Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Abstract

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Keywords: SMPDL3A; crystal structure; enzyme catalysis; nucleoside/nucleotide metabolism; sphingomyelinase; zinc.

FIGURE 1.

Sequence alignment of SMPDL3A and ASMase. The amino acid sequences of the catalytic and C-terminal domains (CTD) of human (h-) and murine (m-) SMPDL3A (80% identity) as well as of human ASMase (29% identity to SMPDL3A) and SMPDL3B (39% identity) are aligned. Cysteines involved in conserved disulfide bonds are highlighted in yellow, metal-coordinating residues are in green, and potential N-glycosylation sites are in gray. The two histidines and the aspartic acid involved in phosphate binding or catalysis are colored in pink. The tyrosine and the glutamine forming the sides of a cleft, which accommodates the adenine base of nucleotides, are shown in blue, as are the corresponding residues of human SMPDL3A. The CTD is delimited by blue brackets. Secondary structure elements of murine SMPDL3A are shown as red cylinders for helices and yellow arrows for strands.

FIGURE 2.

Structure of SMPDL3A. A, the core catalytic domain is shown in green, and the CTD is in blue. Disulfide bonds appear as yellow sticks. N-Linked glycans (white sticks) are only partially displayed for clarity. The two zinc ions in the active site are represented by spheres. N and C termini as well as secondary structure elements are labeled. B, topology diagram of SMPDL3A. The central β-sheets are shown in gray rectangles. Disulfide bonds are represented by yellow connectors. Red circles indicate locations of metal binding or catalytic residues. C, the β-sandwich fold of SMPDL3A (green) is comparable with that of related phosphoesterases including the purple acid phosphatase (PAP, PDB code 1WAR, overall sequence identity = 11%, root mean square deviation = 1.5 Å for 46 corresponding α-carbons from the conserved β-strands only), shown in violet, and calcineurin (PDB code 3LL8, overall sequence identity = 11%, root mean square deviation = 2.6 Å for 46 corresponding α-carbons from the conserved β-strands only) displayed in gold. The loop regions and the CTD of SMPDL3A are hidden for clarity.

FIGURE 3.

Active site of SMPDL3A. Residues that coordinate the two zinc ions (gray spheres) are shown as sticks, as are the two histidines (His-111 and His-149) interacting with a sulfate ion (sticks). The electron density for the water molecule (red sphere) located midway between the zinc ions and for the sulfate ion is displayed as a F_o − F_c-simulated annealing omit map contoured at 8σ. Distances are indicated (Å).

FIGURE 4.

Inhibition of SMPDL3A by excess zinc. The structure of the protein exposed to a high zinc concentration reveals a third zinc ion bound to the active site. Zinc ions (spheres) with their corresponding anomalous difference electron density map peaks (yellow mesh) are shown contoured at 10σ.

FIGURE 5.

Ligands bound in the active site of SMPDL3A. The structures of ligands present in the active site are displayed along with their corresponding electron density F_o − F_c simulated annealing omit maps contoured at 3σ. A and B, phosphocholine (PC). C and D, AMP. E and F, ADP analog (AMPCP). Zinc ions and water molecules are represented by gray and red spheres, respectively. The two residues interacting with the adenine base are shown as sticks and labeled.

FIGURE 6.

Proposed reaction mechanism for SMPDL3A. A, the active site of SMPDL3A is compared with that of calcineurin (PDB code 3LL8). Residues in beige, one zinc ion, and one iron ion (orange sphere) form the calcineurin catalytic center occupied by a phosphate ion (orange central atom). Residues in green and a sulfate ion (yellow central atom) are part of the SMPDL3A active site. Active site residue labels in parentheses refer to calcineurin. B, a comparison of SMPDL3A bound to AMP and AMPCP is shown (only the ligands are depicted). The structures are in the identical orientation to illustrate the difference in the terminal phosphate group between the two ligands. Zinc ions and the nucleophilic water are labeled. C, model of ATP substrate bound to SMPDL3A. An ATP molecule was manually docked into the substrate binding site using the AMP complex structure as a guide. The β-phosphate is positioned for nucleophilic attack by a water molecule (red sphere), whereas the γ-phosphate acts as the leaving group. Potential interactions with protein residues are indicated. D, schematic diagram of the proposed ATP hydrolysis mechanism for SMPDL3A, which involves a nucleophilic attack by a zinc-activated water molecule on the β-phosphate. This is followed by protonation of the departing γ-phosphate by one of the two nearby histidines, likely His-111 with assistance from Asp-79.