Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family

Abstract

The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3',4,4',5,5'-hexakisphosphate [BiPh(3,3',4,4',5,5')P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3',4,4',5,5')P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3',4,4',5,5')P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B-BiPh(3,3',4,4',5,5')P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed "moving metal" mechanism.

Figure 1

Structures of YU142670 (1), d-myo-inositol 1,3,4,5-tetrakisphosphate (2), biphenyl 3,3′,4,4′,5,5′-hexakisphosphate (3), and benzene 1,2,4,5-tetrakisphosphate (4).

Scheme 1. Synthesis of Biphenyl 3,3′,4,4′,5,5′-Hexakisphosphate (3)

(i) K₂CO₃, (2 equiv), 35°C, acetone/water (3:3.5), 0.5% Pd(OAc)₂, 67%; (ii) BBr₃, CH₂Cl₂, dry ice/acetone cooling, 94%; (iii) (EtO)₂P–Cl, CH₂Cl₂, diisopropylethylamine, mCPBA (80%); (iv) TMSBr, CH₂Cl₂, H₂O, TEAB (91%).

Figure 2

Cartoon representation of the crystal structure of INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ (panel A) and INPP5B–Bz(1,2,4,5)P₄ (panel B) complexes. BiPh(3,3′,4,4′,5,5′)P₆, inorganic phosphate, and Bz(1,2,4,5)P₄ are shown as sticks, the Mg²⁺ ion is shown as a green sphere, β-sheets are in yellow, α-helices in red, and loop regions in green. For both complexes, the symmetry related molecule that provides additional interactions to the phosphorylated ligand is shown in cyan with the His-Tag highlighted in dark blue. Only chain A of the INPP5B–Bz(1,2,4,5)P₄ complex is shown for clarity.

Figure 3

Panel A shows BiPh(3,3′,4,4′,5,5′)P₆ bound in the catalytic site of INPP5B, with a molecule of inorganic phosphate positioned between the 3- and 4-phosphates. The green sphere is a magnesium ion (Mg²⁺), and the red spheres are water molecules in the absence of the His-Tag. Panel B shows the BiPh(3,3′,4,4′,5,5′)P₆ bound in the catalytic site of INPP5B, in complex with the His-Tag. The carbon atoms of the two INPP5B molecules interacting with BiPh(3,3′,4,4′,5,5′)P₆ are colored gray (catalytic site) and green (neighboring INPP5B molecule). Panel C shows Bz(1,2,4,5)P₄ bound in the catalytic site of INPP5B. The red sphere indicates the presence of a molecule of water and in the absence of the His-Tag. Panel D shows the presence of Bz(1,2,4,5)P₄ in the catalytic site of INPP5B (carbon atoms colored in gray) bound to the His-Tag of a neighboring INPP5B molecule (carbon atoms are colored yellow).

Figure 4

Overlay of Bz(1,2,4,5)P₄–INPP5B (green) and BiPh(3,3′,4,4′,5,5′)P₆–INPP5B (blue) complex structures showing good overlay between the two molecules and different phosphate groups. The Mg²⁺ (blue sphere) and inorganic phosphate (sticks) from the BiPh(3,3′,4,4′,5,5′)P₆–INPP5B structure are shown, and the numbering of the ligand phosphates are included.

Figure 5

Overlay of the INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ crystal structure with the ligands from the other 5-phosphatase-ligand crystal structures (PtdIns4P, PtdIns(3,4)P₂, and Bz(1,2,4,5)P₄ from INPP5B complexes, SHIP2–BiPh(2,3′,4,5′,6)P₅, and SPsynaptojanin–Ins(1,4)P₂) highlighting the similarities and differences in the phosphate and hydroxyl positions. The 1-phosphate region is shown in blue, the 5- and 6-hydroxyl region in green, and the 4-phosphate region in red. Other phosphates that show a variety of different interactions with the protein are shown in yellow. The Mg²⁺ ion is shown as a green sphere and the inorganic phosphate as sticks.

Figure 6

(A) Overlay of the proposed catalytic residues for INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ (green), INPP5B–Bz(1,2,4,5)P₄ (yellow), INPP5B–PtdIns4P (dark blue) SHIP2–BiPh(2,3′,4,5′,6)P₅ (light blue), and SPsynaptojanin–Ins(1,4)P₂ (red) complexes. Numbering is for the INPP5B enzyme. (B) Overlay of inorganic phosphate from the INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ complex structure with water molecules from INPP5B–PtdIns4P, SPsynaptojanin-Ins(1,4)P₂ apo-SPsynaptojanin, and SHIP2–BiPh(2,3,′4,5′,6)P₅ structures. Only the catalytic residues surrounding the water molecule are shown for clarity. The Mg²⁺ from the INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ complex structure is shown as a green sphere, and the PtdIns4P from the INPP5B complex is shown in blue. (C) Overlay of the phosphate anion and co-ordinating residues from the INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ complex with the ligand and the INPP5B–PtdIns4P complex. (D) Overlay of the active site regions of INPP5B–BiPh(3,3′,4,4′,5,5′)P₆ (gray carbons; Mg²⁺ as a green sphere; inorganic phosphate in green) AP endonuclease–DNA (pink carbons; Mn²⁺ as a purple sphere; DNA shown as a cartoon with the cleaved sugar–phosphate groups shown as sticks). Residue numbering is for INPP5B with AP endonuclease in parentheses.

Figure 7

Superposition of INPP5B-BiPh(3,3′,4,4′,5,5′)P₆ (yellow), INPP5B-PtdIns(3,4)P₂ (magenta), and OCRL (cyan). Amino-acids whose side-chains are shown as sticks are labeled using the same color as the protein to which they correspond. BiPh(3,3′,4,4′,5,5′)P₆ (yellow) and PtdIns(3,4)P₂ (magenta) are represented as thin sticks. Mg²⁺ ions and the putative catalytic water molecule are displayed as spheres. Protein–metal, protein–water, or protein–phosphate interactions are represented by dashes. Spheres and dashes are both colored yellow, magenta, or cyan whether they belong to INPP5B-BiPh(3,3′,4,4′,5,5′)P₆, INPP5B-PtdIns(3,4)P₂, or OCRL structures, respectively. The water molecule (present in the structure of INPP5B-diC₈–PtdIns(3,4)P₂) that attacks the 5-phosphate upon deprotonation in the mechanism we propose is depicted by a red sphere, labeled with a “W”. Free phosphates are labeled using either a yellow or a cyan font whether they were present in INPP5B-BiPh(3,3′,4,4′,5,5′)P₆ or the OCRL structure.

Figure 8

(A) Stick diagram showing participants in a pentacoordinate intermediate based on the AP endonuclease mechanism. After nucleophilic attack of an activated water molecule the trigonal bipyramidal intermediate is stabilized by a series of residues and the active site Mg²⁺ ion. Arg-451 (colored green) was not originally suggested as part of the mechanism, but is located such that it may help stabilize the intermediate and is conserved in all 5-phosphatases. Only schematic involvement of the various residues is depicted. (B) Mechanism for the hydrolysis of the 5-phosphate of Ins(1,4,5)P₃ by INPP5B, based upon the stick diagram and interactions found in (A) together with additional contributing amino acid residues and the movement of the Mg²⁺ ion from site B towards site A. Attack by a water molecule produces a trigonal bipyramidal intermediate that collapses releasing the phosphate anion. The amino acids are color-coded for easier recognition. The anion that forms at the 5-position is probably quenched with a proton originating from a nearby water molecule, possibly Mg²⁺-bound, or from the adjacent protonated 4-phosphate group. This has not been shown on the diagram for the sake of clarity. Key: R = H for Ins(1,4,5)P₃, R = diacylglycerol for PtdIns(4,5)P₂. Only schematic involvement of the various residues is depicted. Negative charges during phosphoryl transfer are not shown.