmC250S/C548S proteins are catalytically inactive as a result of the cysteine-to-serine mutation (an isosteric substitution). All contained an N-terminal His₆ tag and were produced and purified as described previously (17, 19, 23, 26). PhyAsrC252S was dialyzed into 20 mm ammonium bicarbonate (pH 8.0) and lyophilized; PhyAmmC252S/C548S was dialyzed into 100 mm Tris-HCl (pH 8.0), 100 mm NaCl, 1 mm β-mercaptoethanol (BME), and 0.1 mm EDTA (pH 8.0) followed by the addition of glycerol to 20% v/v; and PhyAbb was dialyzed into 50 mm sodium acetate (pH 5.0), 300 mm NaCl, 5 mm BME, and 0.1 mm EDTA (pH 8.0). The protein was used immediately or flash-frozen and stored at 193 K.

Crystallization

Crystallization experiments were conducted at room temperature using sitting-drop vapor diffusion with drop ratios of 2 μl of protein solution to 2 μl of reservoir solution. PhyAsrC252S protein solutions were prepared at 20 mg/ml, and crystals were grown as described previously (29). PhyAmmC250S/C548S protein solutions were concentrated to 4.5 mg/ml using a Millipore Ultracel 10-kDa centrifugal filter, and crystals were grown in 10% w/v PEG 8000, 100 mm Tris-HCl (pH 8.0), 1 mm BME, 4% v/v ethylene glycol, and 20% v/v glycerol. Following a 24-h equilibration, the reservoir solution was supplemented with 100 μl of glycerol. After 30 days, PhyAsrC252S grew rod-like crystals with approximate dimensions of 30 × 30 × 100 μm, and after 10 days PhyAmmC250S/C548S grew rod-like crystals with approximate dimensions of 100 × 100 × 500 μm. In each case, the crystals were soaked in mother liquor supplemented with 1 mm (60 min; see supplemental material) or 10 mm (15 min) Ins(1,3,4,5)P₄ (Echelon Bioscience) or 10 mm (15 min) Ins(1,4,5)P₃ (Sigma-Aldrich). Following the introduction of ligand, the PhyAsrC252S crystals were transferred to mother liquor containing 22 or 25% v/v glycerol (cryoprotectant) and flash-frozen in liquid nitrogen. The PhyAmmC250S/C548S crystals were flash-frozen directly following the introduction of ligand.

Data collection and image processing

Diffraction data (λ = 0.97934 Å) was collected from frozen crystals (100 K) using a Rayonix MX300 CCD detector at beamline 08ID-1 located at the Canadian Light Source (Saskatoon, Canada). The space group and unit cell parameters of the PhyAsrC252S crystals in complex with ligand are equivalent to those of the PhyAsrC252S·InsP₆ structure (PDB 3MMJ), whereas the PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ crystals have a novel P1 unit cell. All diffraction image data were processed interactively with MOSFLM prior to scaling and merging within AIMLESS of the CCP4 program suite, version 6.3.0 (30–33). Data collection statistics are shown in Table 2 and supplemental Table S6.

Table 2.

Data collection and refinement statistics for the PhyAsrC252S·Ins(1,3,4,5)P₄, PhyAsrC252S·Ins(1,4,5)P₃, and PhyAmmC250S/C548S·Ins(1,3,4,5)P₄ structures

Values in parentheses are for the highest resolution shell.

	PhyAsrC252S·Ins(1,3,4,5)P4	PhyAsrC252S·Ins(1,4,5)P3	PhyAmmC250S/C548S·Ins(1,3,4,5)P4
PDB code	4WTY	4WU2	4WU3
Data collection
Space group	P21	P21	P1
a, b, c (Å)	45.8, 138.2, 80.6	46.0, 137.7, 80.0	73.9, 86.7, 124.2
α, β, γ (°)	90.0, 102.3, 90.0	90.0, 102.4, 90.0	107.3, 91.7, 90.0
Wavelength (Å)	0.97934	0.97934	0.97934
Resolution (Å)	43.0–2.10	44.9–2.15	44.5–2.20
	(2.16–2.10)	(2.21–2.15)	(2.24–2.20)
Observed reflections	214,850	175,251	323,812
Unique reflections	57,016	52,695	146,418
Completeness (%)	100 (100)	99.9 (100)	98.1 (96.6)
Redundancy	3.8 (3.7)	3.3 (3.3)	2.2 (2.2)
Rmerge (%)	13.5 (48.1)	12.8 (52.3)	8.0 (16.0)
I/σI	6.2 (2.3)	6.1 (1.9)	7.2 (4.3)
Refinement statistics
No. reflections work set	55,187	50,541	144,299
No. reflections test set	1,797	2,154	2,118
Rwork/Rfree (%)	15.7/17.5	19.7/21.9	19.0/21.3
Asymmetric unit	Dimer	Dimer	Dimer of dimers
Protein atoms	5,057	5,096	19,188
Solvent atoms	663	484	2,262
Ligand atoms	93	83	270
Wilson B (Å2)	27.51	31.04	19.52
Average B protein (Å2)	26.2	30.5	18.4
Average B solvent (Å2)	35.3	35.3	27.4
Average B ligand (Å2)	42.4	35.3	39.4
r.m.s.d. bonds (Å)	0.005	0.008	0.007
r.m.s.d. angle (°)	1.067	1.153	1.106
Ramachandran distribution
Preferred (%)	97.04	95.9	96.28
Allowed (%)	2.46	3.44	2.69
Outliers (%)	0.49	0.66	1.03

Structure refinement and model validation

Phases derived from the PhyAsrC252S·InsP₆ structure (PDB 3MMJ) and wild-type PhyAmm (PDB 3F41) were used to solve the structures by isomorphous replacement and molecular replacement (MOLREP), respectively. The refined structures have continuous electron density for main-chain atoms of amino acids 33–346 of PhyAsrC252S and 46–636 of PhyAmmC250S/C548S, with the remaining residues located at the termini assumed to be disordered. This includes the N-terminal histidine tag of both proteins, residues 28–32 of PhyAsrC252S and residues 31–45 of PhyAmmC250S/C548S. Refinement was performed using REFMAC, version 5.7, within the CCP4 program suite, and interactive fitting of the models to the electron density was performed in COOT, version 0.6.2 (31, 34). PROCHECK and structure validation tools with COOT were used throughout refinement to assess the stereochemistry of the model (35). Unless indicated otherwise, the figures were prepared with CCP4mg, version 2.10.8 (36). The key data processing and refinement statistics for each PhyAsrC252S and PhyAmmC250S/C548S complex structures are presented in Table 2 and supplemental Table S1.

Structure analysis

These and previously determined structures were compared by LSQ superposition using LSQKAB from the CCP4 program suite (31). The main-chain atoms of residues 35–346 of PhyAsrC252S, and residues 47–342 and 343–636 for the N- and C-terminal repeats of PhyAmmC250S/C548S, respectively, were used in overall fold comparisons. Active-site comparisons were made using residues 56–58, 152–154, 189–190, 221–226, 249–262, 304–309, and 311–313 of PhyAsrC252S and residues 448–450, 484–486, 517–522, 545–558, 584–586, and 599–605 of the PhyAmmC250S/C548S C-terminal repeat. Differences in the relative position of the myo-inositol ring of bound ligands of PhyAsrC252S were calculated using the superposed structure coordinates and GEOMCALC in the CCP4 program suite (31, 34).

Identification of hydrolysis products

Hydrolysis of 5 mm InsP₆ (Sigma-Aldrich) was carried out at room temperature in the presence of 10 nm wild-type PhyAsr or PhyAmm (50 mm sodium acetate (pH 5.0), 200 mm NaCl, 1 mm BME, and 0.1 mm EDTA). In the case of PhyAbb, 100 nm protein and 10 mm InsP₆ were used. Aliquots of 200 μl were taken, heat-denatured at 95°C for 2 min, and subjected to HPIC (Waters 1525 binary HPIC pump, Milford, MA) utilizing a CarboPac PA-100 (4 × 240 mm) analytical column (Dionex, Sunnyvale, CA) (37) at room temperature with a post-column reactor flow rate of 0.2 ml/min. Identification of hydrolysis products utilized a standard hydrolysis chromatogram (supplemental methods).

Author contributions

L. M. B. crystallized and solved the 4WTY, 4WU2, and 4WU3 structures, determined the pathway of PhyAmm, and wrote the paper. R. J. G. crystallized and solved the 3O3L structure. C. P. C. determined the pathway of PhyAbb. S. C. M. supervised the work, and all authors analyzed the results and approved the final version of the manuscript.