Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7

Abstract

Background: Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM.

Results: The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding.

Conclusions: Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.

Figure 1

NPP7 substrate structures.

Figure 2

Comparison of NPP family member crystal structures in the absence (magenta) and presence (blue) of ligand. A. Comparison of Xac. NPP crystal structures 2GSO and 2GSU (with AMP). All-atom RMSD for residues within 4.5 Å of AMP is 0.017 Å. B. Comparison of mouse NPP2 crystal structures 3NKM and 3NKP (with LPA). All-atom RMSD for residues within 4.5 Å of LPA is 0.44 Å. Glu308 is labeled to emphasize the sidechain showing the largest difference between the free and bound forms of the enzyme.

Figure 3

Alignment of NPP enzyme crystal structures and hNPP7. Positions with conserved residue identity are shown in blue and marked with asterisks. Positions with conservative substitutions are shown in green and marked with colons.

Figure 4

Comparison of hNPP7 models based on three different template structures. Green ribbons represent hNPP7 residues aligned against residues in the template. Magenta ribbons represent hNPP7 residues aligned against gaps in the template. Zn²⁺ ions are shown as blue spheres and the position of N146 is shown as a spacefilling model. A. hNPP7 based on PDB entry 2GSO[16] (Xac. NPP), 24.7% amino acid identity. B. hNPP7 based on PDB entry 2XR9[5] (Rat NPP2), 24.9% amino acid identity. C. hNPP7 based on PDB entry 3NKM[6] (Mouse NPP2), 25.3% amino acid identity.

Figure 5

Comparison of docked substrate positions. Zn²⁺ ions are shown as light blue spheres. Substrates are shown as ball & stick models. hNPP7 residues selected for mutagenesis studies are shown as green sticks and labeled. A. PAF 16:0. B. LPC 16:0. C. pNPPC. D. SM.

Figure 6

Fluorescence signals from Amplex Red assays of LPC hydrolysis in the presence (grey bars) and absence (white bars) of alkaline phosphatase using assay buffer (AB) or conditioned media from either mock or NPP7-transfected HEK293T cells.

Figure 7

Relative k_cat/K_m values of all mutants for hydrolysis of all substrates. Substrates include LPC (white bars), PAF16:0 (light grey bars), pNPPC (dark grey bars) and SM (black bars). The relative k_cat/K_m values were obtained by dividing the k_cat/K_m value of each mutant with that of wild type enzyme against the same substrate.

Figure 8

LPC 16:0 position in NPP7 relative to mutated residues. Panel A. Structure shown represents an energy-minimized snapshot from the MD simulation. Distances from sidechains to LPC 16:0 are 2.7 Å (E169 to hydroxyl), 2.9 Å (L107 to choline), 3.0 Å (F275 aromatic centroid to choline), 3.0 Å (F80 to nonpolar tail), 7.2 Å (Y166 to nonpolar tail), and 9.0 Å (Y142 to nonpolar tail). The two distances showing the greatest standard deviations when measured at 1 ps intervals during a 2000 ps simulation trajectory were to F80 (2.0 Å) and to Y166 (1.7 Å). Panel B. Molecular dynamics simulations of the LPC 16:0 complex with NPP7 show dynamic motion of the nonpolar tail within the hydrophobic channel as reflected in the distances between the nonpolar tail and the sidechains of F80 and Y166. This dynamic motion results in close interactions at any given time point with either F80 or Y166.

Figure 9

SM position in NPP7 relative to mutated residues. Structure shown represents an energy-minimized snapshot from the MD simulation. Distances from sidechains to SM are 2.4 Å (Y166 to sphingoid tail), 2.5 Å (L107 to choline), 2.9Å (E169 to palmitoyl tail), 3.7 Å (F275 aromatic centroid to choline), 5.5 Å (Y142 to sphingoid tail), and 6.9 Å (F80 to sphingoid tail). Mutation of F141 (yellow) to serine was reported by Duan, et al. to substantially reduce sphingomyelinase activity of NPP7 [15]. Distance between F141 and the palmitoyl tail is 2.8 Å.

Figure 10

Crystallographic positions of LPA species in NPP2/ATX (PDB entries 3NKN, 3NKO, 3NKP, 3NKQ, 3NKR[6]). For clarity, the backbone of only 3NKN is represented as a ribbon colored by secondary structure (yellow for β-sheet, red for α-helix, and blue for turns) and the zinc ions of only 3NKN are represented as cyan-colored spheres. Residues Y214 and W275 are shown as sticks due to the correspondence with F80 and Y166 of NPP7, respectively.