Crystal Structure of the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3B Provides Insights into Determinants of Substrate Specificity*

The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.

Sphingomyelins (SM) 3 represent a major class of phospholipids found in cellular membranes and are particularly enriched in the outer leaflet of the plasma membrane (1). Removal of the phosphocholine (PC) head group from SM leads to the generation of ceramide sphingolipids, whose resting levels in the plasma membrane are extremely low and which have been shown to alter biophysical properties of the membrane and affect cellular processes (2). This reaction is carried out by enzymes called sphingomyelinases (SMases). In mammals, three SMase families can be distinguished: the alkaline SMase is expressed on microvilli in the intestines, where it metabolizes dietary SM (3); the four neutral SMases are localized in the plasma membrane inner leaflet, Golgi, endoplasmic reticulum, nucleus, or mitochondria (4); and the acid SMase (ASMase, ASM, SMPD1) is found in the lysosome where it serves catabolic purposes and can also be secreted to act on plasma lipoproteins or the cell membrane (5). This latter function occurs in response to numerous stresses and modulates signaling events via ceramide production (5).
ASMase is the founding member of a family of three paralogs, along with the relatively poorly characterized enzymes SMPDL3A and SMPDL3B. Interestingly, SMPDL3A is not a SMase but hydrolyzes various nucleotide triphosphates and diphosphates (6). In this regard, an anti-inflammatory role in purinergic signaling in the extracellular space was proposed for SMPDL3A (6), although it was also reported to be found in lysosomes (7). However, its function is still linked with lipid metabolism, because its expression and secretion is stimulated by cholesterol and liver X receptor ligands in macrophages (6,8).
On the other hand, SMPDL3B is a glycosylphosphatidylinositol (GPI)-anchored protein (9 -11) with reported roles in inflammatory processes, as well as in kidney diseases. A negative regulatory role in innate immune signaling was uncovered in murine macrophages, where the enzyme is found on the cell surface and up-regulated upon inflammatory stimuli (11,12). Its knockdown results in enhanced responsiveness to Toll-like receptor (TLR) stimulation and Smpdl3b-deficient mice display an increased inflammatory response. Furthermore, deficiency of this protein in macrophages is associated with alterations in the cellular lipid composition and changes in membrane fluidity.
In the kidney disease focal segmental glomerulosclerosis, expression of SMPDL3B on podocytes is reduced; overexpression of the enzyme prevents actin cytoskeleton disruption and apoptosis associated with this condition (13). Rituximab, a therapeutic anti-CD20 monoclonal antibody targeting B cells but also used in certain nephrological conditions, binds to SMPDL3B (13)(14)(15) and remediates these defects in a SMPDL3B-dependent manner (13). Rituximab similarly protects podocytes from morphological disruption, decreased proliferation, and SMPDL3B protein loss in xenotransplants (14). In contrast, this protein is elevated in glomeruli from patients with diabetic kidney disease where it binds to the soluble urokinase plasminogen activator receptor on podocytes; SMPDL3B knockdown protects podocytes from apoptosis in this condition (16).
The identification of this enzyme at other locations suggests additional functions. For instance, SMPDL3B has been found in pancreatic zymogen granules, an organelle containing digestive enzymes to be released into the pancreatic duct (17); in saliva exosomes, which are secreted vesicles with various functions (18); in the plasma protein-depleted cerebrospinal fluid (19); and in milk (20).
Although these findings provide clues as to the potential roles of SMPDL3B, its precise function and substrate specificity remain unknown. That it is membrane-anchored, bears similarity to ASMase, and possesses membrane-modulating activities suggests that it may act on lipid substrates. On the other hand, its other homolog, SMPDL3A, is a nucleotide phosphodiesterase. The crystal structures of ASMase and SMPDL3A were recently reported (21)(22)(23)(24), but no structural information is as yet available for SMPDL3B.
Here we present the high resolution crystal structure of murine SMPDL3B, which revealed that its substrate binding site is a narrow pocket, quite distinct from its paralogs, a consequence of a unique conformation of a surface loop. The structure of the enzyme in complex with PC showed that this potential product is accommodated inside a narrow pocket surrounded by a "cage" of aromatic side chains, a common feature of choline-binding proteins (25). Although no in vitro enzymatic activity against lipids could be detected, SMPDL3B can cleave other choline-containing substrates including CDPcholine and releases phosphate from ATP and ADP. Functional experiments in murine macrophages further revealed amino acid residues lining the channel leading to the active site of SMPDL3B as potentially important for recognition of endogenous substrate molecules involved in the modulation of macrophage function.

Results
Overall Structure-We determined the crystal structure of full-length murine SMPDL3B, which shares 78% identity with its human counterpart. Our construct excluded the N-terminal signal peptide, as well as the C-terminal GPI attachment signal peptide, which is normally cleaved off to generate a new C terminus, the -site of GPI attachment. The -site is Ala-431 for human SMPDL3B (10), corresponding to Gly-431 in the murine protein. Although the crystallized construct contained four additional residues beyond the -site (residues 19 -435), Gly-431 is the last residue visible in the crystal structure. The structure was solved by single-wavelength anomalous diffraction from two zinc ions in the active site and refined to a resolution of 1.14 Å ( Table 1).
The enzyme comprises a catalytic domain affixed with a small C-terminal subdomain (CTD) (Fig. 1). The catalytic domain consists of two central mixed ␤-sheets of six strands each, surrounded by eight ␣-helices. This architecture classifies SMPDL3B in the calcineurin-like phosphoesterase structural superfamily (PFAM code PF00149). One end of the ␤-sandwich has a wide opening, against which the CTD, consisting of a globular cluster of four ␣-helices, is packed up. The fourth helix of the CTD is terminated by Gly-431. The CTD appears to be a defining feature of the ASMase-like proteins because it is also found in ASMase (5) and SMPDL3A (6), but not in other phosphoesterases. It displays lower sequence conservation than the catalytic domain, and its exact function is unknown, but it may enhance the stability of these proteins in their lysosomal and extracellular environments.
The protein fold is stabilized by a total of three disulfide bridges: two in the CTD and a third at the base of a large loop in the catalytic domain. Although murine SMPDL3B has five potential sites of N-linked glycosylation, only three glycans are discernable in the structure. The two other sites are located on the surface of the protein, suggesting that they potentially can be glycosylated. Human SMPDL3B shares only two of these five sites and has a third potential one, also on the surface of the protein.
Structural superimposition of SMPDL3B with the ASMase catalytic domain and with SMPDL3A ( Fig. 2A) shows that the overall fold and all secondary structure elements are well conserved within this subfamily. The main differences arise in the lengths and compositions of several loops on the "top" face of the proteins, which harbors the active site.
Active Site and Substrate Recognition-At the core of SMPDL3B are found two zinc ions coordinated by the side chains of seven residues located on loops extending from the central ␤-sheets (Fig. 2B). Two histidines, His-135 along with the His-97-Asp-65 pair, complete the active site of the enzyme. All of these residues are perfectly conserved in SMPDL3A and ASMase (Fig. 2B), suggesting that all three family members utilize the same general catalytic mechanism for substrate hydrolysis. Briefly, a phosphate-containing substrate would bind in the active site mainly via strong electrostatic interactions between the zinc ions and the phosphate moiety. A zinc-activated nucleophilic water molecule would then attack the phosphate, resulting in departure of the leaving group, protonated by one of the neighboring histidines (21)(22)(23)(24). The nucleophilic water molecule coordinated midway between the two zinc ions is clearly visible in the high-resolution structure of SMPDL3B (Fig. 2B).
Although the three phosphodiesterases likely employ the same catalytic mechanism, the identity of the phosphate-containing substrate varies widely within this subfamily. ASMase hydrolyzes SM to ceramide and PC. The related lipid phosphatidylcholine, bearing the same PC head group, may also serve as substrate to a lower extent (5). On the other hand, SMPDL3A is inactive against SM and instead hydrolyzes various nucleotide triphosphates and diphosphates (6). The physiological substrate of SMPDL3B, however, is not known. Interestingly, despite the overall structures of the three enzymes being highly conserved ( Fig. 2A), the shapes of the regions surrounding their active sites are markedly different (Fig. 3A). The structure of ASMase in complex with sulfate (24) displays a wide depression emanating from the sulfate and zinc ions at the active site. In contrast, murine SMPDL3A (22) accommodates its nucleotide substrate in a roughly T-shaped cleft, which restricts the type of molecules that can enter its active site. Human SMPDL3A displays a similar T-shaped configuration (21). In all cases, the regions adjacent to the active site of these proteins are surface-exposed depressions.
Strikingly, the structure of SMPDL3B revealed a relatively closed off active site with a narrow boot-shaped cavity accessible through a small circular opening on the protein surface (Fig.  3, A and B). The active site zinc ions are located at the heel of the boot, indicating that substrates must reach deep into this cavity. The unique nature of the SMPDL3B substrate binding site is mainly due to a loop insertion between strand ␤9 and strand ␤10 (L9 -10), which assumes a distinct conformation relative to its paralogs (Fig. 3C) and forms the "tongue" of the boot-shaped substrate-binding cavity. In ASMase, this insertion is essentially absent, leading to its more expansive substrate binding site. In SMPDL3A, this region is shorter by three residues and is pinned back by hydrophobic interactions between Val-322 at the tip of the loop and Ala-65 on a neighboring loop (Fig. 3D). This creates space for the accommodation of nucleobases in this region of SMPDL3A. In SMPDL3B, however, the sequence of L9 -10 differs from SMPDL3A, and this loop is oriented toward the substrate binding site, leaving a small, 9 Å opening for substrate entry.
The shape of the substrate binding site provides clues as to the nature of the phosphate-containing substrate of this enzyme. Given that SMPDL3B could potentially act on lipids at the membrane surface (11), we wondered whether it can accommodate the small PC head group of SM. To assess this, we crystallized the protein in presence of PC and found that it is indeed located in the substrate binding cavity (Fig. 4A). Its phosphate group forms numerous electrostatic interactions with the zinc ions and several active site residues as observed previously in structures of ASMase and SMPDL3A (Fig. 4B). The choline moiety extends into the cavity and interacts with the backbone carbonyl of His-277 and the side chains of Leu-310 and Thr-308, which emanate from L9 -10 of SMPDL3B. This space is occupied by a glycerol molecule from the crystallization condition in the apo structure. Additionally, the choline moiety is surrounded by five aromatic residues: Phe-242, Trp-250, Trp-306, His-277, and His-279, the latter two of which also participate in zinc coordination. This arrangement of several aromatic residues within 4 -5 Å of the cationic quaternary amine has been termed a "cationbox" and is a common choline recognition motif (25).
The dimensions of the PC group just about match the size of the substrate binding site opening, suggesting that it may have to adjust slightly to allow PC access to the buried active site.
Comparison of the PC-bound structure with the apo form, however, revealed no conformational changes, although binding of PC seems to increase the disorder in L9 -10 as interpreted from higher crystal temperature factors (an increase by 18 -64% in alpha carbon temperature factors normalized to the entire protein). Thus, L9 -10 may have some malleability to allow entry of small substrates like PC, or it can perhaps move out of the way altogether to accommodate larger substrates. To assess these possibilities, we turned to mutagenesis and enzymatic assays. Enzymatic Activity in Vitro-It had been previously shown that SMPDL3B possesses phosphodiesterase activity against the artificial generic substrate bis(4-nitrophenyl) phosphate (bNPP) (11). Based on its relatedness to ASMase and its effects on cellular lipid composition and membrane biophysical properties (11), we initially sought to study the activity of the purified enzyme in vitro against PC-containing lipids. Interestingly, no head group hydrolysis was detected with the following substrates: SM in Triton X-100 micelles, SM in neutral liposomes (also comprising phosphatidylcholine and cholesterol), the soluble micelle-forming lipids sphingosylphosphorylcholine (lyso-SM), and platelet-activating factor. These assays, however, were carried out with the soluble form of the protein used for crystallization that lacks the GPI moiety responsible for anchoring SMPDL3B to the cell surface (9,10,11). Thus, it may be that the membrane tethered form of the enzyme is capable of hydrolyzing lipids. On the other hand, SMPDL3B was able to cleave CDP-choline (Fig.  5C), an essential building block for the biosynthesis of PCcontaining phospholipids in the Kennedy pathway (26). Additionally, as with SMPDL3A, we found that SMPDL3B can release phosphate from ATP and ADP (Fig. 5, D and E), but not from AMP.
To assess the role of L9 -10 in substrate binding, this loop was replaced by a glycine residue, GG, or GSG. However, none of these modifications enabled hydrolysis of lipid substrates. Notably, shortening of L9 -10 did increase activity against  CDP-choline, as well as bNPP, and the artificial substrate p-nitrophenylphosphorylcholine (NPPC) (Fig. 5, A-C), suggesting that it does regulate access to the active site. In theory, CDPcholine could be recognized by the protein either with its choline group positioned at the bottom of the cavity, as in the PC bound structure (Fig. 4A), or in the reverse orientation with the nucleotide moiety occupying that location, as occurs with SMPDL3A substrates (21,22). The latter would require a stable rearrangement of L9 -10 because the cytidine moiety from CDP-choline is too large to fit in the observed substrate cavity.
To ascertain between these possibilities, the enzyme was challenged with diadenosine tetraphosphate (AP4A), a substrate with nucleosides at both ends of a tetraphosphate group. Purified SMPDL3A can cleave this molecule, but SMPDL3B is completely inactive against it (Fig. 5F). Moreover, shortening of L9 -10 did not allow hydrolysis of AP4A (although the G loop swap did have a very low rate of activity). This suggests that L9 -10 cannot stably rearrange into a different conformation to accommodate larger substrates like nucleosides. In the case of CDP-choline, its PC moiety would be bound to the active site as observed in the PC bound structure, and for ATP/ADP, only the terminal phosphate would enter the active site. Thus, L9 -10 appears to act as a relatively fixed selectivity filter that through slight "breathing motions" can allow entry of smaller substrates like PC and isolated phosphate ends.
Enzymatic Activity in Cells-The observed activity on nucleotides, including CDP-choline, and the absence of knowledge of the precise physiological substrate lead us to examine other residues in addition to the aromatic cage and the L9 -10 loop that may interact with the substrate. The walls of the channel leading to the active site are lined with amino acids involved in catalysis but also with four other residues (Lys-140, Asn-141, Tyr-198, and Asn-200; Fig. 6A), two of which, Lys-140 and Tyr-198, are highly conserved in vertebrates. We introduced mutations in these residues, as well as in His-135, one of the conserved amino acids contacting the substrate phosphate group. These mutations were designed to avoid disrupting local pro-tein structure, but could potentially abrogate polar contacts with the physiological substrate. SMPDL3B was reported to negatively regulate TLR signaling in murine macrophages, affecting the release of pro-inflammatory cytokines from these cells (11). To test the impact of the mutants in such an experimental system, we generated a series of stable RAW264.7 macrophage cell lines, overexpressing an empty vector control (mock), wild type, or mutant forms of SMPDL3B. Western blot analysis revealed that introduction of all SMPDL3B constructs led to similarly increased protein expression as compared with the endogenous level observed in control cells (mock) (Fig. 6B). The cell lines were subsequently stimulated with the TLR ligand LPS and evaluated for cell-associated enzymatic activity toward the experimental substrate bNPP, as well as for release of the TLR-induced cytokine IL-6. Overexpression of wild type SMPDL3B led to an increase in cell-associated enzymatic activity toward bNPP (Fig. 6C) and concomitantly to a significant reduction of IL-6 release in comparison with control (Fig. 6D), confirming its negative impact on TLR-dependent processes. Individual mutants differed in their ability to hydrolyze the experimental substrate bNPP, with K140M/N141A and H135A almost completely failing to increase detectable activity (Fig.  6C). Interestingly, these are also the only two mutants tested that failed to significantly reduce LPS-induced IL-6 release from RAW264.7 cells in this experimental system (Fig. 6, D and  E). The molecular position of His-135, Lys-140, and Asn-141 in relation to the PC group in the structure, along with the functional data, suggest that these residues are indeed important features for recognition and binding of endogenous substrates affecting the activity of macrophages.

Discussion
The overall structure of SMPDL3B determined here is similar to that of its paralogs SMPDL3A and ASMase. All three possess a highly conserved active site that hydrolyzes phosphate-containing substrates via a zinc-activated water molecule. Despite this, differences in protein localization and the shape of the substrate binding site appear to confer functional differences in these proteins. ASMase has an additional N-terminal saposin domain, a small membrane-and lipid-binding module that enables it to act on its SM substrate on the outer leaflet of the plasma membrane, on intralysosomal vesicles, on lipoprotein particles in the circulation, and at other locations  (5). Additionally, ASMase has a relatively wide and accessible substrate binding site with optimal activity coupled to the conformation and position of the saposin domain relative to the catalytic domain (24). Conversely, SMPDL3A is not known to be membrane-interacting and has a T-shaped substrate binding site. These attributes alter its substrate specificity to soluble nucleoside-diphosphates and triphosphates and implicate it in purinergic signaling pathways (6).
In contrast to its paralogs, SMPDL3B is constitutively attached to the cell membrane exoplasmic face via a GPI anchor (9 -11), and the structure reveals that its substrate binding site is different from either paralog. An extended and conformationally distinct loop, L9 -10, closes off a large portion of the surface near the active site, leaving a small restricted opening. This configuration of the active site might suggest an altogether different substrate specificity for SMPDL3B from either ASMase or SMPDL3A. However, the point of GPI attachment of SMPDL3B is located on the "top" face of the enzyme (Fig. 1) and places the active site opening in proximity to the membrane lipid head groups. Moreover, previous studies implicated SMPDL3B in TLR signaling, where its action on macrophage membranes modulated lipid composition, affecting the levels of SM, other PC-based lipids, and ceramide, concomitantly altering membrane fluidity (11).
This notion is further bolstered by the structure of SMPDL3B bound to PC. The small molecule head group of SM binds in the tight cavity of the active site, and the chemical surroundings are appropriate for choline recognition as multiple aromatic residues around the quaternary ammonium group establish cationinteractions with it. A more anionic environment would have been expected for phosphoethanolamine or other cationic or polar head groups, which would be inappro-priate for choline because it cannot form hydrogen bonds or salt bridges with protein residues.
The difference in conformation of L9 -10 from the corresponding loops in SMPDL3A or ASMase and that it was slightly more mobile in the presence of PC raised the possibility of a switch-like regulatory role for this segment. Although replacement of L9 -10 does increase activity on some substrates tested, there is no indication that a stable conformational change occurs, at least in solution. Instead, the conformation of L9 -10 appears to be more or less fixed, allowing entry of only appropriately sized substrates through small breathing motions. Alternatively, the conformation of L9 -10 may be impacted at the membrane by presence of lipid head groups, because this loop is on the side of the protein likely to face the membrane. This could also apply to the other loops that diverge between SMDPL3B and its paralogs and that are found on the same face ( Fig. 2A).
Based on the crystal structure, we identified amino acid residues potentially required for recognition of endogenous substrates, and evaluated their importance for SMPDL3B-dependent effects in a cellular model of LPS-induced IL-6 release. In addition to His-135, a conserved residue mediating interactions with the phosphate group, we found that a mutant affecting both Lys-140 and Asn-141, two amino acids situated in the channel leading to the active site of SMPDL3B, failed to significantly reduce LPS-induced IL-6 release. The location of these amino acids relative to PC seen in the structure, along with the functional information, makes it likely that these residues indeed specifically participate in substrate recognition.
Taken together, these findings suggest that SM with its small PC head group is a viable candidate substrate for SMPDL3B. Manual modeling of SM based on previous docking of SM onto FIGURE 6. Functional impact of SMPDL3B mutants on LPS-induced IL-6 release in macrophages. A, residues lining the wall of the channel leading to the active site are displayed in orange along with their degree of conservation in vertebrates. Other adjacent amino acids, including the catalytic machinery, are in green. B, Western blot analysis for SMPDL3B, HA, and tubulin in samples from RAW264.7 macrophages stably transduced with an empty vector (mock) or HA-tagged wild type or mutant murine SMPDL3B constructs as indicated. C and D, RAW264.7 macrophages stably transduced with an empty vector (mock) or HA-tagged wild type or mutant murine SMPDL3B constructs were stimulated with 100 ng/ml LPS for 8 h. The cells were probed for enzymatic activity by incubation with bNPP (C), and supernatants were analyzed for IL-6 release by ELISA (D). Experimental data were normalized to mock and represent means Ϯ S.E. from five independent biological replicates. Statistical significance was tested using a one sample t test against a hypothetical mean value of 1. *, p Յ 0.05; n.s., not significant. E, relative enzymatic activity and relative IL-6 release shown in C and D plotted against each other.
ASMase (23,24) shows that in both cases, SM is positioned similarly and can insert into the active site with few clashes (Fig.  7A). With SMPDL3B, only the headgroup of SM would be stably bound, whereas with ASMase, portions of the ceramide moiety may also interact with the protein with the aid of is accompanying saposin domain. Although we did not observe any in vitro hydrolysis of SM in detergent micelles or liposomes or of other soluble lipids even when the loop L9 -10 is replaced, this is likely because the protein requires proper attachment to membranes for substrate hydrolysis to occur.
Alternatively, the physiological substrate could in fact be a non-lipid molecule. We found that the enzyme can hydrolyze CDP-choline, the activated metabolite of PC used in lipid biosynthesis, in vitro. Even though this molecule is usually found intracellularly, it might encounter SMPDL3B under specific physiological conditions, such as inflammatory tissue damage or digestive processes (27). However, the affinity and activity rate of the protein on CDP-choline are rather low (Fig. 5C). We also found activity against ATP and ADP. In all of these cases, however, the means by which substrate molecules are hydrolyzed would be different from SMPDL3A, which also acts on nucleoside diphosphates and triphosphates (Fig. 7B). In SMPDL3A, during the hydrolysis of ATP or ADP, the phosphate preceding the terminal phosphate (␤-phosphate for ATP, ␣-phosphate for ADP) must be coordinated by the zinc ions for attack by the nucleophilic water molecule, and the terminal phosphate (␥-phosphate for ATP, ␤-phosphate for ADP) is the leaving group because of the constraints imposed by the position of the protonating histidine side chain and orientation of the nucleobase in the T-shaped substrate binding site (22). Conversely, in SMPDL3B, because of the block imposed by L9 -10 preventing accommodation of large moieties like complex glycolipids or nucleobases inside the cavity, ATP or ADP phosphate release likely proceeds in a "tail-first" manner in which the terminal phosphate contacts the zinc ions in the active site for nucleophilic attack, while the rest of the molecule remains in the channel leading to them and is the leaving group upon hydrolysis. The suitability of nucleotides as physiological substrates of SMPDL3B, however, is unclear because of their weak, millimolar K m (Fig. 5, D and E) and relatively low activity rate, about 10-fold slower than SMPDL3A at neutral pH (22).
In summary, the structure of murine SMPDL3B highlights molecular details important for catalysis and substrate recognition. This knowledge will further contribute to the understanding of which substrates are bound and hydrolyzed under physiological conditions and, as a consequence, how SMPDL3B influences cellular properties such as lipid composition or inflammatory signaling. The tight cavity forming its substrate binding site, differing from its paralogs, also provides a promising feature for the identification of enzyme-specific inhibitors that could be exploited for modulation of SMPDL3B-dependent functions in relevant experimental systems, including immunity and kidney biology.

Experimental Procedures
Constructs-For expression in Sf9 insect cells, murine SMPDL3B (residues 19 -435; UniProt no. P58242) was subcloned into a derivative of pFastBac 1 (Invitrogen). The vector contained the melittin signal peptide MKFLVNVALVFMV-VYISYIYA followed by a hexahistidine tag DRHHHHHHKL. Swaps of the loop L9 -10 consisted of residues 307-316 (KTTLPGVVDG) replaced by a glycine, GG, or GSG. Expression constructs for mammalian cells were based on a derivative of the entry vector pEntry1A (Invitrogen) encoding the signal peptide of influenza HA followed by an HA tag and the cDNA for murine SMPDL3B (residues 19 -456) as reported previously (11). Individual point mutations corresponding to H135A, K140M, N141A, K140M/N141A, Y198F, N200A, and Y198F/N200A were introduced by site-directed mutagenesis using the QuikChange II system (Agilent). For retroviral gene transduction, cDNAs were shuttled to a derivative of pMSCV-GW containing a blasticidin resistance gene using the Gateway LR Clonase (Invitrogen) system. All constructs and mutants were verified by sequencing.
Protein Production-Soluble SMPDL3B was expressed as a secreted protein in Sf9 cells and purified as previously described for SMPDL3A (22). The protein eluted as a monomer in size exclusion chromatography.
Crystallization and Data Collection-Soluble SMPDL3B at 10 mg ml Ϫ1 in buffer (15 mM Tris-HCl, pH 7.5, 100 mM NaCl) was mixed with an equal volume of well solution consisting of 0.2 M NH 4 NO 3 and 25% PEG 3350. Crystals were grown by hanging drop vapor diffusion at 22°C. For anomalous diffraction experiments, the crystals were soaked in well solution supplemented with 10 mM ZnCl 2 . PC-bound crystals were obtained by preincubating and crystallizing the protein with 20 mM PC. Diffraction data were collected at 100 K on Beamline 08ID-1 at the Canadian Macromolecular Crystallography Facility, Canadian Light Source, and on Beamlines MacCHESS A1 and F1, Cornell High Energy Synchrotron Source. The data were processed with HKL2000 (28).
Structure Determination and Refinement-The structure of SMPDL3B was solved by zinc single-wavelength anomalous diffraction using Autosol in Phenix (29,30) and manually rebuilt in Coot (31). The PC-bound structure was obtained by molecular replacement using Phaser (32) in Phenix. Refinement was performed with phenix.refine (33). Data processing and refinement statistics are presented in Table 1. Structural images were generated with PyMOL (PyMOL Molecular Graphics System, version 1.3; Schrödinger).
In Vitro Enzymatic Activity Assays-Proteins for activity assays were purified as described above, but the final buffer included 10 M ZnCl 2 . To detect hydrolysis of the cholinecontaining substrates SM in Triton X-100 micelles, SM in neutral liposomes (also comprising phosphatidylcholine and cholesterol), sphingosylphosphorylcholine (lyso-SM), plateletactivating factor and CDP-choline, the Amplex Red sphingomyelinase assay (Thermo Fisher Scientific) was slightly modified. Proteins at 5-25 nM for CDP-choline or up to 10 M otherwise were incubated with substrate in buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl) for 1 h at 37°C. The reaction was terminated at 95°C for 5 min, and an equal volume of the second step solution was added as recommended. The second step was carried out at 37°C, and the change in fluorescence (560-nm excitation and 590-nm emission) after 30 min was used to quantify product formation with the help of a PC stan-