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

Alexei Gorelik,Katalin Illes,Giulio Superti-Furga,Bhushan Nagar

doi:10.1074/jbc.m115.711085

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.

Highlights

acid sphingomyelinase (ASMase) is a member of a small family of three paralogs that includes SMPDL3A and SMPLD3B
The structure consists of two mixed ␤-sheets at its core flanked by ␣-helices (Fig. 2B); this architecture classifies it as a member of the calcineurin-like phosphoesterase structural superfamily (PFAM code PF00149, Fig. 2C), which contains nucleotidases and, most notably, the PPP family of serine/threonine phosphatases [28]
This study reports the first structural characterization of a member of the ASMase-like family of enzymes

Summary

Experimental Procedures

Constructs—SMPDL3A from mouse (residues 23– 445; Uniprot #P70158) and human (residues 23– 453; Uniprot # Q92484) were subcloned into a derivative of pFastBac 1 (Invitrogen). Enzymatic Assay with Generic Phosphodiesterase Substrate— Protein at 50 nM was incubated with 1.7 mM bis(p-nitrophenyl) phosphate in assay buffer (100 mM NaCl and 100 mM Tris-HCl, pH 7.5, or sodium acetate, pH 5) at 37 °C. Protein at 1 nM to 2 ␮M was incubated with 4.35 ␮M to 2 mM ATP in assay buffer supplemented with 5 ␮M ZnCl2 at 37 °C. Four volumes of Biomol Green reagent were added followed by incubation and absorbance measurement at 620 nm. The difference in absorbance after 15 min of ATP hydrolysis by SMPDL3A was used to interpolate the amount of phosphate produced via a standard curve. Crystals were soaked in well solution supplemented with 20 mM ZnCl2. Ligand-bound structures were obtained by crystallizing the protein in the presence of 50 mM phosphocholine, AMP, or ␣,␤-methylene ATP (AMPCPP).

Results

PDB code Data collection

Protein km kcat

Discussion